Test
Explain how axis specification in amphibians depends on both cytoplasmic rearrangements and the Wnt pathway.
Axis specification is dependent upon dorsal localization of B-catenin, which induces the formation of dorsal tissue in the early embryo. B-catenin is regulated by activity of GSK3, which degrades B-catenin when activated. Additionally, GSK3 activity is dependent upon the presence of GBP, which inactivates GSK3, such that when GBP is present the GSK3 complex cannot degrade B-catenin. During initial fertilization, GBP is transported with Dsh to the future-dorsal region of the embryo allowing for regional concentration increases in B-catenin in pre-dorsal tissue. Additionally, transportation of Dsh along microtubules acts to inhibit B-catenin degradation, while cytoplasmic transportation of Wnt11 during cortical rotation acts upon the Wnt pathway to increase the production of B-catenin in dorsal tissue. Experimentally, we can observe that GBP and Dsh activity are unable to specialize dorsal tissue without Wnt11 expression. Overexpression of Wnt11 leads to complete dorsalization of the embryo.
Explain the various ways in which bmp signals contribute to the induction of neural crest, as well as the subsequent morphogenesis and differentiation of these cells. Be sure to cite experimental evidence.
BMP signaling plays important roles in both the induction of neural crest cells and in the initial dispersal of neural crest cells from the neural tube. Based on the previously studied role of bmp signaling in amphibian axis specification, researchers suspected that bmp signaling might play a role in the specification of neural tube. Researchers determined that a gradient of bmp signaling specifies neural crest cells - specifically high levels of bmp and non-existent bmp signaling do not specify neural crest cell development, while low, "Goldilocks-levels" of bmp signaling specify neural crest cell development. Experimentally, researchers discovered that embryos that artificially overexpressed bmp failed to develop neural crest cells, while embryos that expressed limited amounts of bmp developed neural crest cells throughout the embryo. During the initial dispersal of neural crest cells, the regulation of bmp expression plays a critical role in the timing of dispersal. Researchers have determined that, while bmp is dispersed throughout the embryo, a variety of other factors are localized to neural crest cells. These factors act to regulate the activity of bmp, which ultimately promotes dispersal of neural crest cells. Specifically, somatic cells inhibit that activity of noggin, which in turn inhibits the activity of bmp. Therefore activation of somatic cells activates bmp activity in neural crest cells by preventing noggins inhibition of bmp. After activation, bmp activates an unidentified protease enzyme that degrades N-cadherin, which under normal circumstances acts as a cell adhesion molecule to attach neural crest cells to the neural tube. In sum: somatic cells activate bmp by inhibiting noggin activity. And bmp facilitates the dispersal of neural crest cells by degrading their N-Cadherin mediated attachment to the neural tube.
What is the function of bindin and what explains its extraordinary variability?
Beads covered in bindin bound only to eggs from the corresponding species.
What are the similarities and differences between Myxobacteria and Dictyostelium, and their mechanisms of morphogenesis and differentiation?
Both are single celled organisms that exhibit a collective multi-cellular response to starvation. Myxobacteria: Factor A to Aggregate Non-motile Spore Positive feedback w/ Factor A All organisms continue to reproduce Dictyostelium (Slime Molds): cAMP to Aggregate Motile Slug Negative feedback w/ pre-spore cells inhibiting cAMP Stalk cells do not reproduce
Distinguish between cell fate and cell commitment. How can one assay these aspects of cellular phenotype? Give specific examples.
Cell fate - Specification that the cell will adopt if allowed to continue normal development without transplantation/alteration of normal development. Determine cell fate by observing cell growth in a neutral medium. Cell commitment - A measurement of the degree to which a cell's fate can be altered. Once a cell is entirely committed, it can be transplanted into a new tissue and will not have its final specialization altered.
What are the major changes that happen upon egg activation?
Changes upon egg activation: -Initiated by fertilization -Fast (Na+) and/or Slow (Ca2+) Block -Ca2+ release from E.R. -Activate NAD+ (create plasma membrane) -Transcribe maternal mRNAs -Start dividing cells rapidly
How does one construct a fate map? How can one analyze cell lineage? What differs between the two approaches?
Construct a fate map by examining the changes in developmental morphology of the embryo over time. Trace regions using vital dyes and fluorescent dyes. Or chimeric embryos with cells from a phenotypically different animal (e.g. different color cells). Lineage is traced by marking an individual cell w/ fluorescent dye. Progeny contain same dye. Lineage is on a single cell and fate map is on a region w/o same resolution as lineage.
What are cytoplasmic determinants? What is the relevance of the yellow crescent found in some ascidian embryos? How could you identify the molecular nature of these deteminants?
Cytoplasmic determinants are developmental factors present within the cytoplasm of a cell that influence the cells development and specialization; cytoplasmic determinants are inherited by daughter cells during cell division.
Define "determination" and "specification" and discuss the three major classes of cell fate specification. Provide experimental evidence that supported the existence of each mode.
Determination: The stage of cell commitment where the cell has adopted a particular cell fate. Neutral medium. Specification: Point where you can't alter cell fate through transplantation. Three classes: -Autonomous: Sea Urchin w/ cell that could be separated and still develop a semi-functional embryo. -Conditional: Frog embryo where blastula cells can be transplanted and adopt specification of their surrounding tissue. -Syncytial: Inject bicoid in Drosophila for 2 headed embryo
Define the terms "differentiation", "cell fate", "cell lineage", "fate map", "morphogenesis", "epithelium", "mesenchyme", "isometric growth", "allometric growth".
