Developmental Bio 2

7. What are three dominant gain of function mutations identified by Ed Lewis and what effect do they have on segment identity in the adult fruit fly?

*Dominant Gain of Function Mutation: Activation of gene in the wrong place can transform segment's identity. The abnormal activity of a gene in an anterior segment gives that segment a more posterior segmental identity.* -Cbx (contrabithorax) T2 -> T3 no wings, 4 halteres. -Hab (hyperabdominal) T3 -> A2 4 legs, larger abdomen. -Mcp (miscadastral pigmentation) A4 -> A5 enlarged area of abdominal pigmentation.

5. What are three transcription factors required for Spemann's organizer function?

*Not, Goosecoid and Lim1* = Transcription factors required for Spemann organizer function

6. What are three recessive loss of function mutations identified by Ed Lewis and what effect do they have on segment identity in the adult fruit fly? (WORKSHEET)

*Recessive Loss of function Mutation: Removal of a gene's activity changes a segment into a more anterior segment* -bx mutation transforms the anterior compartment of the haltere (T3) into a wing (T2). -pbx mutation transforms the posterior compartment of the haltere (T3) into a wing (T2). -bx pbx/bx pbx (double mutant) is a four-winged fly (complete T3 -> T2).

6. What is a key target of ectodermin that promotes the formation of ectoderm at the expense of mesoderm?

*Smad4* is associated with the determination of mesoderm in both of these pathways(BMP and Nodal TGF-B). Ectodermin's ubiquinization of Smad4 promotes the formation of ectoderm at the expense of mesoderm.

7. What are three growth factors that promote ventral development and are targets of the secreted antagonists, Chordin, Noggin, Cerberus, and Fzrb-1 and Dkk-1?

*Xwnt, Xnrs, and BMP* -Frzb1, Dkk-1 ---| Xwnt-8 -Cerberus---| Xwnt-8,Xnrs, BMPs -Chordin, Noggin, Follistatin---| BMPs

4. What proteins in the extra cellular matrix coordinate cell signaling and communication of neural crest cells?

-*CCN (connective tissue growth factor) proteins* physically interact with several extracellular matrix (ECM) proteins and growth factors (BMP, TBFB, FGF2). -CCN proteins are a family of proteins that are Wnt-induced secreted proteins that act as ligands in signaling pathways. -CCN proteins likely constitute a hub for the coordination of cell signaling and communication

19. How does Cerberus block primitive streak formation? What two mesodermal inducing signals cause the primitive streak to form?

-*Cerberus inhibits the proteins BMP4, Xnr1, and Xwnt8*, important for proper head induction. -Cerberus gene encodes a polypeptide that is 270 amino acids in length. -This gene encodes a cytokine member of the cysteine knot superfamily, characterized by nine conserved cysteines and a cysteine knot (3 disulfide bridges) region. -Binds directly to BMP-4 (or Nodal or Wnt), preventing BMP-4 from binding to its receptor. -*As the hypoblast is displaced (removing Cerberus' negative effect), Nodal from the epiblast and FGF from Koller's Sickle induce epiblast cells to internalize and a new primitive streak is formed.*

9. What are the members of the pathway that starts with FGF8 expression at Hensen's node and results in the expression of hairy? What is associated with the wavefront and what is associated with the clock? What turns on Wnt in a gradient from Hensen's Node?

-*FGF8 Wavefront* (with its highest concentration in Hensen's Node) induces Wnt. -Wnt induces *Notch (clock)*at and Notch induces the oscillating expression of hairy.

8. What are two other factors that can result in the dedifferentiation of muscle cells?

-*Thrombin*: +Proteolytic enzyme that is part of blood clotting cascade, but provides an environment for dedifferentiation. +Multinucleate post-mitotic newt muscle cells can be converted to dividing mononuclear cells in culture in the presence of thrombin in culture medium. Cells enter S-phase -*Msx-1*: +Dedifferentiation of muscle cells also involved renewed expression of the homeodomain transcription factor, Msx-1. +Msx-1 prevents myogenic differentiation in mammalian cells. +Msx-1 expression found at sites of inductive cell-to-cell interaction during embryogenesis (as in the limb bud). +Its expression is characteristic of undifferentiated mesenchymal cells that can undergo regeneration.

1st phase and 2nd phase

-1st phase: regulation of segment polarity genes by the pair-rule genes. -The expression of the maternal, gap and pair-rule genes fades away early in development, thus a new mechanism for regulating engrailed and wingless is needed. -Cellularization of embryo has occurred by this stage and its turns out that the mechanism for maintaining wingless and engrailed expression is based on cell to cell communication. -This is why not all of the segment polarity genes are transcription factors.

7. Which Hox genes are involved in limb development?

-23 Hox genes are expressed in the developing chick limb. -*Hoxa and Hoxd* clusters are expressed in both fore and hind limb buds. -*Hoxd9-13* are expressed sequentially nested pattern such that Hoxd9 is expressed throughout but all Hoxd9-13 are expressed in the small posterior region. -Hoxd seems to control A/P (i.e. finger) identity. -Hoxa9-13 are expressed in a nested proximo-distal pattern. i.e. Hoxa9 only in the presumptive upper limb, Hoxa9-11 in the lower limb region and Hoxa9-13 in the upper limb region to give rise to wrist and digits.

10. What is responsible for the segmentation clock? Make sure you know the steps in the Notch/Delta signaling pathway. What turns off this signaling pathway and how?

-A segmentation clock, generated by a Notch-signaling-based oscillator, is central to somitogenesis in vertebrates -Binding of Delta to Notch triggers the cleavage of the intracellular domain of Notch (=> Notch Intra, which goes into the nucleus and activates target genes like hairy). Responsible for somite boundary and polarity. -Lunatic fringe (a glycosyltransferase - inactivates the Notch receptor binding Delta). Notch activation also turns on the HES gene which inactivates Lunatic Fringe, re-enabling the Notch receptor to bind to Delta (accounting for the oscillating clock model). Each wave of hairy (HES) expression that precedes the formation of one somite, may be due to the timing of these feedback loops in the Pre-Somitic Mesoderm.

AP-2 - Biomarker of Neural Crest

-A transcription factor that is inducible by retinoic acid, AP-2, is expressed by crest cells and neurepithelial cells at the neural folds. -AP-2 null mice display cranio-abdominoschisis and severe dysmorphogenesis of the face, skull, sensory organs, and crest-derived cranial ganglia. -AP-2 is not required for neural crest migration, but it does appear to be necessary for the regulation of neural tube closure, and its absence causes abnormalities in crest-derived structures.

5. What is a blastema and what is it formed from?

-Amputation of a limb triggers epidermal cells migrate from the edges of the wound and cover over the wound surface to form a blastema. -The cells of the blastema arise from beneath the wound epidermis, dedifferentiate and start to divide. Blastema cells are derived locally from the mesenchymal tissues of the stump, close to the site of amputation

8. What is a teratogen? What are three categories or types of teratogens? Why should pregnant women avoid all contact with teratogens, particularly during the first three months of pregnancy?

-An agent that can cause malformations of an embryo or fetus. This can be a chemical substance, a virus or ionizing radiation. -Thalidomide, Alcohol -Rubella virus -X-rays -Pregnant women should avoid all contact with teratogens, particularly during the first three months of pregnancy. It is estimated 10% of all birth defects are caused by prenatal exposure or teratogen. first three months because thats when major anomalies occur

11. What is a negative regulator of Wnt in the wavefront/clock pathway?

-Axin acts in a negative feedback loop to turn off Wnt3a. -Before Wnt3a is turned off, Wnt3A has induced Notch. -Before Notch is turned off by Lfringe, Notch has induced hairy.

What is moving from the vegetal pole to the dorsal side during cortical rotation?

-B-catenin - vertebrate homolog of Dros. armadillo -Dsh protein - vertebrate homolog of Dros. dishevelled -Wnt 11 - vertebrate homolog of Dros. wingless

12. What effect does inhibiting all BMP signaling have on the formation of neural tissue in the embryo? What effect does depleting all of the BMP antagonists have on the formation of neural tissue in the embryo?

-BMP depletion causes ventral mesoderm to acquire dorsal fates, animal caps to become neural, and primordial germ cell losses -If BMP antagonists are depleted (Chordin, Noggin, Follistatin, Cerberus = failure of neural and other dorsal development.

2. What are the sites for epigenetic modifications? What are two main mechanisms of epigenetic control?

-DNA or histones. - DNA methylation and histone modifications

5. When is DNA methylation removed and reestablished during the lifetime of the organism? Where it the site of DNA methylation relative to the promoter?

-Demethylation in early embryogenesis (morula/blastocyst) is reversed by a wave of methylation during implantation of embryo. -Post-implantation methylation are stage- and tissue-specific, that define each individual cell type lasting ability over the long period. -CpG islands near promoter

6. What is responsible for the withdrawl of the muscle precursor cells from the cell cycle in the process of forming myotubes?

-Dephosphorylation of Rb protein is associated with differentiation -As vertebrate skeletal muscles differentate, muscle precursor cells withdraw from cell cycle after myoblast fusion to produce myotubes. This withdrawal invovles the dephosphorylation of the Rb (retinoblastoma protein). Dephosphorylation of Rb = G1 arrest, no growth.

23. What genes are expressed by the dermomyotome, sclerotome, and the medial and lateral regions of the myotome?

-Dermomyotome: expresses Pax3, (homobox-containing gene of paired family), gives rise to the myotome (gives rise to muscles). -Sclerotome: expresses Pax 1, migrates ventrally to surround notochord and develop into vertebrae and ribs. -Cells from the medial region of the myotome form mainly axial and back (epaxial ) muscles, whereas lateral myotome cells migrate to give rise to abdominal and limb (hypaxial) muscles. All committed muscle precursors express muscle specific transcription factors in the MyoD family.

22. What effect does knocking out two Hox genes from the same paralogous group have on the embryo?

-Double mutants between Hox genes in the same paralogous group demonstrate a more extreme phenotype when genes from the same paralogous group are knocked-out as opposed to just one or the other. -E.g. Compare the phenotypes of Hoxa11-/- alone and Hoxd11-/- alone to the double mutant Hoxa11-/-; Hoxd11-/-.

8. What relationship did Ed Lewis propose existed between the genes he identified for segment identity and the evolution of the fruit fly from more simple segmented annelids and arthropods?

-Each body segment is distinguished from its anterior neighbor by the activation of a gene. -The more posterior a segment, the more genes are activated. -The genes that regulate segment identity are organized in a cluster, or complex locus, on the chromosome. -The genes in this cluster arose at the same time segments were specialized during invertebrate evolution through gene duplication and divergence.

15. What is responsible for the subdivision of the somite into anterior and posterior halves? What is expressed in the anterior half of the somite? How is Notch/Delta signaling turned off in the anterior half of the somite?

-Each somite is subdivided into anterior (cranial) and posterior (caudal) compartments and this division is already determined prior to the physiological segregation of the somite. -Cranial compartment = Notch repressed -Caudal compartment = Notch activated -Key component in making these compartments = *Mesp2* (Basic Helix-L-Helix transcription factor). Absence of Mesp2 - Cranial identity of somite is lost and the somite is completely caudalized. -Mesp2 expression (red) is restricted to the anterior half of each somite by repressing Notch/Delta signaling in this region. -*Lunatic fringe is activated by Mesp2* which blocks Notch from binding to its ligand, Delta, in the anterior domain of the somite.

11. What is a second ectodermal determinant that is zygotically active? What regulates this gene?

-Ectodermal Determinant: Foxl1e, a zygotic transcription factor, expressed in the cells of the animal half of the late blastula and is required to maintain their regional identity and their development as ectoderm. -In the absence of Foxl1e, animal cells will mix with cells of other germ layers and differentiate according to their new position. -*Foxl1e is transcriptionally repressed by endodermal and mesodermal determinants: Nodal growth factors*.

7. How do eve and ftz establish the segment polarity gene expression responsible for segment and parasegmental boundaries and engrailed expression?

-Engrailed expression is the result of the combinatorial control of pair-rule genes, ftz and eve. Eve: odd #'d segments represses the expression of ftz activates engrailed represses wingless Ftz even #'d segments activates engrailed represses wingless Eve and ftz are expressed in complementary graded patterns of expression in alternating parasegments.

Sperm entry and Dorsal/Ventral axis

-Established by the position of sperm entry. -The sperm brings in a large centriole into the egg. -Sperm's centriole acts as an organizing center for the egg's microtubules. -Dorsal side will be opposite side of sperm entry. -Microtubules from sperm's centriole form arrays under the egg cortex and drive a 30o rotation of the cortex with respect to the internal yolk. -The initial rotation serves to align parallel arrays of radial microtubules, but then membrane vesicles can be transported along these microtubules by motor proteins (kinesin), from the vegetal pole to the dorsal side of the embryo.

20. What does FGF stimulate to bring about primitive streak formation? What does Wnt activate as a result? What is a maternal signal that also activates Nodal?

-FGFs secreted by hypoblasts during avian gastrulation play a role in stimulating a Wnt signaling pathway that is involved in the differential movement of Koller's sickle cells during formation of the primitive streak. -Primitive streak is initiated in the posterior marginal zone by *Wnt-8C* (present in a gradient with high peak at posterior) and Vg-1 (localized at the posterior end of marginal zone). *Vg-1 and Wnt-8C* induce the expression of Nodal in the posterior marginal zone and streak

16. What defines the A/P axis in the chick?

-Gravity defines the A/P axis in chick -As the egg rotates, the embryo develops depending upon the influence of gravity. The egg is directed point down, the PMZ is at the top. The rotation causes the blastoderm to 'tip' in the direction of rotation (somewhat like an 'off-balanced' top).

