Developmental Biology Bio349 Vokes Exam 2

What is the Prosencephalon?

The Forebrain, forming the telencephalon (olfactory lobes, hippocampus, and cerebrum) and diencephalon (optic vesicle, epithalamus, thalamus, and hypothalamus)

neural crest migration

The Sox9 transcription factor is expressed in neural crest cells just prior to their migration.

What is secondary neuralation?

A solid cylinder of cells cavitates to form hollow neural tube.

Combinatorial gene expression defines neuronal subtypes

An overlapping expression of Nkx6.1 and Pax6 produces motor neurons.

Crest migrate through the anterior sclerotome

Anterior: extensive migration Posterior: no migration

Cranial neural crest condense in the pharyngeal arches

Arch 1 creates the oral ecotderm. Arches 2, 3, and 4 are the pharyngeal endoderm. serially reiterated, transient structures bounded by facial epithelia.

Regulatory circuit

As time progresses, Shh increases in concentration (ventrally). As a result, Gli expression also increases (ventrally). The Gli will have to be expressed at a level.

Transcriptional Interpretation of Shh

At T0 low levels of Shh are translated into low levels of Gli, which are not sufficient to induce Olig2 or Nkk2.2. Increasing Shh induces a gradient of Gli that peaks and then retracts (negative feedback by Ptch1). So different levels of response are driven by a regulatory circuit instead of absolute concentrations of Shh.

Commissural axons in the spinal cord

Axons must project to the ventral midline and then cross once (and only once). Netrin-2 gradient and Shh gradient projects downward to netrin-1 gradients at the floor plate.

Generating a transgenic mouse with green fluorescent motor neurons

Because the EBs contain many other ventral cell types - not just MNs, generate a transgenic mouse with GFP labeled MN and derive an ES cell line. When these ES cells differentiate they will be green.

Signaling Molecules in the Ectoderm Correlate with Dorsal Ventral Boundaries

Border of Shh and Fgf8 becomes the distal tip of the upper beak

Neural crest cells are migratory

Born in the dorsal neural tube at the boundary of neural and non-neural ectoderm, the neural plate border. Undergo an epithelial to mesenchymal transition (EMT) and migrate into the periphery. Follow highly stereotyped migratory pathways.

What are the two models for the study of complex eyes?

Chordates and anthropods

Crest initiating migration express RhoB

Crest leaving

What kind of experiment could tell what these cells become?

Dil Lineage Tracing

How does the formation of the neural tube occur?

Dorsolateral hinge points become wedge-shaped, bringing the neural folds into apposition. Non-neural ectoderm moves towards midline.

Treating EBs with Shh and RA gives

ESCs ventral neuronal identities. Shh > Pax6, Nkx6.1 > Olig2 > Lhx3, Isl1 > HB9 note: Irx3 -] Olig2 and Nkx2.2 -] Olig2

Trunk crest only migrate through the anterior half of somites

Embryo is stained for HNK1 (same neural crest marker is a few slides previously). Crest adjacent to the posterior sclerotome migrate to an anterior segment. Motor axons also extend through the anterior somite generating the segmental spinal nerves. Polarity within the somites directs neural crest & motor axons. Somite reversals cause reversal of crest migration & axon projections.

What keeps the NCCs migration through the anterior half of somites?

Ephrin-b1 is expressed in posterior half-somite. Crest cells express EphB3 (stain for HNK3).

How do motor neurons know to project to the dorsal or ventral limb bud?

Ephrins are concentrated in the ventral limb (Ephrin-A2 and Ephrin-A5)

What BMP levels determine the major derivatives of the ectoderm?

Epidermis: high BMP levels Neural crest: moderate BMP levels Neural plate/neural tube: low BMP levels, Sox TFs expressed

Dorsal Ventral Polarity in the Spinal Cord: Sonic Hedgehog

Extracellular Shh binds to the receptor Patched. Patched, unable to block Smoothened because of Shh, will initiate a cascade that will activate the TF Gli.

Cranial neural crest cell migration

First stream originates from r2, r1 and midbrain, second stream from r4, third stream from r6 - r8. Crest from r3 & r5 will join an adjacent stream or undergo apoptosis, generating crest-free zones.

What Happens When Extra FGF8-Shh Boundaries are Generated?

Invert boundary region

How do NCC Switch from Pathway 1 to 2?