Differentiation - The process through which a cell becomes increasingly specialized to preform specific tasks associated with its cell type. Cell Fate - The specialization that one grouping of early embryo cells will adopt as development continues. Cell Lineage - The precise tracing of individual cells prior history, including its parent cells and the tissue that the cell was derived from. Fate Map - A diagram labeling the cell fates of each region within early embryo. Morphogenesis - The biological process that causes an organism to adopt a specific shape by controlling the organized spatial distribution of cells. Epithelium - Tightly connected cells that undergo morphogenesis as a coordinated grouping of cells. Mesenchyme - Unconnected cells that operate as independent units during morphogenesis. Primarily derived from the mesoderm. Isometric Growth - Growth in size of an organism where body proportions remain constant. Allometric Growth - Growth in size of an organism where body proportions are not maintained.
What are the roles of micromeres during sea urchin development? How is this lineage specified, where are the cells found during embryogenesis, and what are some of the molecular mechanisms underlying their commitment to terminal differentiation? Provide experimental support.
During sea urchin development the micromeres coordinate invagination during gastrulation of the embryo, specify the development of non-micromere cells via induction and ultimately form the skeletal and muscular structures of the embryo. The lineage of micromeres is specified autonomously by cytoplasmic morphogens that are transported to the ventral pole during early cleavage. The micromeres are located at the ventral pole of the embryo. The importance of micromere induction was demonstrated experimentally when researchers observed that ablation of the micromere cells in the early embryo resulted in complete animalization of the remaining cells; the organism was unable to develop without the micromeres. Additionally, ablation of the pre-endodermal cells and recombination with micromeres resulted in a developed embryo that roughly matched a normal urchin embryo, although it did not develop any endoderm. Experimental evidence has identified the B-Catenin as the primary determinant of morphogen specificity. Specifically, researchers noted that when treated with LiCl, B-Catenin distributes amongst all cells of the embryo (likely a result of LiCl disrupting the wnt signaling) and the resulting organism develops only endoderm and mesoderm. Additionally, by preventing the transport of B-catenin into the nucleus, researchers created embryos that only developed ectodermal tissue.
Describe some of the major ways in which embryonic cleavage varies among egg types and taxa, providing specific examples. Do different types of embryonic cleavage ever influence adult morphology?
Early embryonic cleavages differ between species in two major regards: the distribution of cytoplasm within daughter cells and the angle of cell division. Radial cleavage is a pattern of cleavage where both the cytoplasmic volume of each daughter cell is constant and cell division occur at parallel or right angels to the polar axis. Spiral cleavage differs from radial cleavage by allowing for cell division where the daughter cells do not divide along parallel or right angels. Discoidal cleavage also differs from radial cleavage because division produces daughter cells with unequal distributions of cytoplasm. Additionally, cleavage can vary in the development of the cleavage furrow: In holoblastic cleavage the cleavage furrow completely separates the two daughter cells, while in Meroblastic cleavage the where only a portion of the cytoplasm is cleaved. Lastly centrolicethal cleavage occurs when the nucleus is replicated without the physical division of each cell.
Explain the embryological origins of muscle cells and the molecular mechanisms underlying muscle cell specification and differentiation.
Embryological Origins: -Muscle cells develop from the sclerotome of the mesoderms. -Early myotome cells specified by determining factors such as Wnt and Shh. -Myoblasts divide and then attach to each other. -Fusion of multiple myoblast to form a large multinucleated muscle fiber. Molecular Mechanisms: -Key genes: MyoD and Myf5 Specification of different muscle types: -Slow and Fast muscle fiber-types. -Adaxial Mesoderm → Slow Muscle Fiber -Lateral Pre-Somitic Mesoderm → Fast Muscle Fiber -Development of Slow Muscle Fiber is dependent upon Shh signaling. Can prevent slow muscle formation by blocking Shh signaling.
What is the difference between endochondral and intramembraneous ossification? What are the major cellular origins and cellular events for these two types of bone development?
Endochondral Bone = Cartilage Intermediate - Majority of Bones - Derived from mesoderm, unless craniofacial. - Cells initially specialized as cartilage cells. - Hypertrophic chondrocytes in the middle: o Recruits blood vessels o Leads transition from cartilage → bone o Migrates from middle to ends. - Sox9 critical to process. Intramembraneous Bone = No Cartilage Intermediate - Frequently craniofacial bones. - Derived from neural crest. - Osteoblast cells aggregate together, expand and secrete ossifying factors - Osteocytes are recruited to the center of the ostefication.
How does ethanol exposure affect neural crest development and what are the public health implications of such findings.
Ethanol exposure has been shown to have two main effects on the development of neural crest cells. First, ethanol exposure increases cell death, specifically for neural crest cells. One experiment noted that exposure to ethanol in chick embryos led to a 3-fold increase in neural crest cell death. Therefore, ethanol exposure leads to fewer migrating neural crest cells. Second, ethanol exposure has also been shown to disrupt the migration of neural crest cells by disrupting several key signaling molecules that normally direct the migration of neural crest cells. Interestingly, researchers have conducted experiments on SHH (a signaling messenger) that demonstrated that restoration of SHH expression after ethanol exposure restored normal phenotype. From a public health perspective, ethanol exposure in utero can result in mental retardation, craniofacial anomalies, heart defects, deficient myelination, and limb or join defects.