12. How does HES or hairy autoregulate its own expression, which contributes to the oscillation of hairy expression during somite formation? How is HES reactivated again to start a new cycle of expression?&&&

-HES1 protein represses its own expression -Wnt, in turn, plays a role in activating the expression of Notch (independent of regulation by HES). Msgn1 = mesogenin1, a transcriptional activator that synergizes with Notch to start and restart the somitic clock. -Hairy (HES) mRNA is expressed every 90 minutes Because under high FGF concentrations at Hensen's Node, Wnt is able to reestablish the Notch signaling to drive new HES (hairy) gene expression after 90 minutes.

24. What is the avian equivalent of Spemann's organizer and how was this experimentally demonstrated?

-Hensen's node -Hensens node grafted from qual embryo to chick host--> new axis induced in host

13. What processes in vertebrate development require hh and wnt signaling? What happens when hh signaling is blocked during the formation of the face in humans?

-Hh signaling===-Nervous System - axonal guidance cue Limb Polarity - patterning appendages Formation of: adrenal cortex, cerebellum, eye, face gastrointestinal tract, hair follicle, kidney, long bone, lung, pancreas, pituitary gland, prostate, skeletal muscle and teeth. -Wnt signaling=== Gastrulation Nervous System Limb Polarity Kidney Formation Muscle Formation Eye, gut, and reproductive system -The molecular target of the teratogen, cyclopamine, is Smoothened. Cyclopamine prevents the human brain from dividing into two lobes, resulting holoprosencephaly or cyclopia. This occurs by blocking the hedgehog signaling pathway during human development.

Separation of Mesoderm (twi/sna), Neuroectoderm (sog) and Dorsal Ectoderm (dpp) due to Dorsal protein localization in the nucleus:

-High Dorsal -> Twist and Snail -> Mesoderm -Intermediate Dorsal -> Sog, Sim, Rho -> Neuroectoderm -Low or No Dorsal -> Zen and dpp -> Dorsal Ectoderm

11. What part of the histone can be modified and influence accessibility of DNA to transcriptional proteins?

-Histone tails are modified, this influences accessibility of the genomic DNA

16. What is the relationship between Hox gene expression regulation in differentiation and in cancer?

-Homeobox genes that are normally expressed in developing tissues and are downregulated in differentiation are often re-expressed in cancer. -Conversely, homeobox genes that are expressed in differentiated tissues are often downregulated in cancer progression.

11. In the reciprocal transplantation experiments of Hox-positive and Hox-negative skeletal stems cells between the tibia and the mandible, which transplantation resulted in a change in Hox gene expression in the grafted cells? What did this suggest about the direction of response of cells with regard to the reprogramming of Hox gene expression?

-Hox a11 is expressed in the tibia but not in the mandible (jaw). -When Hox-expressing tibial bones were injured, the stem cells at the site of the injury re-expressed Hox a11 during wound repair. -Likewise, Hox-free mandibular bones were injured and retained their Hox-free status during wound repair. -When Hox-positive skeletal stem cells from the tibia were grafted into a Hox-negative (mandibular) injury, the grafted cells showed a persistence of Hox a11 expression in the Hox-negative environment of the mandible. -The grafted cells failed to differentiate into osteoblasts and instead formed a cartilaginous callus (scar). -However, Hox-negative skeletal stem cells from the mandible readily adopted Hox a11 expression when transplanted into a Hox-positive tibial injury site. -These grafted cells formed a seamless regenerate of new bone, with no evidence of a cartilage 'scar.'

19. In what order are Hox genes expressed along the Hox complex of genes? How does this relate to the timing of when parts of the axial skeleton are determined during early development?

-Hox genes activated in 3' to 5' order -A/P pattern of Hox gene expression in the somitic mesoderm may be linked to the time spent by cells in the somitic stem-cell region. -Expression of genes that are activated in the stem-cell region is maintained when the cells leave to form the pre-somitic mesoderm. -Temporal pattern of Hox gene expression is converted into positional information.

U 1. Why is the fidelity of expression patterns of Hox genes important during adult stages of an organism?

-Hox genes control embryonic segment identity, and their expression in adults tell cells where it is located within a multicellular organism. -Fidelity of expression patterns of Hox genes is necessary for the normal homeostasis of adult tissues and organs, and mis-expression of Hox genes can readily lead to diseases such as cancer.

17. What gene expression specifies the segmental identity of somites? How early is the identity specified? How was this demonstrated experimentally?

-Identity of somites along A/P axis is specified by Hox gene expression -This specification by position occurs before somite formation itself begins, during gastrulation. -pre-somitic mesoderm from a thoracic vertebrate-forming region is transplanted from a stage 10 chick embryo into the cervical region of a stage 7 embryo - skeleton of recipient embryo at 9 days with thoracic vertebrae developing from donor tissue transplanted to cervical region

2. What is the "organizer"? Is it found only in Xenopus?

-Important in neural induction from the ectoderm and organizing the A/P axis -The Spemann-Mangold organizer, also known as the Spemann organizer, is a cluster of cells in the developing embryo of an amphibian that induces development of the central nervous system. -*The Spemann-Mangold organizer refers to the population of cells in the Xenopus laevis embryo that establishes the dorso-ventral and antero-posterior axes*.[2] While an organizer exists in other species, the term Spemann-Mangold organizer is specifically reserved for the amphibian embryo. in chicks it is Hensen's node

2. How is mesoderm specified in the Xenopus embryo? What classic experiments demonstrated this point?

-In Xenopus, ectoderm and endoderm are specified by maternal factors, whereas mesoderm is induced by signals from the animal and vegetal region. -This model was first developed based on classical recombination experiments. In the first, certain tissues were formed from explants from a Xenopus late blastula. Vegetal tissue also induces mesoderm in the animal cap. must be a signaling process between the two that brings about mesoderm

13. How is somite formation matched with a gradient of FGF-8 expression? Under what conditions is the somite formed?

-In the chick there is an FGF-8 gradient in both mesoderm and ectoderm with its high point at the node and decreasing anteriorly. -FGF-8 mRNA is made only in cells in and around the node, and this RNA is degraded in cells left behind when the node regresses posteriorly. -Somitic formation can occur when FGF-8 reaches a sufficiently low level and a stabilization of c-hairy occurs.

18. What products induce the formation of the primitive streak in chicks? What is repressing the formation of the primitive streak at this early stage? How is this inhibitor removed?

-In the posterior marginal zone, when the *hypoblast is replaced by endoblast* cells, the primitive streak can form. Hypoblast normally represses the formation of the primitive streak. - *Epiblast cells in the PMZ above "Koller's Sickle" express Vg-1 and Wnts. These induce Nodal in adjacent epiblast cells and endoblast forms* -Nodal is involved in cell differentiation in early embryogenesis Plays a key role in signal transfer from Hensen's node, in the anterior primitive streak, to lateral plate mesoderm (LPM) in the Chick Embryo. -*Nodal and FGF cause primitive streak* -Nodal protein function is blocked by Cerberus which is produced by the hypoblast. The hypoblast is replaced by the endoblast (endoderm). Note that hypoblast is retreating anteriorly

T 1. What is the origin of neural crest cells and when do they form?

-Induced at the borders of the neural plate, which at neurulation rise up to form the neural folds and come together to form the dorsal portion of the neural tube. -Cephalic crest cells start their migration at the four-somite stage in humans. Like mesodermal mesenchyme, movements of neural crest cells depend on molecular interactions with the Extra Cellular Matrix.

14. What is responsible for restricting BMP-4 in the late blastula? Why is this important to the patterning of the embryo? Where is the highest point of BMP-4 activity and what antagonists are responsible in limiting the ability of BMP-4 to bind to its receptor?

-Initially, BMP-4 protein is made uniformly throughout the late blastula, and zygotic Xwnt-8 protein is made in the prospective mesoderm. -BMP-4 is restricted to the equatorial zone by *Ectodermin*. -BMP-4 and Wnt-8 are further restricted to the ventral mesodermal regions by dorsalizing signals that are secreted by the organizer in response to Nodal signaling. -Organizer secretes BMP-4 antagonists:Noggin, Chordin, Follistatin - prevent BMP-4 from binding to its receptor A functional gradient is established of BMP-4 activity with its highest point at ventral mesoderm and no activity in dorsal mesoderm.

16. What is intercalation? What may be a cell surface signal by which positional information along the proximal/distal axis could be encoded? What is one direct test of the hypothesis that the level of this macromolecule programs information along the proximal/distal axis?

-Intercalation is the ability of cells to recognize a discontinuity in positional values and generate normal structures. -*Prod1* exhibits a two fold difference in expression levels between proximal and distal blastemas. nAG (ligand for Prod1) is essential for limb regeneration. -Prod1 is a protein that is anchored to the cell membrane. Its concentration on the cell's membrane varies along the proximal distal axis of the limb bud or regenerating limb. -High levels of Prod1 - more proximal cells. -Lower levels of Prod1 - more distal cells. -Over expression of Prod1 leads to the proximalisation of limb structures. -When mesenchyme from two blastemas from different proximo-distal sites are confronted in culture, the more proximal mesenchyme engulfs the distal, whereas mesenchyme from two blastemas from similar sites maintains a stable boundary. -This suggests a graded difference in cell adhesiveness along the axis, which adhesiveness being highest distally -Overexpression of the cell surface protein Prod1 transforms distal blastema cells to more proximal fates

13. In humans, what is the limit of regeneration of the distal limb? What is the origin of the macromolecule responsible for this regeneration and what does it regulate?

-Level from which digits are able to regenerate is limited to the base of the claw or nail, respectively. -Connective tissue cells under the nail organ express Msx1, which regulates BMP-4 expression and is associated with undifferentiated mesenchymal cells. -Regeneration always proceeds in a direction distal to the cut surface, enabling replacement of the lost part.

10. In what way is limb regeneration depend on nerves? What macromolecule from the nerve is limb regeneration dependent on?

-Limb regeneration is also dependent upon the presence of nerves. -If nerves are cut before amputation, a blastema forms, but fails to grow. -It is the amount of innervation and not the type of nerve that matters. -Nerves provide an essential growth factor, *nAG (anterior gradient protein)*, secreted by glial Schwann cells of the incoming nerves and later by the glandular cells of the wound epidermis

14. What is meant by Functional Equivalence of Distant Orthologs and Paralogs? What is an example?

-Many animal toolkit proteins often exhibit functionally equivalent activities in vivo when substituted for one another. These observations indicate that the biochemical properties of these proteins and their interactions with receptors, cofactors, etc. have diverged little over vast expanses of time. -Ectopic expression of either the Drosophila eyeless gene or its homologous mouse gene Pax6 is able to induce ectopic eyes in Drosophila (Halder et al., 1995).

12. What activates the zygotic expression of mesodermal inducers? What role do Xnr levels play in the activation of mesodermal inducers?

-Maternal *VegT activates the zygotic expression of Xnr proteins* and Derriere (TGF-beta ligand). -Xnr proteins are expressed in a graded fashion at the appropriate time for mesoderm induction, which may be responsible for dorsal versus ventral mesodermal fate. -Derriere is required for posterior mesodermal patterning. -*Wnt signaling* activates nuclear B-catenin localization. B-catenin stimulates Xnr gene transcription, thus Xnr levels are highest on the dorsal side where VegT, Xnrs, and B-catenin signals overlap.

11. Which pathway is stimulated by maternal dorsalizing factors? How does this lead to determining cells of the future dorsal region? What is the signal that activates this pathway? What is the ultimate effect of activating this pathway?

-Maternal Wnt11 signal - activates dorsal pathway -Due to cortical rotation, vegetally localized Wnt11 mRNA is moved to dorsal side of fertilized egg. -Dorsal (canonical Wnt) pathway is activated extracellularly by maternal Wnt11 signal. -During cleavage, Wnt11 is secreted by dorsal vegetal cells and activates the Wnt signaling pathway. -B-catenin is freed to move into the nuclei of dorsal cells and transcriptionally activate dorsal cells. -The genes activated by B-catenin are the nodal genes (*Xnr1, Xnr2, Xnr4*).

2. What are the signs of animal/vegetal polarity in the Xenopus egg? What is established by the interaction between these two layers?

-Maternally determined, even before fertilization, A/V polarity is evident. -Pigmented side = animal pole (ectoderm - external germ layer). -Yellow side = vegetal pole (endoderm - internal germ layer). -Interaction between these layers will establish mesoderm.

8. What evidence from classical embryology suggests that signals that induce mesoderm are differentially located along the D/V axis? What type of mesodermal structures are induced by the most dorsal vegetal region, the next most dorsal region, and the most ventral vegetal region of the embryo? What classic experiment demonstrated this?

-Most dorsal vegetal region induces notochord, Next most dorsal induces muscles and skeleton, Most ventral region induces blood to form. -First observed using classical recombination experiments (next slide). Dorsal vegetal cells induce muscle and notchord from animal cap cells Ventral vegetal cells induce blood and associated tissue from animal cap cells a. Most dorsal: notochord, then muscle/skeleton, most ventral is blood b. Exp: After gastrulation, depending on what region of the embryo you cut out, that region will differentiate into very different cells than if you chose a more dorsal region (Roux's experiment)?

