Kent T. Keyser, PhD Director, Vision Science Research Center 626 Worrell Building 975-7225

1. 2. Gastrulation, neurulation and formation of the neural tube, part 2; patterning VS 211 Kent T. Keyser, PhD Director, Vision Science Research Center 626 Worrell Building 975-7225 ktkeyser@uab.edu

2. Learning Objectives • Earliest development • Cleavage, gene transcription • Blastocyst, inner cell mass • Gastrulation • Henson’s node, primitive streak, primitive groove • Resulting layers, adult derivatives • Neurulation • Neural plate formation • Neural tube formation, anencephaly, spina bifida • Neural tube regionalization • Three vesicle stage, five vesicle stage, adult derivatives • DV axis formation • Signals initially from non-neural tissue • Opposing concentration gradients of BMP and SHH • AP axis formation • Hindbrain - rhombomeres defined by patterns of Hox gene expression • Midbrain – isthmus, Wnt, Otx2, Gbx2, FGF8 • Forebrain - prosomeres, zona limitans, Otx2, Emx, Pax6

3. How do the cells of the developing embryo “know” what part of the nervous system to form? The position of a cell determines its exposure to different inducing factors, and thus its fate. There is a combination of permissive induction and coordinated action by signaling molecules. These signals are turned on by the transcription of specific genes. Examples of inducing factors are the signals from the mesoderm that induce the neural plate, and the signals from the notochord that initiate formation of the neural tube and dorsal ventral patterning. Competence is the ability of a cell to respond to inductive factors. The key seems to be genes turning on and off in cells under control of inducing factors. Competence requires both signaling molecules (external to the neural cell) that can be either freely diffusible or bound to a surface, and the associated receptors (intrinsic to the neural cell) that respond to the inducing factors. Their activation regulates gene expression.

4. Dorsal Ventral organization Ventral: Floor plate cells are at the midline above the notochord (chordal mesoderm). Motor neurons are generated lateral to the floor plate and interneurons dorsal to motor neurons. Dorsal : Roof plate cells populate the PNS midline. Neural crest cells migrate from the dorsal midline of the neural tube. Cells lateral to the roof plate differentiate into dorsal sensory neurons.

5. Crucial signals come from non-neural tissue The notochord is the source of the ventral neural inducer. Notochord removal results in loss of floorplate, and lack of motor neurons. Notochord or floorplate graft onto dorsal neural tube induces ventralization, including ectopic motor neuron formation.

6. Ventral: Sonic Hedgehog (SHH) Signals from mesodermal notochord underlying the neural plate. Dorsal: BMPs Signals from non-neural cells of the epidermal ectoderm that flank the lateral margins of the neural plate. Floor and roof plate cells (specialized glial cells) also begin to express BMP4 and SHH Opposing concentration gradients

7. Ventralizing signal SHH is a morphogen: a type of inductive signal that can direct different cell fates at different concentrations. SHH is synthesized as an inactive precursor that is cleaved to produce two secreted proteins: 19 kDa N terminal N-SHH 25 kDa C terminal C-SHH N-SHH mediates signaling. Most is tethered to the surface of cells in the notochord and floor plate by the addition of a cholesterol moiety to the C terminal. Small amounts diffuse, resulting in a concentration gradient.

8. SHH acts as both local and long range signal. Initially expressed by the notochord and then by floor plate hinge cells. The 2-3x greater concentration at floor plate gives rise to motor neurons. SHH binds to patched (PTC), which disinhibits smoothened (Smo), allowing activation of gene transcription

9. Pax expression is also induced by SHH. The pattern shifts with neural tube development. Pax 3,7 expression is initially across the entire neural plate. Pax 6 is then induced ventrally while Pax 3,7 expression is restricted to the dorsal neural tube. Pax 3 is required for neural tube closure.

10. Dorsal signal There are multiple subtypes of BMP, each of which may be important for different cell types. Initially BMP4 is expressed by flanking ectoderm, and then by roof plate cells. BMPs induce differentiation of progenitor cells, which migrate ventrally.

11. Neural crest cells also migrate from the dorsal neural tube. For these cells the migration path determines cell fate. At the level of the spinal cord, cells that migrate ventrally aggregate to become autonomic and sensory ganglia. NC that migrate dorsally become pigment cells.