Why might facultatively multicellular organisms be limited evolutionarily in their degree of cell and tissue specialization, as compared to obligately multicellular organisms?
Facultatively multicellular organisms have to function as single cells, so can't specialize too much.
What is the clinical significance of neural tube closure and what has been learned about the mechanisms underlying this process in Xenopus, mouse and humans?
Failures that occur during neural tube closures can result in frequently fatal neural tube defects in newborns. The three categories of NTDs are: anecephaly (failure in the anterior neural tube), spinal bifida (failure in the posterior neural tube), and craniorachischisis (failure of the entire neural tube to close). Meckel-Gruber Syndrome is the most frequent cause of NTDs, which results from a mutation in the PCP protein Meckelin. Additionally, research has found defects in primary cilium and PCP signaling can cause NTDs. Lastly, a recent study conducted by the Nishwander lab revealed interesting findings for the role of folic acid during neural tube closure. Currently, it is recommended that all women take folic acid vitamins during pregnancy to prevent NTDs; epidemiologically, folic acid decreases incidences of live births with NTDs. The Nishwander lab showed experimentally that mice, which were heterozygous for certain NTD genes, had higher incidences of NTDs when given folic acid; a surprising result that opposed the role folic acid plays in DECREASING NTDs.
What are the major anterior-posterior regions of the neural tube and their fates in the adult?
Forebrain -Telencephalon ->Cerebrum ->Olfactory Lobes ->Hippocampus -Diencephalon ->Thalamus ->Epithalamus ->Hypothalamus ->Optical Vesicle Midbrain -Mesencephalon ->Midbrain Hindbrain -Metencephalon ->Cerebellum ->Pons -Mylencephalon ->Medula
Explain how several specific morphogenetic behaviors contribute to gastrulation in the frog embryo and describe the experimental support for a particular molecular basis for one of these behaviors.
Gastrulation in the frog embryo occurs in four main steps: invagination, involution, convergent extension and epiboly. Invagination of the frog embryo occurs opposite the site of sperm. The cells regulating invagination, called bottle cells, undergo apical constriction where the apical-facing aspect of the cell tightens "like a purse string" and promotes an inward buckling of the membrane. Experimentally, researchers noted the ability of cells from the dorsal blastopore lip to induce invagination when transplanted into regions of the embryo that do not undergo invagination during normal development. Involution of the frog embryo occurs when involuting marginal cells migrate along the outer animal hemisphere cells, effectively "towing" a string of cells towards the interior of the embryo. These leading edge cells propel their migration by traveling upon extracellular matrix proteins. Convergent extension follows involution, occurring when cells at the blastopore lip merge together in a manner similar to cars merging during traffic. The process of numerous, small individual cell movements causes a dramatic overall lengthening of the forming archenteron. Molecularly, convergent extension operates through lamellipodia extensions regulated by PCP and Disheveled signaling. Lastly, epiboly occurs when animal cap cells and non-involuting cells spread out over the exterior of the embryo. This process is mediated by the flattening of cells such that cells transition from a cuboidal to a squamous shape.
What are germ layers and what are three examples of major derivatives to which each germ layer gives rise?
Germ layers - Groups of cells that form 3 distinct regions of the embryo & give rise to specialized cells and organs. Ectoderm - Epidermis and nervous system Mesoderm - Heart, kidney, gonads, bones and muscle. Endoderm - Lungs and digestive system.
What are homeotic mutants and what are some of the features of the genes underlying these mutant phenotypes?
Homeotic mutants exhibit a dramatic alteration in the identities of segments of the adult fly; what should normally develop as legs develops as an additional pair of wings. The genes underlying these mutations are homeotic selector genes. These genes are present on a single fly chromosome in two distinct regions, one that regulates the anterior segments of the fly and one that regulates the posterior segments. Interestingly, the arrangement of genes along the chromosome match the anterior-posterior arrangement of the segments they impact such that genes at the 3' end of the chromosome match the most anterior segments while genes at the 5' end match the more posterior segments. Homeotic genes are regulated by both pair-rule and segment polarity genes. Homeotic mutants result from mutation in homeobox-binding transcription factors.
Homeotic genes in Drosophila are known for causing dramatic changes in appendage identity when mutated. Compare the organization and phenotypes of homeobox genes in Drospholiaand vertebrates, using the vertebral column as an example.
In Drosophila: -Linear array of hox genes that is split between two chromosomes -Exhibit temporal and spatial co-linearity. -Spatial = Location -Temporal = Timing In Vertibrates: -Still temporal and spatial co-linear. -Multiple copies of genes regulate a single region; so, mutations in one gene will be compensated for by its copy genes → smaller phenotypic differences. -Multiple gene knockout → Dramatic phenotype change. -Knockout → Anterior shift; Overexpression → Posterior shift
How are the anterior-posterior axes specified in the Drosophila oocyte, prior to fertilization?