6. What boundaries are found in the hindbrain that affect the migratory path of neural crest cells?

-Neural crest cells are highly invasive and migratory and interact with the pharyngeal endoderm, paraxial mesoderm and facial ectoderm to construct the tissues and organs of the vertebrate head. -Neural crest cells at the level of the hindbrain migrate laterally into the adjacent *pharyngeal pouches*. -There these cells direct the development of these processes into parts of the face: jaw, lower and upper lips, nose and parts of the ear.

15. What is the nodal signaling pathway and what is it responsible for doing?

-Nodal is involved in cell differentiation in early embryogenesis..regulates mesoderm formation -Plays a key role in signal transfer from Hensen's node, in the anterior primitive streak, to lateral plate mesoderm (LPM) in the Chick Embryo. -"Nodal" relates to the function of this class of ligands, that *being the determination of mesoderm that will become the notochord*. The notochord is mesoderm that is organized into a rod due to Hensen's Node. -Nodal and Nodal-related proteins are secreted signaling proteins that are essential for the induction and patterning of the mesoderm. -Tightly regulated to ensure that the right amounts are present in the right place at the right time, as well as the correct strength of the Nodal signal.

10. What effect does nucleosomes have on transcription and why? Where does transcription normally take place?

-Nucleosomes are obstacles to transcription because DNA is tightly wrapped around -Transcription happens outside of condensed chromatin

14. What effect do oncogenes and mutated tumor suppressor genes have on cyclin and p53?

-Oncogenes: excess cyclin and excess inhibitors of p53. -Tumor suppressor genes: no inhibitors of cyclin and no promoters of p53.

10. What important growth factor that we have seen play a role in development may be the target of thalidomide? What genetic evidence supports this theory?

-Oxidative stress upregulates expression of BMP. -BMP represses FGF (blocking the outgrowth of the limb bud). -Suppression of target genes controlled by -FGF that promotes cell proliferation. -Results in the inhibition of the limb outgrowth.

J 1. What is the function of pair-rule genes during early embryonic development?

-Pair-rule genes define the parasegmental boundaries of an embryo by activating segmentation genes. -Primary pair-rule gene (e.g. eve, h) expression patterns are established by maternal and gap genes. -Pair-rule gene expression is refined through interactions with each other and with secondary pair-rule genes (e.g. prd, slp1).

19. What are Polycomb Group Genes? What are Polycomb Response Elements? How does these affect the expression of genes in the Bithorax Complex?

-Polycomb Group Genes: gene products are proteins that can remodel chromatin such that transcription factors cannot bind to promoter sequences in DNA (repression). PcGs play a role in silencing HOX genes through modulation of chromatin structure -Polycomb Response Elements (PRE): chromosomal (DNA) regulatory elements that recruit PcG factors to chromatin in vivo and mediate epigenetic inheritance of silent and active states throughout development. Located within the Bithorax complex -*Bithorax Complex (BX-C): 340kb region which specifies the identities of several posterior abdominal segments: contains 3 Hot genes observed in this paper, each with corresponding PREs.* + HOX genes: specify the anterior- posterior axis and segment identitiy during early development of Drosophila. Gene products are transcription factors which must be present in specific gradients throughout the embryo to facilitate proper development.

14. How do we know that positional value along the proximal/distal axis is at least partly retained in the blastema?

-Positional value along the proximo-distal axis is at least partly retained in the blastema. -If the blastema is transplanted to a neural location that permits growth, such as the dorsal crest of a newt larva, it gives rise to a regenerated structure appropriate to the position from which it was taken.

20. What does "posterior dominance" of Hox gene expression mean? Where is the primary effect of a knock-out of gene expression in each paralogous group observed?&&&

-Posterior dominance (posterior prevalence) of Hox gene effects -More posteriorly expressed Hox genes tend to over-ride the function of more anterior Hox gene whenever they are co-expressed. -Deletion of Hox d3 shows structural defects in C1 and C2 (where this gene is expressed the strongest), but there are no effects in more posterior structures (despite the fact that Hox d3 is also expressed in this posterior region).

13. What is the molecular function of Retinoic Acid in the cell?

-RA is derived from the liposoluble Vitamin A (retinol). -RA is its acidic metabolite, which is a ligand for transcription factors of the retinoic acid receptor (RAR) nuclear receptor superfamily. -Maternal retinol transferred across the placenta is the major retinoid source for embryos of placental species. -Both synthesis and degradation of RA is highly controlled to allow RA to finely control the differentiation and patterning of various stem/progenitor populations in the embryo. -Retinoic acid inducible enhancer factors (transcription factors) -Belong to the superfamily of steroid/thyroid nuclear receptors. -Binding of RA to its receptor converts transcription factor from repressor to activator of target genes.

15. What effect does blocking the degradation of RA have on the developing mouse embryo? What enzymes are responsible for RA degradation (catabolism)?

-RA is metabolized and degraded by mitochondrial enzymes, *CYP26* into the more polar metabolites, 4-hydroxy-RA and 4-oxo-RA, which eliminated by excretion. -Mutations in genes that encode these catabolic enzymes result in *neural tube defects and truncation (C), head truncation, due to neural crest and hindbrain alterations* (G, I).

7. What effect does phosphorylating Rb have on these cells?

-Rb is phosphorylated during limb regeneration -In the former muscle cells of a regenerating newt limb, Rb protein is inactivated by phosphorylation, allowing the cells to re-enter the cell cycle and divide. -If Rb is phosphorylated it is deactivated and frees TF to be expressed and free cells to go into S phase

6. What is the acrosomal reaction and why is this important to the development of the embryo?

-Receptor proteins on the sperm plasma membrane contact the sea urchin jelly coat and vitelline membrane, resulting in the release of acrosomal enzymes (see next slides). -The acrosome is a vesicle close to the plasma membrane of the tip of the sperm's head. -The acrosome contains soluble proteolytic enzymes and an inner membrane protein (bindin).(Species-specific bindin receptors on the vitelline envelope are only able to recognize bindin molecules from the same species.) -Behind the acrosome, sperm stores actin which will polymerize during the acrosomal reaction. -The acrosomal reaction is triggered when the sperm meets the egg -This reaction releases hydrolytic enzymes that digest material surrounding the egg -Acrosomal process covered in bindin adheres to receptors on vitelline layer (species specific) +Sperm/egg membranes fuse, sperm nucleus enters +Na+ channels open in the plasma membrane resulting in Na+ influx, depolarization +Depolarization sets up fast block to polyspermy.

12. What is Retinoic Acid (Vitamin A) metabolized from? In what foods is this precursor found? Thus, what regulates the natural levels of Retinoic Acid in the human body?

-Retinoic Acid is the product of the metabolism of B-carotene -Retinal: plays critical role in vision. -Retinoic Acid: serves as an intracellular signal that affects transcription of a number of genes. -Vitamin A does not occur naturally in plants, but many plants contain carotenoids. -Beta-carotene can be converted into Vit A within the intestines and other tissue.

S 1. What are the three zones of the somite and their fates in the organism?

-Sclerotome Vertebrae and rib cartilage Tendons -Myotome Musculature of back, ribs, and limbs -Dermatome Dermis of the back

3. Where is engrailed expressed with regard to the parasegment? What is engrailed's function in the embryo/larva/adult?

-Segmentation genes are activated in a 14 stripe pattern, in response to pair-rule genes. -One stripe corresponds to one parasegment. -Engrailed is a segmentation gene that is expressed in the anterior part of each parasegment, -Engrailed functions to define cell lineage boundaries that compose a compartment -Engrailed is expressed throughout the life of the fly, maintaining cell-lineage boundaries that define a compartment.

Egg activation

-Sharp increase in calcium initiates the cell cycle by acting upon proteins that control the cell cycle. -The Mammalian egg is kept in Metaphase II by maturation-promoting factor (MPF) a complex of: p34cdc2 kinase and cyclin B1. MPF is responsible for inducing spindle fiber assembly, chromatin condensation and nuclear envelope break-down. -The calcium wave activates a kinase which results in the degradation of cyclin, which in part results in the inactivation of MPF. This allows entry of the female chromatin into Anaphase II.

4. When do the somites form? What is the paraxial mesoderm and what role does it play in somite formation? How is it related to the presomitic mesoderm?&&&&

-Somites form on either side of the notochord in paraxial mesoderm anterior to the regressing Hensen's node. Somitic formation can occur when FGF-8 reaches a sufficiently low level. -Within the mesodermal layer either side of the notochord (axial mesoderm) lie the mesoderm strips. The region that will later form segments is called presomitic mesoderm (PSM). This PSM is being "patterned" by two molecular activities described as the "clock" and the "wavefront". Many different molecular factors are involved in this patterning effect: Hes7, FGF, Sprouty4, Notch, Shh

3. What is DNA methylation and what role does it play in epigenetic regulation of gene expression? What enzyme is responsible for DNA methylation?

-Target Cytosine in CpG islands near promoter 5-methylcytosine is most common -Largely erased and re-established between generations in mammals. -methyl transferase

9. How did pregnant women come in contact with thalidomide in the 1950's and 1960's? Why was this drug removed from the market?

-Thalidomide was chiefly sold and prescribed during the late 1950s and early 1960s to pregnant women, as an to combat morning sickness (3-8 weeks gestation) and as an aid to help them sleep. Before its release, inadequate tests were performed to assess the drug's safety, with catastrophic results for the children of women who had taken thalidomide during their pregnancies -Wide range of malformations of the limb, ear, eye, internal organs, and central nervous system have been documented as associated with thalidomide. Causes limb skeleton reductions = amelia or phocomelia. Phocomelia is concentrated primarily on reducing the forearm. Defects observed in humans, monkeys, rabbits, chicks, and zebrafish, however, thalidomide does not cause limb defects in rats.

X 1. What is regeneration? For what organism is regeneration easier?

-The ability of the fully developed organism to replace tissues, organs and appendages by growth or repatterning of somatic tissue. Plants - grow new plant from single somatic plant cell. -Starfish, planarians (flatworms), and Hydra - give rise to a whole animal from a small fragment of original animal. -Regeneration may be easier for organisms that reproduce asexually (budding or fission).

7. What is the cortical reaction and what happens to the embryo as a result? Why is this important to the development of the embryo?

-The depolarization (neutralization of charge difference) causes voltage-sensitive Ca2+ channels to open in the egg endoplasmic reticulum (ER). -This reaction induces a rise in Ca2+ in cytoplasm that stimulates cortical granules to release their contents into the perivitelline space. -Cortical granules fuse w/ membrane +Enzymes released break down separation between vitelline and plasma membranes. +Plasma membrane polarity returns to normal +Fertilization envelope formed = slow block to polyspermy (follows repolarization) -These changes cause formation of a fertilization envelope (raised and hardened vitelline membrane due to influx of water, inactivates bindin receptors on vitelline membrane) that functions as a slow block to polyspermy.

2. How do cells remember their position along the three body axes?

-The differential expression of Hox genes in cultured fibroblasts (ex vivo), is maintained as identical to the expression pattern in the adult cells. -Gene expression programs of fibroblasts from numerous anatomical sites confirmed that fibroblasts express distinct patterns of Hox genes that indicate the unique position of the cell along three developmental axes.

15. With regard to the proximal/distal axis, what is the blastema doing during limb regeneration? What is one experiment that demonstrated this?

-The regenerating limb in some way reads the positional value at the site of amputation and then regenerates all positional values distal to it. -Epimorphic regeneration involves the retention of embryonic processes, such as the ability to specify new positional values. -Grafting a distal blastema to a proximal stump will induce the stump (mostly) to generate a normal limb and the distal blastema forms the wrist and hand. -This is accomplished by re-establishing positional values at the site of amputation and then regenerates all positional values distal to it. -The ability of cells to recognize a discontinuity in positional values is illustrated by grafting a distal blastema to a proximal stump. -Epimorphic regeneration involves the retention of embryonic processes, such as the ability to specify new positional values.

18. What is the molecular target of retinoic acid embryopathy? What genes appear to be misregulated as a result of retinoic acid exposure?

-The target of embryopathy are neural crest cells. -Hoxb1, b2 and Krox-20 are misregulated

O 1. What are two functions of the Vegetal Cells in the Xenopus embryo?

-They differentiate into endoderm. -They signal the marginal cells for form mesoderm.

8. What are the three transcription factors associated with the organizer and the four secreted proteins associated with the organizer? Which of these are antagonists of Wnt and which are antagonists of BMP?

-Three TFs: 1. Brachyury 2. goosecoid 3. Xlim1 -Four SPs: 1. Xnr3 2. chord 3. noggin 4. Cerberus -Wnt antagonists: *Dickkopf1 and Cerberus*. -BMP antagonists: *Chordin, Noggin*, Follistantin and *Cerberus*.

11. What region of the embryo secretes Wnt antagonists? What region of the embryo secretes BMP antagonists?

-Thus, Wnt antagonists (Cerberus, Dickkopf1, and Frizzled-related) are predominantly secreted from anterior endoderm and mesoderm underneath the future head. -BMP antagonists are released from mesoderm beneath both head and trunk

Q 1. What are the ventralizing signals in the Xenopus embryo? What is necessary to promote dorsal development?