FoxD3 (essential for generating NCC) stays on in Pathway #1. FoxD3 represses a gene called Mitf, which is a master-regulator of Melanocyte cell fate. So FoxD3 somehow has to be switched off after neural crest specification to drive melanocyte cell fate. The mechanism underlying this switch is not yet known. Mitf1 is also essential for melanocyte stem cells. As we age, these cells are lost and hair turns gray.

What genes have a detrimental effect of long-term control folic acid diet versus high FA diet on % penetrance on NTD? No effect? Beneficial effect?

Frem2, Grhl2, and Shroom3 - no effect Sp2H and Zic2 - beneficial effect L3P and Grhl2het - DETRIMENTAL EFFECT

How is the vertebrate eye formed?

From an evagination event from the forebrain

How does the closure of the neural tube in the hindbrain occur?

Fusion in mouse hindbrain moves through a zipper-like process.

Characterizing gene expression

Genes not expressed in the placode: pax6, pptix, pax2, and sine oculis. Genes expressed in the placode: notch, hes, prospero, eyes absent.

How does the closure of the neural tube in the midbrain occur?

Involves buttoning rather than zippering, from the extension of cellular bridges from non neural ectoderm that lies above the neural tube in this region

What is the Rhomencephalon?

Hindbrain, forming the metencephalon (cerebellum and pons) and the myelencephalon (medulla)

Stripe assay

In culture, migrating neural crest cell avoid ephrin-b1. Mold allows stripes of proteins to be made. Ephrin-b1 is clustered and bound to the surface of the culture dish, making it active

Eph signaling is important in segmentation of crest cells

Inject lipophilic dye (DiI) into the neural tube, crest are labeled before they initiate migration. Culture an explant with soluble ephrin-b1 (blocking Eph signaling).

EphA4 expressing axons project dorsally

Ligands in the ventral limb (Ephrin-A2 and Ephrin-A5)

Motor axon projections at limb levels

MMC axons project to axial muscle. LMC only present at limb levels. LMCL projects to dorsal musculature. LMCm projects to ventral musculature.

What is the Mesencephalon?

Midbrain

Neural tube closure in mammals (3 sites)

Most of the neural tube closes by a "zipper-like" process The two neural folds fuse at three points. Failure to close the neural tube results in anencephaly and spina bifida.

Regenerative Medicine: Generating Motor Neurons from Stem Cells

Mouse ESCs > Embryoid bodieds "EBs" > ... ... + RA > Dorsal Neural Progenitors ... + Shh + RA > Ventral Neural Progenitors

What is an example of a mutation preventing folic acid availability in cells causing neural tube defects in mice?

Mthfd1l is an enzyme expressed in the dorsal neural tube that makes folic acid available to the cell. Mthfd1l mutants cannot process folic acid.

neural crest formation

Neural plate border is the region where neural and non-neural ectoderm meet. It is not committed: cells can become neural, ectodermal or neural crest. high Wnt and BMP > epidermis low Wnt > Bmp > placode cells high Wnt > BMP > neural crest high Wnt > Neural cells

What is primary neuralation?

Neural plate folds to generate hollow neural tube.

What is DiI lineage tracing?

Non-genetic lineage tracing techinques that can inform our understanding of terminal cell fates

Describe the differences in eye morphology

Organisms with eyes are bilateria. There are the deuterostomia, the ecdysozoa, and the lophotrochozoa. Drosophila have a compound eye while humans have a single chambered eye.

What genes each mark the forebrain, midbrain, hindbrain, and caudal neural identity?

Otx2, En1/2, Krox20, and hoxb8 respectively

Pdgf is a cranial neural crest chemoattractant

PDGF is a secreted protein

Trunk NC has 2 Major Migratory Paths

Path 1 and Path 2

What is conserved throughout eye development?

Pax6 Signaling Cascade is Conserved in eye development. Homologous genes descend from a common ancestor and can maintain their function.

Is Pax6 necessary for eye development? Is it sufficient?

Pax6 pathway members are necessary and sufficient for eye development in Drosophila

Pharyngeal arches generate specific skeletal elements

Pharyngeal arch 1: Facial bones, Malleus, Incus, Meckel's cartilage Pharyngeal arch 2: Stapes, Styloid process Pharyngeal arch 3: Hyoid cartilage Pharyngeal arch 4: Laryngeal cartilage

Turducken Experiments: Intrinsic Patterning within Neural Crest

Quck Duail

Where is RA expressed?

RA is expressed in caudal paraxial mesoderm (marked by raldh2)

Where is RA released from and what does it do?