In drosophila, a hierarchy of gene expression that specifies both the anterior-posterior regions of the embryo and the identities of each embryo segment. The regulation of gap gene expression is most critical to the specification of anterior-posterior axes. Specifically, distribution of bicoid mRNA to the anterior region of the embryo and distribution of nanos mRNA to the posterior region of the embryo specifies the axes. Bicoid is transported from nurse cells to the oocyte along microtubules via kinesin ATPase transporters before being transported to the anterior pole via dynein ATPase transporters. Nanos is localized to the posterior of the oocyte through binding association to Oskar, which is transported to the posterior oocyte via kinesin ATPase transport along microtubules. Once bicoid and nanos mRNAs are localized to the anterior and posterior regions of the oocyte both mRNAs are transcribed to produce bicoid and nanos proteins. Bicoid protein diffuses gradually from the anterior region and represses the transcription of caudal and amplifies the transcription of hunchback. Likewise, nanos protein diffuses gradually from the posterior region and represses the transcription of hunchback. Caudal and hunchback ultimately act as transcription factors to specify the anterior and posterior cells of the embryo.
What is the acrosome reaction? How is it initiated and what does it accomplish? Do spermatozoa of all species have acrosomes?
Initiated by sperm cell making contact with species-specific carbohydrates in egg jelly. Reaction: Exocytosis of sperm acrosomal membrane, release of protease enzymes and exposure of binding molecules, polymerization of actin filaments. Process is mediated through changes in Ca2+ ion concentration and pH.
What are the cellular derivatives of the neural crest and to what tissues and organs do these cells contribute? What taxa development neural crest cells and why are they considered to have been so important in the evolution of these taxa?
Neural crest cells contribute to a variety of tissues and organ systems. Specifically cells from the neural crest will migrate to and form the peripheral nervous system (both neurons and glial cells), the adrenal medulla, melanocytes, facial cartilage, and the dentine of the teeth. From an evolutionary perspective, neural crest cells are only found in vertebrate organisms. Many biologists believe that the development of neural crest cells has facilitated the formation of more complex organ structures in vertebrates that are not observed in invertebrates. For example, neural crest cells form the specialized beaks found on birds. The development of specialized beaks can give the organism an evolutionary advantage.
Are all vertebrates equally good at regeneration? Speculate as to why this is or is not the case.
No, just look at differences between salamanders and humans. Why: • Differences in stem cell availability, maintenance of a stem cell state. • Different abilities to revert differentiated cells to a pluripotent state.
What is a normal table of development, and why are such tables important for developmental biology research?
Normal tables of development are species-specific visual diagrams of an organism's developmental morphology at set time points. Comparisons with wild-type and mutant embryos. Standardize protocols.
Why is it important to have a block to polyspermy? What are the differences between the fast and slow blocks to polyspermy and which sorts of organisms exhibit one mode vs. the other?
Polyspermy causes non-diploid organisms :( Fast block - Frogs and many invertebrates. Rapid change in membrane potential, caused by rapid influx of Na+ ions following fertilization. Slow block - Sea Urchins and most mammals. Initiate by marked increase in intercellular Ca2+, which promotes the fusion of cortical granule to the egg membrane. Release compounds into space between membrane and vitelline envelope. Cortical granule serine protease - Cleaves protein bonds between two membranes. Glucosaminoglycans - Absorb water. Peroxidase enzymes - Cross-links to strengthen. Hyaline - Protective coating for entire egg.
What embryological experiments suggest roles for tissue interactions within the ectoderm during the initial induction of the neural crest? What data support a role for Wnt signaling in this process?
Scientist initially identified the existence of tissue interactions during neural crest cell formation through a series of in vitro isolation experiments. These experiments demonstrated that several neural crest cell markers were not in isolated neural tube tissue - indicating that, by itself, neural tube tissue was unable to induce neural crest cell formation. However, when tissue from the neural tube was co-cultured with tissue from non-neural epiderm, markers for neural crest cell induction were expressed. These results indicated that the induction of neural crest cells was dependent upon neural tissue, epidermal tissue and the interactions that take place between the two tissues. Wnt signaling was also identified to play a role in neural crest cell induction. Researchers noted that, when isolated, neural tube tissue could not develop neural crest cells - a result similar to the above experiment. However, when neural tube tissue was bathed in wnt-rich media, neural crest cells formed and dispersed away from the neural tube. This result indicated a role for wnt signaling during neural crest cell induction. Further experiments validated this result. Specifically, insertion of beads that inhibited wnt activity prevented the formation of neural crest cells, and knocking down the expression of frizzled (a wnt receptor) via morpholino injection led to a loss of pigmentation (neural crest cell derived) in zebrafish.
Explain how an initial asymmetric localization of maternal determinants is translated into reiterated parasegments after fertilization.
The initial gradients of Bicoid and Nanos mRNAs translate into reiterated segments through a hierarchy of gene activity that occurs. The hierarchy of gene activity follows the pattern that Maternal Effect genes regulate Gap genes, Gap genes regulate Pair-Rule genes, and Pair-Rule genes regulate Segment Polarity genes. Maternal effect genes, such as Bicoid, act as transcription factors to specify the production of Gap genes throughout the oocyte. Experimental evidence suggest that the production of certain Gap genes is regulated by the concentration of maternal determinants such that some Gap genes are expressed when concentration is high, some are expressed when concentration is low, and some when concentration is between high and low. Additionally, once Gap genes are produced they interact with other Gap genes to further refine their region of expression. Gap genes, such as hunchback, regulate the expression of Pair-Rule genes. Specifically, Gap genes act as transcription factors by binding to enhancer and repressor regions of Pair-Rule genes to regulate their transcriptional expression. Importantly, each Pair-Rule gene is regulated by several Gap genes such that an individual Gap gene is not solely responsible for the expression of a single Pair-Rule gene. Pair-Rule genes go on to regulate the expression of Segment Polarity genes, which reinforce embryo segmentation and specify the cells of each individual segment.