-Ventralizing signals: BMP and Xwnt-8 -To make dorsal parts of the embryo, these ventralizing signals are inhibited by antagonist to both BMP-4 and Wnt

9. What genetic observations support the model of segment polarity gene expression maintenance?

-Wg expression disappears in en mutant embryos. -En expression disappears in wg mutant embryos. -Wg and en maintain each other's expression. -Wg and en are expressed in completely different cells, so cell-cell communication must occur.

12. Be sure you know the molecular function of the products of segment polarity genes and how they function in their respective pathways.

-Wingless secreted growth factor -Frizzled wingless receptor (transmembrane protein) -Disheveled Activated by frizzled/wnt and then inactivates GSK-3 -B-catenin Transcription factor that is normally repressed by GSK-3 -Hedgehog secreted growth factor -Patched hedgehog receptor (transmembrane protein) -Smoothened G-protein coupled receptor

8. What happens as a result of epiboly in this embryo?

-With epiboly (second panel), the upper half of the yolk becomes covered by a blastoderm with a thickened edge (germ ring), and a shield-shaped region is visible on the dorsal side of the blastoderm (third panel). -As a result of epiboly, the embryonic cells form a shield around the yolk

6. What is histone modification?

-covalent post-translational modification (PTM) to histone proteins which includes methylation, phosphorylation, acetylation, and ubiquitylation -The PTMs made to histones can impact gene expression by altering chromatin structure or recruiting histone modifiers. -Histone modifications act in diverse biological processes such as transcriptional activation/inactivation, chromosome packaging, and DNA damage/repair

10. What evidence suggests that Hoxa11/dll and Hox a13/d13 act synergistically?

-extreme phenotype as opposed to if one or the other was knocked out.

12. What effect does histone methylation have on transcription?

-generally condenses chromatin - methylations of amino acids in histones that can either increase or decrease transcription of genes -H4K20me3, H3K27me3, H3K9me3=closed chromatin -H3K4me3, H3K36me3= open chromatin

16. What sorts of abnormalities are associated with embryonic exposure to RA (isotretinoin) in humans?

-malformations involving craniofacial, cardiac, thymic, and CNS structures

2. What do neural crest cells interact with as they migrate laterally? Through which part of the somite do neural crest cells migrate laterally?

-movements of neural crest cells depend on molecular interactions with the Extra Cellular Matrix. -Due to ephrins and Eph receptors (RTK receptors), the motor axons will only migrate through the anterior half of the somites, and the neural crest cells will migrate only through the anterior half of the somites as well

12. What other cell types exhibit a Hox memory?

-smooth muscle cells -skeletal muscle cells -Mesenchymal stem cells in bone marrow (MSCs) +MSCs derived from different bones have distinct developmental potentials and this is correlated with anatomical site-specific differences in Hox gene expression.

17. What experiment demonstrated that cells of the Nieuwkoop Center are required for the development of dorsal and anterior structures?

1) When cells of the Nieuwkoop Centre are grafted onto the ventral side, twinned embryos with two 'dorsal sides' are produced. 2) When cells of the ventral side are grafted onto the dorsal side, normal embryos are produced (no effect). Therefore, signal from the Nieuwkoop Centre must be required for developing dorsal and anterior structures.

4. What are two types of regeneration?

1. *Morphoallaxis* has little growth and depends upon re-patterning of tissues. New boundary regions are first established and new positional values are specified in relation to them. 2. *epimorphosis* depends upon growth of new and correctly patterned structures. +vertebrate limb regeneration +Involves cell dedifferentiation and growth, and is therefore epimorphic regeneration. New positional values are linked to growth from the cut surface

What are the five components of nodal regulation?

1. Activation from a secreted inactive precursor protein by proteolytic cleavage. 2. Prevention of Nodal from binding to its receptor, by either binding Nodal (Cerberus-1 and Lefty 1,2) or to its receptor (Lefty 1,2). 3. Nodal's receptor are Activin I and II. Nodal signal is amplified by the phosphorylation of Smads, which enter the nucleus to regulate the expression of target genes. Smads are intracellular proteins that transduce extracellular signals in TGF-Beta pathways. Smads enter the nucleus and act as transcription factors. 4. MicroRNAs have been observed to regulate the translation of Nodal mRNA (as well as Lefty mRNA). 5. The target genes of Nodal signaling act as either positive or negative feedback signals for Nodal expression.

4. What are the "organizer's" three main properties?

1. It induces neural tissue in the overlying ectoderm. 2. It imparts more dorsal characteristics to the mesoderm of the marginal zone (i.e. "dorsalizes mesoderm"). 3. It induces a secondary gut ("dorsalization of the endoderm").

5. What are two ectodermal determinants and how do they function?

1. Maternal E3 ubiquitin-ligase *Ectodermin*, which is translated from maternal mRNA located at the animal pole of the fertilized egg and becomes localized in nuclei throughout the animal cap. Ectodermin adds ubiquitin to *Smad4 (regulatory protein activated by BMP signaling)* promotes ectodermal development).*Ectodermin's ubiquinization of Smad4 promotes the formation of ectoderm at the expense of mesoderm.* Smad4 is a common signal transducing element in both BMP4 and TGF-B(Nodal) pathways. 2. *Foxl1e*, a zygotic transcription factor, expressed in the cells of the animal half of the late blastula and is required to maintain their regional identity and their development as ectoderm. In the absence of Foxl1e, animal cells will mix with cells of other germ layers and differentiate according to their new position. Foxl1e is transcriptionally repressed by endodermal and mesodermal determinants: Nodal growth factors.

23. What are two ways birds and mammals differ from Xenopus (at the gastrula stage)?

1. The spherical amphibian blastula (just before gastrulation), is a flat layer of epithelium called an epiblast in birds and mammals 2. The equivalent of the amphibian blastopore is the primitive streak

24. What three signals influence the partition of the somite?

1.) Shh (from the notochord and ventral floor of neural tube) specifies the ventral region of the somite and sclerotome development. 2.) Low levels of Shh and signals from dorsal neural tube (Wnt) and overlying ectoderm specifies the dermomyotome. 3.) BMP4 specifies the lateral plate mesoderm.

2. How is mesoderm specified in the Xenopus embryo? What classic experiments demonstrated this point? 3. What are the signals that induce the following cell types or layers: ventral mesoderm, ectoderm, endoderm, and dorsal mesoderm?

2. Ectoderm and endoderm are specified by maternal factors, whereas mesoderm is induced from ectoderm by signals from the ventral region (vegetal and animal cap communicate). This model was first developed based on classical recombination experiments. In the first, certain tissues were formed from explants from a Xenopus late blastula. Vegetal tissue also induces mesoderm in the animal cap. must be a signaling process between the two that brings about mesoderm 3. a. Ventral Mesoderm - BMP4 b. Ectoderm - Ectodermin c. Endoderm - VegT and Vg1 d. Dorsal Mesoderm - Nodal genes induced by B-catenin Note: Vg1, BMP4 and Nodal are secreted signaling proteins.

3. What are the two genetic controls of somite formation?

A combination of a "clock" and a "wavefront" of genetic controls. -Clock = 90 minute cycle of c-hairy expression -Wavefront = the gradient of FGF expression.

The difference between the Nieuwkoop center and the Spemann Organizer

A key function of the Nieuwkoop center is to induce the formation of dorsal mesoderm. It turns out that the most dorsal mesoderm also functions as a signaling center, known as the Spemann Organizer. The difference between the Nieuwkoop center and the Spemann Organizer can be seen in transplantation experiments. Both signaling centers induce a new D/V axis. However, cells in the *Nieuwkoop center are incorporated only into the endoderm*, whereas the cells of the *Organizer are found in the mesoderm* of the newly formed axis. Thus, *the Organizer is the dorsal mesoderm tissue induced by the Niewkoop center*. It is called the "organizer" because it can "organize" host cells into a new D/V axis. This implies that the organizer (dorsal mesoderm) produces factors that can pattern the mesoderm along the D/V axis.

15. Why are over 85% of cancers are found in the epithelium?

A major feature of cancer is the failure of tumor cells to differentiate properly. Since epithelial cells are constantly being renewed by division and differentiation of stem cells, if there is a cancer causing mutation in these regenerating cells, a tumor can result.

4. What is the effect of activation of the Toll receptor protein? What are the proteins involved in this pathway and what is the ultimate result?

A similar gradient of nuclear localization of dorsal occurs. Dorsal sits in the cytoplasm of syncytial blastoderm and is moved by cactus. Cactus kinase phosphorylates cactus, making cactus tagged for degradation and dorsal is allowed to go into the membrane. Toll/spatzle confirmation releases cactus kinase -• Dorsal protein moves into nucleus of *syncitial blastoderm* and acts as a transcription factor • Now secreted or transmembrame proteins and their corresponding receptors are used to influence gene expression

Segment Polarity Genes

Activated by pair-rule genes. Expressed in 14 transverse stripes, one stripe corresponding to a "parasegment". Segment polarity genes: engrailed, wingless, and hedgehog.

6. What is the relationship between Hox genes expressed in cells in adult tissue and those Hox genes turned on in those cells during their development in the embryo?

Adult palmoplantar (palm of hand, sole of feet) fibroblasts express HoxA13, which activates the same downstream genes that originally promoted embryonic limb development and instructed adult epidermal cells to differentiate toward the palmoplantar fate. The same mechanism employed to pattern distal limb outgrowth early in development is used again to specify distal epidermal differentiation in adulthood.

25. What does Hensen's node do after the head process is formed?

After head process forms, Hensen's node moves backwards and the primitive streak begins to regress (cranially)

Species specific binding

After making its way through the jelly coat, the sperm makes contact with the vitelline envelope. Species-specific bindin receptors on the vitelline envelope are only able to recognize bindin molecules from the same species. The sperm and egg plasma membranes fuse, and the sperm nucleus enters the cytoplasm of the egg. It is not known whether the increased sodium permeability is due to the binding of the first sperm or to the fusion of the first sperm with the egg. The depolarization of the egg membrane blocks any additional sperm from fusing with the egg membrane.

12. How many days does it take for a chick to hatch after the egg is laid?

After momma hen lays her egg, she has to wait 21 days for it to hatch

12. What is the archenteron and what will it become?

Archenteron = new cavity made from the invagination of the future endodermal cells --> will become the future gut

5. How did a comparison of different segmented invertebrates inspire Ed Lewis to propose a model to explain the genetic control of segment identity in the fruit fly?

As Ed Lewis saw progressive specializations of the segments (ex: leg development from worm --> annelid --> centipede), he proposed that this occurs b/c of duplication & divergence of ancestrial genes (annelid 1 gene, centipede 2 genes; related b/c one is copy of the other)

10. What is promoted by BMP and Wnt antagonists together? What is promoted by BMP antagonists alone?

BMP and Wnt antagonists promote development of a head with a brain, whereas BMP antagonists alone induce a trunk with a spinal cord (notochord).

12. What are the dpp/sog homologs in vertebrates? What is the curious difference between the d/v axis in vertebrates and invertebrates?

BMP mirrors dpp; dpp/sog pairing is conserved in vertebrates (BMP-4/chordin). The dorsal and ventral positions on the axes are flipped from vertebrates to drosophila (due to placement of notochord)

Antero-posterior 2

Bicoid protein-gradient switches hunchback on at high concentration--> hunchback activates and represses gap genes like Kruppel, knurls, giant--> gap-gene products and gap genes interact to sharpen expression boundaries--> axis id divided into unique domains containing different combination of transcription factors

14. How is hh and wnt signaling connected to cancer in the adult vertebrate?

Both Hh and Wnt pathways are typically dormant in adult tissues, but can be activated under certain conditions. Aberrant activation of these pathways by mutations in key pathway regulators contributes to the genesis and progression of several types of cancers.

11. Which body or organ system has the longest period of formation during human gestation?

CNS

5. Why is Hox maintenance important for cells in the epidermis?

Cells in superficial layer of the skin (epidermis), which are completely replaced every 28 days must know where in the body they are located - should they make scalp hairs or thick hairless skin of the palm of the hand. Ongoing Hox expression in the adult skin fibroblasts provide this important positional memory.

7. Where in the blastoderm do cells that give rise to: both endoderm and mesoderm, mesoderm only, and ectoderm, come from?

Cells that lie right at the blastoderm margin, called the mesendoderm, can give rise to both endoderm and mesoderm. Cells that lie four to six cell diameters from this margin, give rise to the mesoderm. Ectoderm comes from the animal region of the blastoderm.

How was the compartmental constriction of cell lineage first observed in Drosophila? Describe the genetic manipulation that was used and what it allowed investigators to produce? What was the significance of their observation in engrailed adult mutants?

Compartmental constriction was first observed in drosophila when researchers used x-rays to induce a mutant that knocked out engrailed expression (engrailed normally controls the compartmental boundaries) i. Start with flies heterozygous for minute (grow fast) & engrailed (knocked out) irradiate it see what happens if no engrailed expression ii. Found that: cells that grow faster & lack engrailed do NOT respect compartmental boundaries c. Significance of the observation: need engrailed to have compartmental boundaries

16. Why is the specification of anterior versus posterior halves of the somite important to neural development?

Cranial/caudal division of the somite will be used by neural crest cells and motor nerves as guidance cues to generate the periodic arrangement of spinal nerves and ganglia. In vertebra formation, each vertebra is derived from the anterior half of one somite and the posterior half of the preceding one.