RA is released from the paraxial mesoderm and induces caudal neurons, but also requires something else from the mesoderm.

What causes (some) dorsal axons to migrate?

Repulsion

Slits & netrins in vert commissural axons

Robo1 is highly expressed and blocks netrin-mediated chemoattraction through interacting with DCC's cytoplasmic domain. Therefore, net chemorepulsion through Slit binding Robo1.

Overcoming repulsion

Robo3 becomes highly expressed. It cannot block DCC, but does block Robo1. Therefore, net chemoattraction through Netrin binding DCC.

Repulsed again

Robo3 is down regulated. Robo1 blocks DCC. Slit has chemorepellant effect. The combinatorial action of multiple guidance mechanisms drives proper pathfinding.

The spinal cord is segmented

Sensory and motor neuron axons form in segments

Vertebrate facial prominences

Seven structures give rise to the vertebrate face: single median nasal prominence, and 3 pair of elements derived from the first pharyngeal arch

Sonic Hedgehog

Shh, acts as a ventral morphogen, secreted from the notochord (ventral to the NT), which extends along the neural tube.

Repulsion: Robo/Slit in Drosophila commissural axons

Slit (ligand) in the midline Robo (receptor) on axons In Slit muts, axons never leave the midline.

Sox9 and RhoB Induce Neural Crest Differentiation and EMT

Sox9 activates NC marker HNK but cells are still in the neural tube Sox9 + RhoB activate NC marker HNK and cells exit the neural tube RhoB alone - no NC. Cells seem to be migrating - but apoptose

How does the cephalopod eye form?

The Cephalopod eye is formed from the internalization of a placode that ultimately becomes the retina. This is an example of convergent evolution.

Describe the multiple sites of neural tube closure?

The initial fusion of the two neural folds happens in the cephalic region. The neural tube zippers closed towards the anterior neuropore and the posterior neuropore. Failure to close the neural tube results in anencephaly and spina bifida.

How does the expansion of the early embryonic brain occur?

The occlusion of the neural tube allows pressure to swell the lumen

Path 2 cells take a dorsolateral route between the epidermis and dermis

The second path generates melanocytes. They migrate between the dermamyotome (generating musculature and the dermis) and the surface ectoderm.

Genetic Fate Mapping: Applied

Tissues of the mouse skull vault include regions that are neural crest as well as mesodermally derived

Neural crest cells are pluripotent

Vertebrate specific cell type that makes: Peripheral nervous system neurons and glia Melanocytes Endocrine organs such as the adrenal medulla Outflow tract of the heart Bones/cartilages of the skull Connective tissue in the face Cornea

What is the synthetic pathway of Retinoic Acid (RA)? How does it act, once made?

Vitamin A (Retinol) via ADH RoDH> Retinal via RALDH > Retinoic Acid (RA) via Cyp26 > 4-oxo RA (catabolism) or RA > RAR RXR dimeric TF

Why is folic acid essential to neural tube closure?

Vitamin B9 (folic acid) supplements during early pregnancy decrease the prevalence of neural tube closure defects by as much as 70%. Folate receptor is expressed in dorsal neural tube.

Path 1 cells travel ventrally through the anterior sclerotome

Will generate sensory and sympathetic neurons. These are the first trunk crest to migrate. Migrate through the sclerotome, which generates the vertebral skeleton.

Lim1

activates EphA4

Neural crest cell trajectories

circle is the cells original location, line with the arrow shows the migratory path. Soluble ephrin-b1 not only disrupts segmentation but also directionality (the cells are 'lost').

Homologous genes

descend from a common ancestor and can maintain their function

Lateral motor column (LMC)

only present at limb levels

Medical motor column (MMC)

projects to axial muscle contains Isl1, Isl2, and Lhx3 extends to the Dermamyotome (FGFs)

LMCL

projects to dorsal musculature (Sema-3F) contains Isl2, Lim1

LMCM

projects to ventral musculature (Ephrin A5) contains Isl1, Isl2

Isl1

represses EphA4

How doe the formation of neural folds (neural plate) occur?

thickening of the midline ectoderm into a columnar epithelium. Neural folds bend at the medial hinge point, containing wedge-shaped cells, generating the neural groove. Microtubules and actin drive elongation and apical constriction, respectively.

Axon guidance

• Pathway selection: axon chooses route through the environment to its target • Target selection: axon recognizes target & initiates synaptogenesis • Address selection (refinement): synapses are strengthened and lost