What are the embryonic and adult derivatives of mesoderm?
The major adult and embryonic derivatives of the mesoderm are: 1) The intermediate mesoderm, which forms the kidneys and gonads. 2) The chordamesoderm, which forms the notochord. 3) The paraxial mesoderm, which forms the head and somites. 4) The lateral plate mesoderm, which will form the extra-embryonic, splanchnic and somatic tissue. The somites of the paraxial mesoderm will eventually form the scleratome, myotome, dermatome, syndatome and endothelial tissue.
How do the early events of embryonic cleavage differ between amphibian, avian, and mammalian embryos?
The patterns of embryonic cleavage differ dramatically between amphibian, avian and mammalian embryos. Cleavage of the amphibian embryo is holoblastic, meaning that the cleavage furrow divides the cells completely during each division. Initial division of the amphibian embryo is radially symmetrical; and while division of the yolk is not as dramatic as is seen in fish or avian embryos, the yolk is divided unequally between cells. Cleavage of the avian embryo occurs in the oviduct prior to encasing of the embryo in an eggshell. Division of the embryo is telolecithal, meaning that the cleavage furrow does not completely divide the cells during division. Initial cleavage is discoidal, with a large yolk cell forming the base of the embryo, and several, rapidly dividing animal pole cells comprise a disc that sits atop the yolk. At this time, some blastoderm cells absorb water to form the subgerminal cavity, which later helps to form the area pellucida where most of the actual embryo is formed. The mammalian embryo undergoes holoblastic rotational cleavage, where the second cleavage has one cell divide equitorally and the other cell divide meridionally. Several characteristics of mammalian cleavage are unique. Specifically, the timing of division is significantly slower than observed in other animals, and cells do not divide synchronously. Additionally, the mammalian embryo undergoes compaction, where cell adhesion proteins (cadherins) are expressed promoting the tight grouping of cells.
How are expression domains of segment polarity genes established and maintained?
The segment polarity genes are established through transcriptional regulation from Pair-Rule genes. Specifically, Even-skipped, Fushi tarazu, and Paired (all Primary Pair-Rule genes) promote the expression of Engrailed; while Odd-skipped, Runt and Sloppy-paired (all Secondary Pair-Rule genes) inhibit the expression of Engrailed. In turn, Engrailed expression marks the anterior compartment of each parasegment. At the same time, cells that do not receive Even-skipped or Fushi tarazu effectors express wingless, which specifies the posterior compartment of each parasegment. This expression pattern is maintained after initial expression by interactions between Engrailed and Wingless expressing cells. Engrailed acts upon frizzled receptors (Wnt). Wingless acts upon Patched receptors (Hedgehog).
What is the amphibian grey crescent and what is its relationship to the sperm entry point and future embryonic axes? What embryological experiments revealed a role for cortical rotation and "grey crescent cytoplasm" in axis specification?
Upon fertilization the amphibian embryo undergoes a dramatic morphological rotation towards the site of sperm entry that reveals an inner band of grey cytoplasm opposite the site of sperm entry. Ultimately, cortical rotation and grey crescent cytoplasm mark and specify the dorsal region of the amphibian embryo by regulating localizing GPB, Wnt11 and Disheveled proteins to the future dorsal region of the embryo. GPB will inhibit the degradation of B-Catenin, which will lead to specification of dorsal tissue. Experimentally, researchers noted that when cortical rotation was prevented with UV radiation the resulting embryo formed into a belly piece, which lacked clearly defined axes. However, if cortical rotation was inhibited and then artificially rotated 90-degrees, the resulting embryo developed normally. This experiment highlighted the importance of cortical rotation. As a follow-up experiment, Spemann and his colleges noted that when an initial embryo was divided into two separate embryos, only embryos that contained grey crescent tissue developed normally, while separated embryos that lacked grey crescent tissue developed into belly pieces. These experiments established that cytoplasmic determinants from the grey crescent region were essential for specification of the embryo axes.
What are some of the major mechanisms required for cellular communication during development? Give examples of how two of these pathways are associated with human disease or cancer.
Wnt, Hedgehog, TGFB, Receptor Tyrosine Kinase and Extra-cellular Matrix Holoprosencephaly - Mutation in Shh Ligand that interacts with the Patched receptor. FAP - Wnt pathway, mutation in APC complex causes permanent inactivation of B-Catenin degradation complex. Leads to build up of B-Catenin which affects DNA transcription.
Define the terms "genetic buffering", "developmental robustness", "canalization", "developmental plasticity" and "epigenetic landscape".
Genetic Buffering - Inherited mechanisms that keep a trait constant and decrease variance around the mean. Narrow Developmental Robustness - The result of genetic buffering, causing invariance of the phenotype in the face of genetic or environmental perturbation. Canalization - Genetic buffering that has evolved under natural selection to stabilize the phenotype and decrease its variability. Developmental Plasticity - Responsiveness of development to external, environmental influences or perturbations. Epigenetic Landscape - Metaphor of development that imagines an organisms development as a marble rolling down a perturbed surface.
Describe the mechanisms of gonidial and somatic cell development in Volvox. Be sure to include insights derived from mutant phenotypes as appropriate.