Consequence of fast block polyspermy

Depolarization of the egg's plasma membrane (Na+ influx) Release of a wave of Ca2+ ions from intracellular vesicles into the cytoplasm: Initiates activation of the egg as well as the slow block to polyspermy.

21. What is the direction of homeotic transformation in Hox loss of function mutants?

Direction of transformation is the affected segment resembles the segment that is anterior to it.

3. Why are eggs from Toll- mothers dorsalized? Where is the Toll receptor distributed on the surface of the egg? How is it activated?

Dorsal is no longer localized in the nucleus, creating a dorsalized embryo. Toll is uniformly distributed on the surface of the egg, but the active ligand, spatzle is located in the anterior perivitelline space and is secreted by follicle cells. Spatzle is the ligand for toll that creates a gradient of activation. -A processed C-terminal fragment of Spatzle is what binds to Toll. Spatzle is uniformly distributed in the perivitelline space but accumulates in its activated form in the ventral region. The serine protease, Easter, cleaves Spatzle, releasing the C-terminal fragment. Snake (serine protease) activates Easter, and Snake is ultimately controlled by Pipe (an enzyme restricted to the ventral follicle cells).

5. When and how is the dorsal gradient made, where does the dorsal protein go in the cell and why?

Dorsal moves into the nucleus of the syncytial blastoderm (gradient made then) and acts as a transcription factor. With the cellularization of the blastoderm, secreted or trans-membrane proteins and their corresponding receptors are now used to influence gene expression. Dpp, zen, sog, rho, sim, snail and twist all respond to a difference in the concentration of dorsal in the nuclei. -• Spatzle protein activates toll receptor on ventral side, dorsal protein enters ventral nuclei, giving ventral to dorsal gradient

13. What are the tissue fates of those cells controlled by Dpp/Sog and BMP-4/Chordin in invertebrates and vertebrates, respectively?

Dpp creates amniserosa/dorsal epidermis (visceral mesoderm) and Sog creates neurogenic ectoderm (muscle). BMP-4 creates blood/muscle (epidermis) and Noggin chordin creates the (notochord) CNS; molecular signals are highly conserved between Drosophila and Xenopus

16. Have duplications within the HOX complex genes been observed? Cite the rare example.&&&&&&&

Duplications within several prominent toolkit gene families have been surprisingly rare in the course of animal diversification relative to duplications of other gene families. -arrangment of the Hox genes of the Drosophila and human clusters -Genes comprising Abd-B have duplicated & have different purpose in limb development

2. What does each of the 14 stripes of a segmentation gene correspond to with regard to engrailed expression?

Each of the 14 stripe boundaries (parasegments) are marked by engrailed expression at the anterior end

5. With regard to cell lineage restriction, what is engrailed's function in the fly?

Engrailed is expressed throughout fly life b/c it's responsible for maintaining cell lineage boundaries that compose a compartment (specifically expressed in the posterior part of the compartment) -Engrailed is expressed in the anterior region of each parasegment (= posterior domain of each segment) at germband extension. Engrailed delimits the anterior border of each parasegment.

V 1. What is "Epigenetics" and how does it differ from "Genetics"?

Epigenetics: heritable alternative states of gene activity that do not result from altered nucleotide sequence

3. What do adult fibroblasts maintain after extensive in vitro passage in tissue culture?

Extensive in vitro passage of adult fibroblasts in culture showed that these cells maintained their distinct Hox expression patterns for >35 cell generations.

14. What transcription factor found in neural tissue is induced by FGF?

FGF activates the TF Churchill (Zn-finger protein), which in turn activates the neural specific transcription factor, Sox-2, the earliest definitive marker of neural plate in the chick.

FGF signaling pathway - early requirement of Neural induction

FGF may inhibit BMP signaling pathway causing the inhibitory phosphorylation of Smad1 (phosphorylation on a different site that causes Smad1 not to go into the nucleus)

8. What ultimately initiates or starts this cycle of gene expression (hint: it has high concentration at Hensen's node).

FGF-8

4. What evidence suggests that tissue culture results relate to the stability of fibroblasts during an organism's lifetime?

Fibroblasts isolated from young versus old human donors - decades apart in age - show little difference in the expected pattern of position-specific Hox expression.

In the imaginal disk, what two other gene products (in addition to engrailed) are involved in maintaining the cell lineage restriction in the fly?

Ftz and eve (regulate the expression of engrailed & wingless) i. Cells need Ftz in order to express Engrailed

L 1. What two fields did Ed Lewis combine that lead to the discovery of the Hox genes?

Genetics and Evolution

9. How do gradients of Wnt and BMP affect the formation of the head and tail along the A/P and D/V axes?

Head: requires low levels of Wnt, Nodal, and BMP. Tail (Ventral Posterior): requires high level of Wnt and BMP.

Hensen's node

Hensen's Node organizes mesoderm into notochord and somitic mesoderm

15. What are the three ways chromatin can be condensed to repress transcription?

Histone modifications (e.g. methylation)(Histone modifying enzymes alter histone tail modifications), DNA methylation(DNA methylases), chromatin binding proteins(Recruitment of chromatin binding proteins, Polycomb proteins, Heterochromatin Protein)

5. How were homeotic genes in vertebrate organisms discovered?

Homeobox sequence was used as a probe to search other non-insect genomes for similar Master Switch (Homeotic) Genes

When are Hox genes first expressed?

Hox genes are expressed in the mesoderm at an early stage of gastrulation when these cells begin to move internally away from the primitive streak and towards the anterior.

10. What is an important feature of the anterior boundary of Hox gene expression in mammals?

Hox genes show a sharp anterior border of expression and a much less defined posterior border. -Lot of overlap by every region but each has distinct pattern of Hox gene expression. -Most anterior somites express Hoxa1 and Hoxb1 only. -Posterior regions express all Hox genes. -The anterior head, forebrain and midbrain do not express Hox genes but have other homeobox genes (etx & otx.)

18. What is the relationship between the Nieuwkoop Center and Spemann's Organizer? What is the Organizer's determinant and what controls its transcription?&&&

In Xenopus embryos, the dorso-ventral and antero-posterior axes are established by the Spemann-Mangold organizer. According to the prevalent model of early development, the organizer is induced by the dorsalizing Nieuwkoop signal, which is secreted by the Nieuwkoop center. -Goosecoid expressed at the same time and location as Spemann's organizer. -Goosecoid transcription under control of B-catenin and Smads.

3. What do all Homeotic Genes have in common in their protein structure and sequence?

In the last exon (3') of the transcript: The Homeobox (180 nucleotide region of highly conserved sequence) that encodes the Homeodomain (60 amino acid DNA binding motif = helix-turn-helix.

17. How can specific Hox genes expression be used as molecular biomarkers for pancreatic and ovarian cancer, and why?

Isolate RNA from tumor and use reverse transcriptase and hybridize against a microarray. Competitive microarray- compare normal versus non normal arrays. -some hox genes are upregulated/ some are downregulated in certain cancers and are unique to different cancers.

17. In what form is Retinoic Acid marketed to the public today and for what use?

Isotretinoin (13-cis-retinoic-acid; brand name Accutane), which is often used to treat severe acne, is such a strong teratogen that just a single dose taken by a pregnant woman may result in serious birth defects

4. In what two ways are Nodal and BMP-4 pathways different? In what ways are the same? Which pathway directs ventralization of the mesoderm and which directs the dorsalization of the mesoderm? What impact does this have on the associated ectoderm?

Like Nodal, BMP4 is a secreted protein in TGF-B superfamily that binds to a receptor that starts a signal transduction pathway response. Note however, that BMP4 utilizes a different set of Smads in the process of signal amplification. As a result different target genes are activated. *BMP4 promotes the ventralization of the mesoderm.* * Nodal promotes dorsal mesoderm induction.* Ventral:ectoderm binds BMP4; becomes epidermis (skin) Ectoderm: cannot bindBMP4; takes default path to become central nervous system

7. What is an endodermal determinant and how does it function?

Maternal Endodermal determinant: VegT and Vg1. -VegT activates the Xnr-1,2,4 proteins (Xenopus nodal-related proteins) that are mesoderm-inducing signals. VegT acts as a transcription factor to activate genes specifying endodermal fate.

13. What is meant by Ancestral Genetic Complexity? What is an example?

Morphologically disparate and long-diverged animal taxa share similar toolkits of body-building and body-patterning genes. -The finding that: Eyeless gene in Drosophila Small eye (Pax6) of the mouse Human Aniridia (Pax6) are encoded by homologous genes. Eye morphogenesis is under similar genetic control in both vertebrates and insects, in spite of the large differences in eye morphology and mode of development.

3. What are body parts that a salamander can regenerate? What are parts that mammals can regenerate?

Newts can regenerate complete tails and limbs and some internal organs, it can also regenerate dorsal crest, limbs, retina, lens, jaw and tail. Mammals can regenerate the liver, antlers of male deer, fractured bones, tips of digits and ribs

6. How do the inductive properties of the organizer change during gastrulation? How is this change reflected by differential gene expression?

Organizer transplant from: -Early gastrula induces a full 2nd embryo. -Mid-gastrula induces trunk & tail (no head). -Late gastrula induces only tail. -In the early Xenopus gastrula, the vegetal portion of the organizer that is fated to produce prechordal plate mesoderm expresses *TF-XOtx2*, characteristic of anterior structures. -The more dorsal part of the organizer is characterized by expression of *TF-Xnot*, and can induce trunk and tail structures but not heads.

20. What role does DNA methylation play in the silencing of BX genes by Polycomb? What role does the chromatin loop play in silencing a BX gene through Polycomb Group Proteins?

PRE (Polycomb repressive element) is recognized first by PHO-RC, which recruits PRC2. PRC2 methylates local nucleosomes. The accumulation of methylated histone tails on H3 helps attract, bind and stabilize PRC1 binding. The chromatin loop juxaposes the Polycomb group complexes and the transcribed region of the gene itself, so further modification (methylation of H3K27) could shut down the entire gene

2. What are some organisms that can regenerate limbs or parts of their bodies?

Plants, starfish, planarians and hydra

13. What are proto-oncogenes and tumor suppressor genes? What are their normal functions (as wildtype genes)? What effect do these genes normally have on cyclin and p53?

Proto-oncogenes: gene that if it contains a mutation will become an oncogene (promotes a cancer cell state). GOF mutation. Normally functions to regulate the growth and differentiation of a cell, possibly playing a role in cell cycle checkpoints. Sufficient cyclin. Inhibitors of p53. Tumor suppressor genes: inactivation or deletion of both copies is required for cell to become cancerous. Also normally involved in DNA repair, cell cycle checkpoints and triggering apoptosis. LOF mutation. Inhibitors of cyclin. Promoters of p53.

17. How does retinoic acid change positional information along the proximal/distal axis? How was this demonstrated? What may be the target genes of RA in this process?

RA is present in developing vertebrate limbs and can alter positional values in the chick's limb. Exposure to RA changes the positional value of the blastema to more proximal ones, such that elements proximal to those cut as well as those distal will be generated and most likely inducing the overexpression of Prod1 and some Hox genes. By making RAR-delta/thyroxine chimeric receptor (Where RAR's DNA binding domain is activated by thyroxine), results in a movement of transfected blastemal cells to more proximal regions in the intercalating regenerating blastema. Prod1 is a likely candidate for mediating this change

19. How does retinoic acid exposure change the Hox code expression pattern in the neural crest cells of the hindbrain? Based on the difference mentioned above, between the trunk and cranial (cervical) neural crest cells, how could the activation of these genes in cranial neural crest cells have a negative effect on the embryo?&&&&&

RA is synthesized in mesoderm by RALDH2. This RA diffuses to the hindbrain neuroectoderm where it induces Hox expression patterns and rhombomere segmentation. It extends expression of Hox genes boundaries' anteriorly. It imitates a gain of function mutation and acts as a phenocopy.

9. What effect does the removal of Hox a11 and Hox dll have on the development of the vertebrate limb?

Radius and ulna missing

Role of Retinoic Acid in the Somitic Clock

Retinoic Acid (small secreted molecule) has an antagonist effect on FGF action. RA forms a gradient in the opposite direction to FGF and by antagonizing FGF, prevents the pre-somitic region from continually getting longer. RA is synthesized in the somites and diffuses both posteriorly and anteriorly. -causes genes to be expressed in more anterior segments

14. What other important signaling molecule is in a gradient complementary to that of FGF-8? What effect does this have on somite formation? What is the "determination front"?

Retinoic acid allows for somite formation, and Hox genes are specified. Wnt is turned on with FGF-8 which activates the The Notch/Delta signal transduction pathway is responsible for somite boundary and polarity. Notch receptor and its ligand (DSL) make up signal transduction pathways that regulate Hairy1 (HES1) which sets up the caudal half of the somite.

14. What effect does inactivating the synthesis of RA have on the developing mouse embryo? What two enzymes are the target for this experiment?

Retinol is oxidized into retinaldehyde which is then oxidized into retinoic acid. Embryonic requirements of RA (retinoic acid) are evident in the *facial, eye, and forelimb abnormalities* in embryos lacking *Rdh10 (C) and Raldh2 (E)*.

12. How is Shh regulated during the development of the frog? During what developmental stage is Shh able to be expressed and when is it repressed?