12. The take home point patterning is that the inductive signals are initially expressed by non-neural cells, and the opposing concentration gradients of these signals are key in inducing ventral and dorsal neurons in the spinal cord. The basic dorsal/ventral signaling systems are maintained throughout the full length of neuraxis, but shifted in the rostral-most neural tube. • However, because additional genes are expressed across the A-P axis, neurons induced by SHH at same the D-V level have different identities depending on location along A-P axis • Motor neurons • Dopaminergic neurons • Serotonergic neurons • Ventral forebrain interneurons

13. Anterior-Posterior Patterning As with DV patterning, A-P identity results from gradients of inducing molecules. BMP and Wnt signaling is blocked in the anterior. In the posterior, BMP, Wnt, and RA work via the Cdx family of proteins and activate specific Hox genes. FGF8 mRNA is produced at the posterior tip of the embryo and degrades towards the anterior. The resulting decrease in protein production generates the FGF8 gradient.

14. Hindbrain patterning The hindbrain arises from the rhombencephalon and is organized in segmental units by HOX genes. These segmental units are known as rhombomeres. Cranial nerve identity is dependent on rhombomeric location.

15. Genes responsible for patterning are evolutionarily conserved. HOMC in drosophila is the homologue of Hox in mouse and human. Hox genes are expressed in rhombomere-restricted domains within the hindbrain. The gene expression patterns provide a mechanism for specifying the unique identities of each rhombomere. hhmi.org/research/ investigators/moens.html

16. Segmental programming - NOT just one gene per segment. Hindbrain patterning is one of the best examples of cooperative expression. There are repeated patterns of gene expression (the same kind and order in fly and human). The initial expression is “sloppy”, but becomes more restricted during development, to the point that the expression boundaries represent rhombomere boundaries.

17. Midbrain (SC, tectum) The midbrain is patterned by signals from the isthmus region, a secondary organizing center at the junction of the mesencephalon and metencephalon. The isthmus controls the division between the midbrain and hindbrain. There is cooperative expression and gradients of Wnt1, Otx2, engrailed, and FGF8. For example, KO of Wnt results in loss of the midbrain and the rostral part of the hindbrain (future cerebellum), while loss of Otx2 results in anencephaly

18. Early transplantation of the isthmus induces ectopic midbrain/cerebellum development. If the mesencephalon is reversed at a later stage, the En gradient and cytoarchitecture, including retinal axon innervation is reversed.

19. FGF8 is also a critical signal. It acts through homeodomain proteins, and can act as an organizer. FGF8 may be induced when Otx2 and Gbx2 mutually inhibit one another at the isthmus. The Otx2/Gbx2 interaction maintains FGF8 expression patterns.

20. Forebrain Early in the development of the forebrain, it is divided into “compartments” called prosomeres. P1-p3 comprise the diencephalon. P4-p6 are longitudinally divided into the hypothalamus (ventral) and the telencephalon (dorsal). The telencephalon is further subdivided into cortex, palladum, and striatum.

21. Neuromeric structure of the brain with the hypothetical inductive events superpositioned on them. (A) The mesencephalon/metencephalon boundary is positive for both Fgf8 and Wnt1 gene expression. The p2/p3 border is thought to be the source of sonic hedgehog protein. (B) In situ hybridization of a 3-day chick embryo for Fgf8 expression. One of the major areas of expression can be seen at what will become the boundary between the midbrain and hindbrain. (A After Bally-Cuif and Wassef, 1995; B courtesy of E. Laufer, C-Y. Yeo, and C. Tabin.)

22. SHH and BMPs are expressed in combination, and coincide with Otx2 gene expression. The p2/p3 boundary corresponds to the zona limitans, a source of SHH. The zona limitans may be critical in patterning the forebrain region. Regional identity of cortex is patterned by gradients of Pax6 and Emx2.

23. This model shows the expression patterns of some of the main factors involved in forebrain and midbrain identity. The number of factors increases over time, and are subsequently restricted spatially.

24. Earliest development • Cleavage, gene transcription • Blastocyst, inner cell mass • Gastrulation • Henson’s node, primitive streak, primitive groove • Resulting layers, adult derivatives • Neurulation • Neural plate formation • Neural tube formation, anencephaly, spina bifida • Neural tube regionalization • Three vesicle stage, five vesicle stage, adult derivatives • DV axis formation • Signals initially from non-neural tissue • Opposing concentration gradients of BMP and SHH • AP axis formation • Hindbrain - rhombomeres defined by patterns of Hox gene expression • Midbrain – isthmus, Wnt, Otx2, Gbx2, FGF8 • Forebrain - prosomeres, zona limitans, Otx2, Emx, Pax6