Three mutant phenotypes: Gonidialess (gls): Mutant doesn't develop gonidia Normal gene promotes asymmetric division Late Gonidia (lag): Normal gene prevents somatic features in gonidia Somatic Regenerator A (regA): Normal gene prevents gonidial features in somatic cells Mutant multiple green gonidia all over Dispersal and eversion of gonidia to form new Volvox
What is chemotaxis? How do spermatozoa "find" eggs using such a mechanism and what is the evidence for species-specificity of this process?
"Find the Egg!" Speract and Resact in Sea Urchin Mechanics: Resact --> RGC receptor on Sperm --> cGMP production from GTP --> Opening of Ca2+ channels to direct sperm.
Explain the mechanism underlying rapid cycling during initial cleavage divisions and what changes at the mid-blastula transition.
-Cell cycle is only synthesis and mitotic division. No growth phases. -MPF regulates initial divisions, and maintains synchronization. -Cyclin-B synthesized after S-phase, degrades in M-phase. -Cyclin-B activates Cyclin-Dependant Kinase, which phosphorylates essential mitotic proteins. @ Mid-Blastula transition, regulators of Cyclin-B synthesis are used up over time. So Growth phases added, non-syncronized divisions, and cell transcribes own DNA -> RNA.
Citing experimental evidence, describe how the embryonic melanocyte lineage is specified relative to other lineages and what are the consequences of this fate specification for subsequent morphogenesis?
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Citing experimental evidence, describe new data suggesting a distinct adult lineage of melanocytes.
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Describe the developmental anatomy and molecular mechanisms of somitogenesis, citing experimental data where possible.
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Describe the embryological evidence that implicates particular tissues in dorsal-ventral neural tube patterning and the molecular evidence that implicates Shh signaling in this process. Are there other molecular mechanisms that contribute to neural tube patterning?
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Explain how cell fate specification relates to transcription factors and the molecular mechanisms of transcriptional regulation.
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Explain the principle of genomic equivalence and its relevance to developmental biology. What are some exceptions to this principle that occur during normal development?
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Gastrulation of amniote embryos appears superficially very different from that of amphibian embryos. Nevertheless, one can recognize homologous tissue regions and molecular mechanisms during gastrulation of all these vertebrates. Explain how similar cellular and molecular events contribute to gastrulation in amphibian, avian, and mammalian embryos. In describing the relevant tissues, be sure to define the species-specific equivalents.
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Hilde Mangold and her graduate advisor Hans Spemann won the Nobel prize for their discovery of the amphibian organizer. What were the key experiments that demonstrated the existence of the organizer, what are it's properties, and what are the molecular bases for organizer activities?
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How do chromatin configurations and chromatin remodeling factors affect transcription during development?
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How does Shh signaling contribute to the specification of mesodermal derivatives? How has this been shown?
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Imagine that you have discovered a new transcription factor, ntf, in your favorite biomedical model organism and you find by RT-PCR that ntf mRNA is expressed differentially between stages and tissues. How could you further assess the temporal and spatial dynamics of ntf transcription during development? How might you identify the cis regulatory regions or specific cis-regulatory elements that control ntf expression? Suppose that you want to identify the suite of genes that ntf regulates during development. How would you proceed, assuming your model organism is a mouse? Would your approach differ if your model organism is a dung beetle?
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Imagine you are interested in how different species of birds develop differently shaped beaks (e.g., sandpipers vs. sparrows). Given that beak development requires a series of inductions between craniofacial mesenchyme and overlying ectoderm, propose one or more experiments to test whether species differences reside in changes in the mesenchyme, ectoderm, or both. As a follow-up, propose how you would identify the molecular bases for the observed differences in cell behaviors.
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The OMIM (Online Mendelian Inheritance in Man) database lists disorder #603643 Situs inversus totalis with cystic dysplasia of kidneys and pancreas: "Balci et al. (1999) described sib fetuses in which prenatal diagnosis was made of situs inversus totalis with cystic dysplasia of the kidneys and pancreas. One fetus was female and one male. They suffered from severe cystic dysplasia of the kidneys and pancreas with no abnormality of the liver and also showed situs inversus, bowing of the lower limbs and clavicles, severe intrauterine growth retardation, and oligohydramnios. The parents were first cousins and had no cysts of kidney, liver, or pancreas detected by ultrasonography." Based on your extensive knowledge of developmental genetics, propose a candidate gene or pathway that might be responsible for some or all of the phenotypes observed, and justify this choice of candidate. Using a developmental model organism, propose an experiment (with appropriate controls) to test your candidate gene or pathway, explaining how alternative experimental outcomes would support or refute your hypothesis.
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The zebrafish fin exemplifies a highly regenerative tissue, which includes bone, skin, nerves, pigment cells and other cells types. In principle, regenerative tissue could arise from latent stem cells (pluripotent or not), from previously differentiated cells that are activated to repopulate the regenerate (making more of themselves), or from previously differentiated cells that de-differentiate and subsequently contribute to multiple new cell types. Propose experimental strategies to distinguish among these hypotheses for regenerated bone, pigment, and glia.
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What are polar lobes, how do they promote normal development, and in what taxa are they often found? Propose an approach to identify candidate molecular mechanisms for polar lobe activity and and two experimental approaches by which you could test these candidates.
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What are three potential mechanisms for regulating gene activity and how might each of these be achieved?
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What data indicate that all three germ layers of amphibians are specified in the blastula and that the mesoderm is already regionalized by these early stages? What are the differences between dorsal and ventral mesodermal derivatives and what molecular and cellular interactions are required for their specification?
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What evidence suggests roles for Wnt and Fgf signals in regulating the regeneration of vertebrate tissues?