Shh is highly methylated (through epigenesis) in the froglet (transcriptionally repressed), but not in the tadpole. Xenopus laevis tadpole can regenerate amputated limbs but, after morphogenesis, the young frog cannot. If a limb is amputated in the froglet, only a spike of tissue develops

13. What impact do Xnrs have on Smad2? What does this lead to on the dorsal side of the embryo? (Hint: How does the high level of Xnr's lead to the specification of the Spemann organizer on the dorsal aspect of the embryo?)

Signaling via Xnrs stimulates phosphorylation and activation of *Smad2*. This leads to the specification of the Spemann organizer on the dorsal aspect of the embryo.

16. What are three different signals emanating from different parts of the blastula and what effect does each have?

Signals emanate from different parts of the blastula: Vegetal region gives signals that induce mesoderm and establish Spemann's organizer. Another set of signals gives the ventral mesoderm its character. At the same time the organizer emits yet another set of signals that counteract the ventral signals (enabling the mesoderm adjacent to the organizer to give rise to somites.)

6. What gene responds transcriptionally to high levels of dorsal protein?

Snail and twist are activated at the cellular blastoderm stage transcriptionally by dorsal protein. Both genes are required for ventral invagination of the presumptive mesoderm.

10. How is the Sog gradient refined and what cell type does it specify?

Sog definitively limits dpp by destroying it. Tld is expressed in response to dorsal and it cleaves sog which protects dpp. Tsg enhances the ability of dpp to exist in its domain by dislodging latent dpp diffusion. It is expressed throughout neuroectoderm

5. What is released from Hensen's node that suppresses somite formation in the presomitic mesoderm?

Somitic formation can occur when FGF-8 reaches a sufficiently low level.

11. What gene that is expressed during normal limb development is also expressed in regenerating limb?

Sonic hedgehog is expressed in posterior margin of blastema, as it is in the limb bud.

Dorso-ventral 1

Spatzle protein activates Toll receptors on ventral side--> Dorsal protein enters ventral nuclei, giving ventral to dorsal gradient

3. What experimental evidence (by Spemann and Mangold) demonstrated the "organizing" ability of the dorsal lip of the blastopore?

Spemann Organizer (dorsal blastopore lip) grafted to the ventral side of the marginal zone results in a twinned embryo. The second embryo can have head, trunk and, sometimes, a tail but will be joined to primary embryo along the axis.

5. What effect does fertilization have on the D/V axis with regard to the position of sperm entry?

Sperm entry can occur anywhere in the animal hemisphere. It causes the outer layer of cytoplasm, the cortex, to loosen from the inner dense cytoplasm so it can move independently. Cortex is gel-like layer rich in actin filaments. -Upon fertilization, 2nd meiotic division of female nucleus is complete. Polar body is formed to preserve the cytoplasm for the embryo. Egg and sperm nuclei fuse and dorsal determinants (at vegetal pole) rotate (cortical rotation) opposite the site of sperm entry. -a. Position of sperm entry becomes the ventral side opposite end becomes the dorsal side -b. Due to cortical rotation effect after fertilization, put into effect by dorsalizing factors (responsible for rotation)

3. What are the three features of the phylotypic stage of vertebrates?

Structures that are common to the phylotypic stage of the vertebrates are: 1) the notochord (an early mesoderm structure along A/P), 2) the somites (blocks of mesoderm on either side of notochord which form the muscles of the trunk & limbs), 3) the neural tube- ectoderm above notochord form a tube (brain and spinal cord).

16. What is the Nieuwkoop Center? What effect does each cleavage division have on this center? After the second cleavage division what cells contain the Nieuwkoop Center?

The Nieuwkoop Center sets up D/V polarity in the blastula and is essential for normal development. The 1st cleavage cuts through the site of sperm entry and the Nieuwkoop Centre. The 2nd cleavage splits the embryo into 4 cells (2 with the Nieuwkoop Centre and 2 without).

2. What genetic evidence suggested that the maternal genome established the D/V axis during oogenesis?

The existance of maternal effect mutations that will disrupt the D/V patterning of the embryo; maternal genotype is important.

9. What is transdifferentiation? How was this tested? What was the one cell type that was able to undergo transdifferentiation?

The fate of the green transplanted cells could be traced in the newly regenerated limb. Test: produce transgenic axolotls that express GFP in all their cells. Cells of the dermis could contribute to both new dermis and new cartilage skeleton

15. What is meant by the Deep Homology shared across the HOX complexes in diverse organisms? Cite an example.

The formation and differentiation of many structures such as eyes, limbs, and hearts - so morphologically divergent among different phyla that they were long thought to have evolved completely independently (but didn't) - are governed by similar sets of genes and some deeply conserved genetic regulatory circuits. -Whereas ordinary homology is seen in the pattern of structures such as limb bones of mammals that are evidently related, deep homology can apply to groups of animals that have quite dissimilar anatomy: vertebrates (with endoskeletons made of bone and cartilage) and arthropods (with exoskeletons made of chitin) nevertheless have limbs that are constructed using similar recipes or "algorithms".

12. What effect does injection of B-catenin mRNA into ventral vegetal cells have on the Xenopus embryo? What about doing the same with Vg-1?

The function of B-catenin in this context is to regulate gene expression by entering the nucleus and displace repressors bound to certain regulatory regions (activates transcription). -a. Beta-catenin in ventral vegetal cells these cells become dorsal mesoderm, & embryo appears as a mirror image b. Injecting Vg-1 into ventral vegetal cells same thing

7. How does dorsal protein act like a concentration dependent transcriptional activator? What genes are activated under different concentrations of nuclear dorsal protein?

The nuclear gradient in dorsal protein is interpreted by the activation and repression of other genes such as twist (high nuclear dorsal protein) and decapentaplegic [dpp] (absence of dorsal nuclear protein). Zygotic target genes respond to different levels of nuclear dorsal protein. -• Dorsal protein gradients decide which genes will be expressed • No dorsal: dpp expression (ectoderm) • Low nuclear dorsal: sog expression (neuroectoderm) • High nuclear dorsal: twist, snail (mesoderm)

7. What is a nucleosome and of what is it composed?

The nucleosome is the fundamental subunit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a histone octamer. Each histone octamer is composed of two copies each of the histone proteins H2A, H2B, H3, and H4.

17. What acts as the Nieuwkoop Center in the chick?

The presumptive primitive streak (or posterior marginal zone) acts like a Nieuwkoop Center in that it can induce a new axis if transplanted to another embryo.

6. What provides the prospective identity of the somite?

The temporal order in which these cells leave the Node region and entered the pre-somitic mesoderm provides the prospective identity of the somite (its Hox code!).

4. What part of this DNA binding domain interacts directly with bases in the DNA?

The third helix in this motif is called the recognition helix - interacts directly with the bases at the binding site in the DNA.

7. What happens to the pharyngeal arches (branchial arches) during the formation of the face in human embryos? What is responsible for these changes?

There these cells(neural crest cells) direct the development of these processes into parts of the face: jaw, lower and upper lips, nose and parts of the ear.

Terminin 1

Torso receptor activated by Trunk at ends of egg

13. Through which pathway in the overall FGF signaling pathway is neural induction controlled at an early stage of embryogenesis?

Transcription factor Churchill (Zn finger protein)

21. What happens when the primitive streak reaches its anterior extension? What is the purpose of this new structure? What cells form this structure?

When the primitive streak has reached anterior extension, a condensation of cells appear, called Hensen's node. Hensen's node is a site of cell invagination and is the major organizing center for the chick embryo, equivalent to Spemann's organizer in amphibian embryos. It is formed from cells derived from Koller's sickle and the epiblast.

5. The ability of cranial or cervical neural crest cells to give rise to cartilaginous cells and the membranous bones of the head depend on the absence of what genes?

While both cranial and trunk neural crest cells have the ability to develop into pigment cells, glial cells, and several types of peripheral neurons, only cephalic neural crest cells give rise to mesectodermal derivatives such as cartilaginous cells and the membranous bones of the head. The ability of the cranial neural crest to develop into the facial skeleton is dependent on the absence of *Hox genes*.

3. Do trunk and cervical neural crest cells give rise to identical cell types? Why not?

While both cranial and trunk neural crest cells have the ability to develop into pigment cells, glial cells, and several types of peripheral neurons, only cephalic neural crest cells give rise to mesectodermal derivatives such as cartilaginous cells and the membranous bones of the head. The ability of the cranial neural crest to develop into the facial skeleton is dependent on the absence of Hox genes.

9. What identity does Wnt11 promote?

Wnt11 promotes dorsal identity

13. How was it shown that the Wnt receptor is directly required for dorsal structures to form? What component of the Wnt pathway is moved into the dorsal region as a result of cortical rotation? What part does this component play in the Wnt/B-catenin pathway?

a. (knockout exp) Inhibiting expression of Wnt receptor loss of dorsal structures b. As a result of cortical rotation, Dsh is moved into the dorsal region i. Dsh acts directly downstream of the Frz receptor (that Wnt11 binds to)

14. What are the molecular elements that are responsible for establishing the D/V axis of vertebrate embryos? What role does each play? What is the function of each relative to the Wnt/B-catenin pathway?

a. *B-catenin* (when maternal B-catenin mRNA is degraded in the oocyte -> ventralized embryo, which is rescued by injecting fertilized egg with B-catenin mRNA.) Releases transcriptional repressors bound to the regulatory region of targets genes. b. *Dishevelled*: protein can be tracked during cortical rotation and is observed to be in vesicles that are carried along microtubules to the dorsal side. (Binds to the intracellular side of Wnt receptor (frizzled) and prevents the degradation of B-catenin in the cytoplasm). c. *Wnt11*: located specifically at the vegetal pole prior to cortical rotation and is moved to the dorsal side where it activates the Wnt/B-catenin pathway. d. *GSK-3*: Glycogen synthase kinase 3, serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acids. Phosphorylation of B-catenin inhibits B-catenin's ability to enter the nucleus and de-repress transcriptional target genes. The formation of the Nieuwkoop Centre is caused by the suppression of ventralizing signal including GSK-3. e. *VegT*: a T-box transcription factor, is localized to the vegetal cortex and upon cortical rotation is released in a gradient fashion into the embryo to regulate mesoderm development. *VegT activates Wnt expression*. f. *Nodal genes*: key factors in mesoderm induction in all vertebrates. Members of the TGF-B family of signaling molecules. Target genes of the Wnt/B-catenin pathway.

13. With regard to the chick blastoderm, what are the following structures: subgerminal space, area pellucida, area opaca, hypoblast, Koller's sickle and the posterior marginal zone.

a. *Subgerminal space* = space (underneath central region of blastoderm) that forms as a result of blastodisc cleavage, from which the epiblast & hypoblast form b. *Area pellucida* = central region of the blastoderm c. *Area opaca* = outer region of the blastoderm d. *Hypoblast* = cells that form the floor of the subgerminal space (forms the extra-embryonic structures - the afterbirth stuff) e. *Koller's sickle* = sliver on the blastodisc where the invagination will begin (similar to shield region in zebrafish) i. Boundary between area opaca & area pellucida at posterior end of embryo ii. Defines position where primitive streak will begin f. Posterior marginal zone = just epiblast region

4. What are Vg-1 and Veg-T, when are they made and where are they found in the Xenopus egg/embryo? How does Vg1 mRNA become localized in the Xenopus oocyte?

a. *Vg-1*: maternal determinant of embryonic cleavage; comes from TGF-beta family of GFs Vg-1 is synthesized during early oogenesis and is localized to the vegetal cortex of the oocyte. By early after fertilization, Vg-1 has moved to the vegetal cytoplasm to drive animal/vegetal identity. i. Vg-1 mRNA becomes localized in xenopus by recruiting kinesin II to transport it along microtubules becomes localized in a wedged region between the nucleus & vegetal cortex b. *Veg-T*: maternal TF that promotes vegetal identity (endoderm); activates mesoderm inducers (like nodals & TGF-betas) found on vegetal side

10. What are the planes of the first three cleavages in the frog egg? The late blastula contains what three zones?

a. 1st cleavage: in plane of cortical rotation (animal/vegetal axis) b. 2nd cleavage: 90 degrees to the first, still in A/V plane c. 3rd cleavage: perpendicular (equatorial), between the A/V poles d. Late blastula contains 3 zones: i. Animal cells alone --> gives rise to Ectoderm ii. Vegetal cells alone --> gives rise to Endoderm iii. Animal + Vegetal cells (marginal zone) --> gives rise to Mesoderm

28. How is the head fold made? What is the ultimate function of the pouch that is made as a result?

a. All three germ layers in the head fold ventrally to form the head fold (in a similar manner to making a sock puppet)! b. Creates a pouch lined with endoderm becomes the foregut & pharynx

30. What is the function of the following in the chick: amnion, chorion, allantois, and yolk sac.

a. Amnion = surrounds fluid-filled amniotic sac, protects egg from mechanical stress b. Chorion = lies outside amnion just beneath the shell c. Allantois = receives waste products & acts as site of gas exchange d. Yolk sac = surrounds the yolk, which embryo uses for nutrition

9. What is the organization of the Antp and BX complexes (number, order and name of genes). What is their order from 3' to 5' on the DNA? How does this correlate with the pattern of expression of these genes along the A/P axis of the fly?

a. Antennipedia complex (Antp): on the 3' end of the 3rd chromosome i. lab - labial ii. pb - proboscipedia iii. Dfd - Deformed iv. Scr - sex combs reduced v. Antp - antennipedia b. Bithorax complex (BX): 5' end i. Ubx - ultrabithorax ii. abd-A - abdominal-A iii. Abd-B - Abdominal-B c. The order of these genes along DNA correlates with gene expression along A-P axis: Ex - lab & pb affect the anteriormost areas of the fly, while abd-A and Abd-B affect the posteriormost regions

26. What happens as a result of the regression of Hensen's node? What specific cells structures are made as a result?

a. Because Hensen's node regresses, primitive streak is arrested as a result, it sets in motion activation of somites and brain formation & notochord (b/c arrest happens cranially)

9. How is the polarity of the frog egg identified before fertilization? In which hemisphere does the sperm enter the egg? What is completed after fertilization? How is the egg's cytoplasm preserved during this process? What two important changes take place once the egg nucleus has completed meiosis?

a. Before fertilization, differential pigmentation distinguishes the animal end (darker) from the vegetal end (lighter). b. After fertilization, nuclei fuse and egg orients it's A-P axis in relation to the point of sperm entry by rotating its membrane (with the use of microtubules) c. Once egg completes meiosis: i. Dorsal/ventral axis is established ii. cleavage

6. What happens to the blastomeres at the margin of the blastoderm of the zebrafish?

a. Blastomeres at the margin of the blastoderm merge and collapse into the yolk cells & become the yolk syncytial layer b. This yolk syncytial layer then spreads vegetally by epiboly

11. Where are the cells that will give rise to the mesoderm and endoderm? How do they move during Xenopus gastrulation? What happens to the ectoderm?

a. Cells that give rise to endoderm: at the vegetal pole (composed of vegetal cells alone) b. Cells that give rise to mesoderm: along the sides (marginal zone), b/c composed of animal + vegetal cells c. During xenopus gastrulation: i. vegetal cells move inward to make the endoderm ii. Marginal cells (A+V) move along the ectodermal line that forms the inner mesodermal lining -Mesoderm and endoderm converge and begin to move inwards at dorsal lip of the blastoporeThis extends inwards along A/P axis. Ectoderm (blue) spreads to cover embryo -Dorsal endoderm (yellow) separates mesoderm (red) from the space between the yolk cells = ARCHENTERON (future gut cavity) Lateral mesoderm spread ventrally to cover inside of archenteron.

4. What is a compartment? In which segmental compartment is engrailed expressed in the wing?

a. Compartment = contains all the descendants of the cells present when the compartment is set up during embryogenesis b.posterior

7. Describe the process of cortical rotation. What effect does this have on the arrangement of microtubules in the sub-cortex?

a. Cortical rotation occurs within first 90 min. of fert b. Cortex rotates ~ 30 degrees (due to dorsalizing factors that later induce dorsal lip formation) c. As a result, microtubules arrange according to cortical rotation, *positioning plus ends away from site of sperm entry* (ventral side) and act as tracks to move mRNAs & proteins

15. What are six features of the Xenopus embryo by the end of gastrulation?

a. Dorsal mesoderm is beneath dorsal ectoderm b. Mesoderm is spread to cover gut c. Epiboly - ectoderm covers embryo d. Yolk cells are internalized (food source) e. Dorsal mesoderm develops into: i. Notochord = rod along dorsal midline ii. Somites = segmented blocks of mesoderm along notochord

9. When are Hox genes first expressed in mammalian development? What do these genes specify during development?

a. During gastrulation (very early in development) b. These genes specify differentiation of different segments of the vertebrae in mammals. Once gastrulation begins, Anterior-Posterior polarity in all vertebrates becomes specified by the expression of Hox genes (homologous to the homeotic gene cluster of Drosophila).

10. In the chick, where does the endoderm and mesoderm come from and how are they internalized?

a. Early cleavage in the chick embryo produces a small opening - the subgerminal space - which later widens to become a cavity. i. At the point that the subgerminal space becomes big enough, the momma hen lays the egg, and the egg develops two layers surrounding the subgerminal space: 1) the epiblast & 2) the hypoblast ii. The epiblast invaginates forms the endoderm & mesoderm iii. Whatever does NOT invaginate (remains outside) becomes the ectoderm

13. What are the cellular fates of the ectoderm, mesoderm and endoderm?

a. Ectoderm i. Epidermis ii. Nervous system iii. Cornea & lens of eye iv. Lining of mouth & rectum v. Nervous system b. Mesoderm i. Heart, blood, blood vessels ii. Kidneys iii. Muscles iv. Bones (skeleton) v. Dermis of skin vi. Ovaries/testes c. Endoderm i. Lining of gut & branches ii. Lining of excretory ducts, bladder iii. Lining of lungs, trachea iv. Lining of reproductive ducts v. Liver vi. Pancreas

P 1. What are the cell types that develop from ectoderm, mesoderm and endoderm?

a. Ectoderm: Nervous system, Epidermis (skin), Eyes, Adrenal medulla, Tooth enamel, Epithelium of: mouth, anus, pineal, & pituitary glands b. Mesoderm: Notochord, Skeletal system, Muscular system, Excretory system, Circulatory & lymphatic systems, Lining of body cavity, Adrenal cortex, Dermis of skin, Reproductive system c. Endoderm: Epithelial lining of digestive tract & respiratory system, Lining of urethra, urinary bladder, & reproductive system, Liver, Pancreas, Thymus, Thyroid & parathyroid glands

3. When does the embryo begin to transcribe its own genome? Why is the embryo able to divide during that period when it doesn't transcribe the genes from its own genome?

a. Embryo transcribes its own genome at the mid-blastula stage (after 12 cleavages) i. "But how does it know to divide for the first 12 cleavages?" 1. Maternal mRNAS (distributed along animal/vegetal axis) tell the embryo to divide until it can transcribe the genes itself

10. What segment polarity genes are involved in the maintenance of segment boundaries, what are their molecular functions, and what is their role?

a. Engrailed = TF b. Wingless = secreted signaling peptide c. Hedgehog = secreted GF d. Patched = Hh receptor (transmembrane protein)

11. Be able to describe the effect of hh binding to an adjacent cell that has a patched receptor on the activation of intracellular proteins and transcription. Be able to contrast this with what happens in the absence of hh binding to patched.(WORKSHEET?)

a. Engrailed-expressing cell: i. En expression produces expression of Hh Hh gets secreted from En-expressing cell b. Wingless-expressing cell: i. Hh from other cell binds to Patched receptor on Wg-expressing cell activates Smoothened G-protein to activate Ci induces Wingless (& Patched) expression Wingless gets secreted to En-expressing cell --> c. Engrailed-expressing cell: i. Wingless binds to Armadillo receptor induces En expression d. If Hh does NOT bind to Patched, the cycle is cut off: we no longer get Ci activation, no wingless expression, and no engrailed expression either (or Hh for that matter)

15. How do the ingressing epiblast cells spread across the blastoderm once they have passed through the primitive streak? What will these cells give rise to?

a. Epiblast cells converge on the primitive streak, then move inward and spread out anteriorly and laterally underneath the upper layer form mesenchyme b. Ingressing cells give rise to mesoderm & endoderm c. Cells that remain outside ectoderm

4. What are the six events in embryology?

a. Fertilization b. Activation: metabolism increase, cytoplasm movement, prepare for cleavage c. Cleavage d. Gastrulation e. Mesoderm formation f. Organogenesis

5. What happens during fertilization and activation? What triggers the onset of embryonic development?

a. Fertilization: nuclei of egg & sperm fuse together to make the zygote b. Activation: metabolism increases, different parts of the cytoplasm respond motionally to sperm entry, materials for cleavage are synthesized c. Onset of embryonic development is triggered by sperm's contact with the egg

5. How is this early distinction of cell type in the embryo of the zebrafish like that of the human blastocyst?

a. Flattened peripheral cells (zebrafish) are similar to the human trophoblast cells --> which eventually become the placenta & amniotic ssac (afterbirth stuff) b. Rounded cells (zebrafish) are sim. to inner cell mass in humans becomes embryo/fetus

6. What is the molecular nature of gap, pair-rule and segment polarity genes? How does the environment in which segmentation genes work differ from that of the gap and pair-rule genes?

a. Gap and pair-rule genes: act when the embryo is a syncytium (both TFs) b. Segment polarity genes: act when the embryo has cellularized (Engrailed is a TF; Wnt & Hh are secreted factors) i. These are long-acting compared to the gap & pair rule genes

10. What are the functions of genes in the Antp Complex versus the function of genes in the BX complex?

a. Genes in the Antp complex: i. Specify segment identity in the head & first thoracic segment b. Genes in BX complex: i. Specify segment identity in 2nd & 3rd thoracic segments & abdominal segments

14. How was the organizer (Spemann's organizer) discovered? What is its significance to gastrulation?

a. Han Spemann discovered that when you remove the dorsal lip from the blastopore and transplant it onto another blastopore (onto the ventral side), you get two conjoined twins! b. Spemann's organizer is significant to gastrulation b/c: induces development of the CNS! (central nervous system)

4. What are the body segments of the adult fruit fly and what are some important physical characteristics of these segments (allow you to distinguish one segment from another)?

a. Head - eye/antennae, mouth (gnathal) parts b. Thorax i. T1 - legs ii. T2 - legs + wings iii. T3 - legs + halteres c. Abdomen (A1-A10) i. Female 1. A1-A6: tan pigmentation 2. A7-A8: dark pigmentation 3. A9-A10: anus ii. Male 1. A1-A4: tan pigmentation 2. A5-A8: dark pigmentation 3. A9-A10: anus iii. **Male has MORE DARK pigmentation on his abdomen

7. What is the molecular function of all homeobox genes? What is a paralogous group? What is an ortholog?

a. Homeobox genes: Hox genes are a subset of the homeobox genes encoding TFs that serve as "master switch" genes to turn on expression of certain genes in the nucleus -Most homeobox genes are not Hox genes. b. Paralogous group = composed of the most similar members of each hox gene cluster c. Hox genes = general term used for mammalian genes containing homeobox (conserved 180bp region of gene encoding the homeodomain, 60 AA DNA-binding motif) i."homeobox genes" = genes that have a homeobox; later called hox genes (hox genes contain the homeobox & encode a homeodomain) -Paralogs are genes related by duplication within a genome. E.g. Ubx and Abd-B. -Orthologs are genes in different species that evolved from a common ancestral gene by speciation. E. g. Antp (flies) and Hox B6 (humans).

2. What was the definition Bateson (rediscoverer of Mendel's laws, by the way) gave to the phenomenon called, "homeosis"?

a. Homeosis = a structure resembling the likeness of another b. (Ex: A homeotic mutant called antennipedia causes the antennae of a fly to look like legs), owl mom, frog fish and sponge

12. What is a Hox code and why is this important in specifying somite identity during development?

a. Hox code = combination of specific hox genes in a certain segment that determines the segment to be a certain morphological type -Various downstream genes respond to unique combinations of Hox genes expressed along the A/P axis. b. This is important during development because it produces differentiation of different body segments

6. Compare the number and similarities shared between Drosophila and Mammalian Hox complexes.

a. Hox complex in mammals: i. 4 copies of hox complex per haploid set ii. More posterior complexes (duplications of most posterior paralogous group) -Mouse and human genomes contain four copies of the Hox complex per haploid set, located on four different chromosomes:Hoxa through Hoxd in the mouse, andHOXA and HOXD in humans (on chromosomes 2, 7, 12, and 17).

9. In what family are mesoderm inducers found? What is the overall function of members of this family? What are examples of this family found in the Xenopus embryo?

a. Mesoderm inducers are part of TGF-beta family b. TGF-beta proteins function in many processes in adult & embryo: cell growth & differentiation, apoptosis, cellular homeostasis, & other cellular functions c. Ex's: Xenopus nodal-related proteins, Vg1(secreted protein), activin(receptor for TGF-beta factor), derriere protein(posterior mesoderm), & BMPs(bone morphogenetic proteins (ligand) )

N 1. What are the three fundamental processes in development and what do they mean?

a. Morphogenesis = origin of shape and form in an organism b. Differentiation = process of cells developing differently than other cells due to differential gene expression & specializing for different functions c. Growth = increase in size, requires input of matter, food

18. What are neural crest cells, what do they do and what four structures do they give rise to?

a. Neural crest cells = derived from dorsal aspect of neural tube; come from edges of neural folds after neural tube fusion i. They detach & migrate as single cells between mesodermal tissues ii. Give rise to: 1. Sensory & autonomic NS 2. Skull 3. Pigment cells 4. Cartilage

16. What is neurulation and what are its four steps?

a. Neurulation = neural tube formation b. *Neural plate: ectoderm located above notochord & somites* c. Edge of the neural plate forms neural folds that rise toward the midline d. Folds fuse to form neural tube e. Neural tube sinks below epidermis i. Anterior neural tube --> becomes brain ii. Mid & posterior neural tube become spinal cord

10. When does the mid-blastula stage occur and what is it significance? What triggers this transition?

a. Occurs after 1st 12 cleavages and the embryo contains 4096 cells b. Significance: blastomeres no longer think of themselves as isolate cells; change cell-cell contact with one another - know they're part of a whole and cell division becomes asynchronous c. After that point, if you pluck off an embryo, you'd miss structures d. Transition triggered by ratio of DNA:cytoplasm

8. How is the maintenance of segment polarity gene expression regulated differently than the establishment of segment polarity gene expression?

a. Pair rule genes set up the parasegmental boundaries (establish segment polarity gene expression) b. On the other hand, maintenance of segment polarity genes is accomplished by cell-cell communication i. Through wingless-expression cell & engrailed-expressing cell (after eve & ftz have gone away)

2. What is the phylotypic stage in development and when do all vertebrate embryos pass through this stage? Where does the stage get its name?

a. Phylotypic stage of development: after gastrulation, when embryos of different species look (more or less) like each other -The head is distinct and the neural tube (forerunner of the nervous system) runs along the dorsal midline of the antero-posterior (head to tail) axis. -Immediately under the neural tube runs the notochord, a signature structure of chordates, flanked on either side by the somites (from which muscles and skeleton will develop). b. Gets its name from the fact that all animals in the PHYLUM chordata look like one another during that stage

22. How is the prechordal plate mesoderm and head process formed?

a. Prechordal plate formation: after the primitive streak has elongated all the way (anteriorly), cells from Henson's node migrate in the opposite direction (posteriorly) along the midline underneath the epiblast gives rise to prechordal plate mesoderm & head process i. Like zipping top-down (the opposite way)!