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What experimental data indicate roles for a novel ligand-receptor pair in salamander limb development and regeneration? Propose an experiment to test the relevance of these genes to mammalian regeneration.
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What is Fanconi anemia and what does it tell us about the importance of genomic stability during normal development?
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What is an induction? What is the difference between "permissive" and "instructive" inductions?
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What are the major derivatives of endoderm?
1. Digestive System a. Esophagus b. Stomach c. Small Intestine d. Large Intestine e. Colon f. Rectum 2. Others a. Liver b. Pancreas c. Gallbladder d. Lungs
What evidence indicates that Drosophila pole plasm specifies the pole cells and that these pole cells later become the adult germ cells?
1. Evidence that Pole Cells → Germ Cells a. A series of pole cell transplantation experiments generates flies that have a combination of indigenous and exogenous germ cells. b. Crossing flies produce offspring that inherit genetic information from the transplanted pole cells. 2. Evidence that Pole Plasm specifies Pole Cells a. Irradiate the embryo to disrupt any pole cell specifying information in the initial embryo. b. Introduce pole plasm (posterior cytoplasm) from a non-irradiated embryo and observe a restoration of normal pole cell development.
In what way(s) is the ZPA reminiscent of the amphibian blastopore lip? What is the ZPA and what experimental evidence indicates that Shh mediates the polarizing activity of these cells? What are the molecular and cellular consequences of Shh activity during limb patterning?
1. First identification of ZPA came from the observation that, when transplanted, the ZPA can induce "mirror image" limb bud development. a. Similar to the amphibian blastopore lip in its ability to specify the axes of limb development and the number of limb digits. 2. Shh a. Shh identified through in situ staining of the developing limb, where Shh was identified at the ZPA. b. Infecting limb with Shh expressing retrovirus led to mimic of ZPA transplant experiments. c. More recent experiments have identified that Shh is transferred to new cells via filopodia. 3. Shh effects a. Shh mutants don't have digit growth. b. Additional experiments that stained for time-dependent and location-dependent expression of Shh identified that the most posterior digits of the limb expressed high concentrations of Shh while anterior digits did not express Shh.
What data suggest the existence of cytoplasmic determinants for specifying the germ line and what is now known of their molecular nature and function?
1. Germ Plasm: Specialized form of cytoplasm that is found in pre-germ cells and is inherited by daughter cells during cell division. 2. Example (1) - Nematode embryos have visible pole plasm containing cells that specify into germ cells and protect chromosomes from fragmentation. If you disrupt the distribution of pole plasm in the early embryo through centrifugation, you disrupt the eventual specification of germ cells and typically somatic cells contain un-fragmented chromosomes. 3. Example (2) - Drosophila embryos have pre-germ cells, called pole cells, specified at the posterior end of the embryo. If you irradiate an embryo and introduce pole plasm from the posterior end of a non-irradiated embryo, you can restore normal pole cell development to the irradiated embryo. 4. Molecular: Germ plasm contains transcription factors and proteins that influence the development of germ cells.
In the neural crest, reiterated bmp signaling influenced several critical events. In the tetrapod limb, how do similarly critical events depend on reiterated fgf signaling, and how is such signaling established and maintained? Be sure to include descriptions of salient experiments.
1. Identification of role: a. Initial experiments using in situ staining identified fgf expression in future sites of limb growth. b. From there, researchers noted that an fgf-coated bead was able to act as the apical epidermal ridge (AER) when you removed the AER. c. Fgf alone was able to induce additional limb development. 2. Wnt expression in lateral plate mesoderm turns on fgf10 expression in lateral plate mesoderm. Fgf10 then turns on wnt3a expression in the AER, which activates fgf8 in the AER.
What are some of the cues used by primordial germ cells during their migration to the gonad?
1. In mouse development, primordial germ cells first arise from the gut endoderm at the posterior end of the embryo. PCGs migrate anteriorly to the gonadic ridges of the mesoderm. 2. Identifying molecular cues in Zebrafish: a. Germ cell specifying factors (vasa and nanos mRNA) are randomly distributed to pre-germ cells. b. Intermediate mesoderm signals the aggregation of germ cells @ 1st waypoint. Experiment: Block the development of Wt1 intermediate mesoderm → no migration of germ cells. c. Molecular Cues: i. Stromal Derived Factor 1 (sdf1) ii. Sdf1-receptor (cxcr4) iii. Experiment: In situ staining shows a sdf1 pathway that guides cxcr4-expressing PCGs to final destination. iv. Experiment: MO knockdown of sdf1 or cxcr4 disrupts migration of PCGs.
Many organs develop asymmetrically. Explain some of the cellular and molecular mechanisms underlying left-right axis specification that allow such asymmetries to develop.
1. Key genes found to regulate left-right asymmetry; they stain only on one side of the embryo. a. Cerebrus b. Nodal c. Pitx2 d. Overexpression of these genes (notably Pitx2) leads to a randomization of left-right specification. 2. Activity of motile cilia in Henson's Node / Kupffer's Vesicle establish the left-right asymmetry. a. Drive the dispersal of Shh b. Ca2+ changes that result from directional flow. c. Sensory cilia can also detect the directional flow of fluids to aid in establishing the detection of left-right asymmetry.
What experimental data demonstrate the critical role for thyroid hormone in amphibian metamorphosis? What are the molecular and cellular mechanisms underlying tissue level responses to thyroid hormone? Are these responses the same across tissues, and why do you think this might or might not be the case?