8. What are as many as four changes that take place with the activation of the egg?

a. Sharp rise in calcium in egg cytosol --> increases cellular respiration & protein synthesis by egg b. Nuclei fuse c. Chemical signals from cortical reaction cause H+ to be transported out --> pH increases

9. To what is the "shield" stage analogous? What is the fate of the earliest cells to involute? What follows the presumptive endoderm during involution? What is the spatial fate of these mesendodermal cells?

a. Shield stage in zebrafish is analogous to the Spemann Organizer in xenopus b. Earliest cells to involute *endoderm* c. As mesendoderm becomes internalized, the ectoderm continues to undergo epiboly until all of the yolk is inside d. The mesendodermal cells migrate under the ectoderm *toward the animal pole* forming main axis of the zebrafish -Mesodermal and mesendodermal cells roll under the blastoderm margin in the process of involution and move into the interior under the ectoderm.

2. What are some advantages of the zebrafish for genetic analysis and developmental studies?

a. Short life cycle (12 weeks), so can see gene expression of offspring relatively quick b. Takes 3 months to have sexually mature adult

9. What are two gene products that limit dpp protein to the dorsal apex of the embryo? How do they act on dpp?

a. Sog - binds to dpp and blocks dpp from binding to its receptor. This stops the ventral diffusion of dpp. b. Tsg - binds dpp, acts like sog. Tsg enhances the ability of dpp to exist in its domain by dislodging latent dpp diffusion c. Tolloid - releases dpp when bound to sog.Tld is expressed initially in the same cells as dpp. Tld is expressed in response to dorsal and it cleaves sog which protects dpp Tld is an enzyme that enhances dpp and is a protease. Tld may activate dpp by cleaving it

17. What is the fate of the cells of the somites, lateral plate mesoderm and ventral mesoderm?

a. Somites become --> future dermis b. Lateral plate mesoderm --> heart, kidneys, gonads, & gut muscles c. Ventral mesoderm --> blood-forming tissues

8. Explain the spatial and temporal co-linearity of the Hox genes in Drosophila and mammals.

a. Spatial colinearity = order of genes ~ order of corresponding segments on the A-P axis b. Temporal colinearity = 1st paralogous group expressed before the second, expressed before the 3rd, etc... -Anterior-most gene is expressed first (Hensen's node starts @ top, & then moves posteriorly) c. Both drosophila & mammals hox genes show spatial & temporal colinearity -The Hox genes in mammals are arranged in the same order as their expression pattern along the anterior-posterior axis;- most 3' gene (labial) being required for the production of the most anterior structures, and the most 5' gene (AbdB) specifying the development of the posterior structures.

Q 1. What are two ways homeotic selector genes differ from the segmentation pattern genes that you have learned about so far in this course?

a. Specify segment identity (not segment number) b. Control activity of other genes c. Required through development to maintain pattern of gene expression

6. To what part of the Xenopus egg is sperm entry restricted? How is the effect of sperm entry mediated?

a. Sperm entry restricted to animal pole b. Effect is mediated by cortical rotation, accomplished by dorsalizing factors)

4. What is the sphere stage of the zebrafish embryo and what two cell types are distinguished at this stage and which will give rise to the embryo?

a. Sphere stage: where 1000 cells lie on top of the yolk mass and we see two developing layers: outer and deep 1. Outer layer: flattened cells 2. Deep layer: rounded cells (eventually become the embryo itself)

14. Where does the primitive streak form relative to the posterior marginal zone?

a. The primitive streak forms out of the Koller's sickle region, the darker, denser area of cells in the posterior marginal zone b. It forms anteriorly over half way across the area pellucida (like zipping up a onesie!!)

29. How is the rest of the gut formed? How is the heart formed and from which germ layer?

a. The sides of the embryo fold together to form the rest of the gut b. As this folding happens, two opposing cavities come together to form the heart (which immediately begins beating upon fusion) lying ventral to the gut

11. Do neural ectoderm and mesoderm have the same expression boundary for a given Hox gene? Which is more anterior than the other?

a. They have different expression boundaries b. Neuroectoderm is more anterior than the mesoderm

10. Where does the D/V axis lie relative to the point of sperm entry and the gray crescent?

a. Ventral side: point of sperm entry; dorsal is opposite b. Gray crescent : dorsal side

What genetic observations support the model of segment polarity gene expression maintenance?

a. Wingless expression halts in embryos with engrailed mutants b. Engrailed expression halts in embryos with wingless mutants c. Wingless and engrailed maintain each other's expression d. Wingless and engrailed are expressed in completely different cells, so cell-cell communication must occur

3. What polarity is evident in the zebrafish egg? Where is the zygotic nucleus located in the fertilized egg? How does yolk affect the first cleavage divisions?

a. Zebrafish egg has animal & vegetal ends b. Nucleus and cytoplasm located at the animal end c. Yolk affects the first cleavage division b/c it does not penetrate the vegetal half of the embryo ; blastomeres are only confined to the animal end

R 1. Why are both the chick and zebrafish embryos good model systems from the stand point of observing the stages of development?

a. Zebrafish: i. Transparent skin ii. Vertebrate iii. Short life cycle (12 weeks) iv. Used as model system for genetic defects in certain blood & cardiovascular disorders b. Chick i. Very similar to human ii. Can remove egg shell to easily see stages of development

8. As a result of cortical rotation, what two gene products become localized at the equator opposite the site of sperm entry? How does the 2nd cleavage division affect the distribution of these gene products?

a. xWnt11 (homolog of wingless) & Dsh (Disheveled)..Dishevelled is a phosphoprotein that acts directly downstream of frizzled receptor (mediates the Wnt signal). b. 2nd cleavage division: only 2 of the 4 blastomeres will contain these determinants these will become the dorsal side of the embryo

Cerberus

antagonist to BMPs, Nodal-related proteins, and Wnt proteins in prospective anterior tissues. Cerberus is expressed in the organizer and the anterior endoderm, and it is involved in the suppression of mesoderm fate and induction of anterior structures, in particular the head. Ectopic expression of the Transcription factor Xmsx-1 (target gene of BMP4 pathway) or BMP4 itself in the anterior endoderm suppresses head structures and the expression of Cerberus.

7. Which gene product is expressed in the presomitic mesoderm in a 90 minute cycle?

c-hairy 1 and c-hairy 2 are expressed in pre-somitic mesoderm in a posterior to anterior sweep that lasts for a 90 minutes cycle (time it takes form a new pair of somites

16. What are the three characteristics of euchromatin? What are the three characteristics of heterochromatin?

euchromatin: high histone acetylation, low DNA methylation, H3-K4 methylation heterochromatin: low histone acetylation, dense DNA methylation, H3-K9 methylation

3. What did Bateson believe homeosis was key to understanding?

evolution

11. What is the chick blastodisc? When does the chick egg become fertilized (relative to the events of egg maturation and egg laying)?

i. Blastodisc = chick blastoderm (flat) ii. Chick egg is laid 20 hours after fertilization (so when we eat an egg we are eating a fertilized egg. Bon appetit!) -The large yolky egg of the hen is fertilized and begins to undergo cleavage while still in the hen's oviduct. Cleavage is confined to a small patch of cytoplasm on the top of the yolk, which forms the blastodisc or blastoderm. During passage down the oviduct, the egg is surrounded by albumen, shell membranes and the shell. At the time of laying, the blastoderm is 20,000-60,000 cells.

Antero-posterior 1

mRNAs: bicoid forms anterior to posterior gradient; hunchback uniform; nanos and caudal uniform--> anterior to posterior gradient of Bicoid protein formed, hunchback mRNA translation suppressed in posterior region by Nanos, caudal mRNA translation repressed by Bicoid

2. Do different organisms have the same number of somites?

no, they different numbers

14. What role does acetylation play in epigenetic regulation?

opens up chromatin, chromatin remodeling also opens up chromatin

8. What is the relationship of the paralogous groups to their spatial expression along the proximal/distal axis of the vertebrate limb?

their order is the same in the genome as it is moving distal -Hox a and d- forelimb -Hox c and d- hindlimb

4. What effect does DNA methylation have on chromatin packing?

typically represses gene transcription

Dorso-ventral 2

ventro-dorsal gradient of intranuclear Dorsal protein forms-->ventral activation of twist, snail, and repression of decapentaplegic--> Decapentaplegic expressed dorsally--> gradient of Decapentaplegic activity patterns dorsal region--> torso-ventral axis divided into prospective mesoderm, neurectoderm, epidermis, amnioserosa

20. What effect did RA exposure have on the expression patterns of Hox 2.9(b1), 2.8(b2), 2.5, Krox-20 in mouse embryos?

• 2.9 and 2.8: extends more anteriorly • 2.5: no effect • Krox-20: expression disappears

11. What are the four different cell types specified by the dpp/sog pathway along the d/v axis?

• Amnioserosa, dorsal epidermis, neurogenic ectoderm, mesoderm (muscle)

27. What signals are necessary or need to be inhibited to make the following in the chick: anterior mesoderm, lateral plate mesoderm, the head.

• Anterior mesoderm: BMP antagonists (Noggin, Chordin, Cerberus) • Lateral plate mesoderm: High BMP • Head: Low Nodal, BMP, and Wnt (depends on antagonists - Dkk1, Cerberus)

9. How can the modification of histones themselves contribute to modifying chromatin packing?

• Can either condense or open up the chromatin

21. What genetic evidence was discovered fifty years ago that support the importance of the PRE's (Fab-7, Mcp, bxd, Bx) in regulating the genes of the Bithorax complex?

• Discovered that each BX complex transcript is regulated by at least one PRE • Genetic screening showed that mutants for a PRE would overexpress the Hox gene it affects

8. What gene responds transcriptionally to the absence of dorsal protein? Where does this protein go during this late period in the syncitial blastoderm? What stops this protein from diffusing far ventrally?

• Dpp (decapentaplegic) • Diffuses ventrally with the highest point at the dorsal apex of the embryo • Within an hr of cellularization of blastoderm, dpp is limited to a 5-7 cell wide strip at dorsal apex of embryo. • Restrictuion due to: interactions with other proteins, interactions with different forms of dpp receptors, and interactions with collagen in the ECM

18. How do the sharp boundaries of Hox gene expression differ between their expression in the central nerve cord versus the somites? What is a Hox code?

• For a given Hox gene, the anterior boundary differs between the nerve cord and somites • Hox code - unique pattern of gene expression that specifies the identity of a region

8. What region of the histones are sites of chemical modification?

• H3 and H4 usually the modified proteins • Generally modified on lysines, arginines, serines, and threonines o Tails are lysine and arginine rich, so this is usually where the histones are modified

21. What early embryological marker does the normal boundary of Hox 2.9 correspond to?

• Hensen's node

13. What amino acids are targets for chemical modification of histones and where do they reside in the histone protein? What are some of the modifications found on these amino acids?

• Histone modifications (methylation) by histone modifying enzymes • DNA methylation • Chromatin binding proteins o Polycomb proteins o Heterochromatin Protein

22. What are the current names for Hox 2.9, 2.8, 2.5?

• Hox 2.9 = Hoxb1 • Hox 2.8 = Hoxb2 • Hox 2.5 = Hoxb9

18. What effect does the loss of function of the Polycomb gene have on the expression of Abd B (last gene in the Bithorax Complex)? What change in gene expression is observed? How would this have affected the embryo?

• Loss of function of Polycomb: Abd-B is expressed over the entirety of the embryo (because all Hox genes are expressed in every segment) • Change in expression: A8 segment is exhibited as the identity of all body segments

M 1. What are the two functions of the Drosophila Toll gene?

• Maternal control of Dorsal/ventral patterning • Affects fly's immunity to gram positive bacteria and fungal infections

22. How is the epigenetic regulation of Bithorax genes change during embryogenesis?

• Over time, H3 acetylation extends posteriorly

17. What role does epigenetics play in the differentiation of cells during development? Why do you think this is important?

• The DNA sequences of pluripotent and unipotent cells are exactly the same, but with different patterns of DNA methylation. Clearly, this plays a significant role in whether or not cells can differentiate into multiple types.

15. What are 3 Wnt inhibitors and what role do they play in patterning the embryo?

•*Cerberus* oAntagonist to BMPs and Wnt oExpressed in organizer and anterior endoderm oSuppression of mesodermal fate + induction of anterior structures •*Frzb* •*Dickkopf* oBoth of the above bind Wnt proteins