1. Researchers noted that injection of TH in amphibian tadpoles induced early metamorphosis and could cause metamorphosis in isolated tissues (specifically the tail and hind-legs). 2. At a molecular level: a. Thyroxin (T4) is processed into T3 by deiodinase. b. T3 interacts with the TR and RxR dimer to recruit an activator of gene transcription. 3. While thyroid hormone can affect many cells throughout the body, the response of each cell is going to vary depending upon slight differences in protein composition: a. Differences in Deiodinase, RxR and TR varieties. b. Differences in Transcription Factors and Histone Accessibility
Discuss the similarities and differences between amphibian and insect metamorphosis.
1. Similarities a. Transition from larval to adult stage. b. 20E is structurally highly similar to TH receptor. 2. Differences: Amphibian a. Some salamanders do not undergo metamorphosis; acquires sexual maturity in the larval stage. b. TH dependent. 3. Differences: Insect a. Several varieties of life-cycle metamorphosis. i. Ametabolous - Increase size, w/ molting. ii. Hemimetabolous - Smaller changes in body plan. iii. Holometabolous - Butterfly b. Complete reabsorption of larval body plan to form the adult organism. c. Imaginal discs d. Hormones: A pro-metamorphosis (ecdysone) and pro-juvenile (juvenile hormone) hormone.
What are three potential mechanisms that might contribute to the phenomenon of developmental robustness in response to genetic or environmental perturbation? Be sure to explain the empirical support for these mechanisms.
3 Potential Mechanisms of Developmental Robustness: 1) miRNA 2)Hsp90 3)Mathmatical Prediction of Robustness
Distinguish between the different types and phylogenetic distributions of vertebrate kidneys. What are some of the key signaling molecules needed for particular processes in kidney development?
3 Varieties of Kidney Types: 1. Pronephros a. Single glomerulus. b. Cells migrate caudally to form pronephric duct. c. Functional in Fish and Amphibian Larvae 2. Mesonephros a. Multiple nephrons and tubules. b. Final kidney of Fish and Amphibians 3. Metanephros a. 1000s of nephrons b. Final kidney of amniotes. Molecular Factors: Glial Derived Neurotropic Factor - Required to initiate branching of nephric duct. Wnt11 & Wnt9b - Involved in branching and more basic kidney formation
What is a forward genetic screen? How were forward genetic screens employed to isolate the genes involved in Drosophila early pattern formation?
A forward genetic screen is an experimental method of identifying developmental genes by identifying randomly generated mutant phenotypes and then determining the mechanism through which they generate the observed phenotype. Forward genetic screens were prominent in the Morgan lab. Experimentally, chemical mutagens introduced random mutations into adult flies, which were then breed to isolate flies with visible mutant phenotypes. After mutant flies were generated the gene could be named, and the mutation could be further characterized through genetic mapping and molecular cloning experiments.
What is a gene regulatory network (sensu Davidson and colleagues) how is one eluicidated? What is a GRN module, and what are three regulatory motifs that characterize different modules in the sea urchin micromere/skeletogenesis GRN?
A gene regulatory network is a pathway of gene activation and inhibition that specifies gene expression during development. A GRN module is a frequently observed motif that operates independently within the overall GRN to regulate a specific process of development. Within sea urchins we observe three GRN modules: (1) Double negative gates, modules where an initial gene inhibits the activity of a main inhibitory gene. By blocking the main inhibitors activity multiple genes that the main inhibitor normally represses become active. (2) State stabilization, modules where the expression of each gene stabilizes the expression of other genes that accomplish the same developmental role, through interlocking positive double feedback loops. (3) Differentiation gene module, where feed forward loops act to coordinate the expression of downstream genes in a manner that is robust to noise.
What is an adaptive polyphenism? Give an example and propose an approach that might be used to identify the developmental or genetic mechanisms underlying it.
Adaptive Polyphenism - Production of discreet morphs in respons to particular environmental conditions. Example: Tadpole development of carnivore morphology when in the presence of shrimp. Identify developmental or genetic mechanisms: - In Situ stain for transcription of candidate genes. - $$$ Microarray for key genes at this time frame of development. - Knockouts and morpholinos to verify effects of gene activity.
Explain how events beginning prior to fertilization specify the dorsal-ventral axes of theDrosophila embryo.
After the initial anterior-posterior segregation of bicoid and nanos mRNA, specification of the dorsal-ventral axes begins with the movement of a singular oocyte nucleus to the dorsal anterior region of the embryo. Once relocated, the nucleus produces gurken, a messaging proteins that specify nearby dorsal follicle cells. The distribution of gurken to dorsal follicle cells ultimately specifies the dorsal-ventral axes of the embryo through a cascade of gene activity. Specifically, gurken interacts with torpedo receptors to inhibit the production of pipe in dorsal follicle cells. In ventral follicle cells, pipe is synthesized because gurken is not present to activate torpedo receptors. Pipe then interacts with an unknown factor and nudel to activate Gd through cleavage. Gd activates Snake, which activates Easter, which activates Spatzle (each signal is activated through cleavage of a precursor to produce an active signaling molecule). Spatzle then binds to the toll receptor within the oocyte membrane leading to the production of Tube and Pelle. Tube and Pelle allow for the dissociation of Cactus from Dorsal, which allows Dorsal to enter the nucleus and alter gene expression to specify the ventral region of the oocyte.
