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Classical descriptions of the hypothalamus divide it into three rostro-caudal domains but little is known about their embryonic origins. To investigate this, we performed targeted fate-mapping, molecular characterisation and cell cycle analyses in the embryonic chick. Presumptive hypothalamic cells derive from the rostral diencephalic ventral midline, lie above the prechordal mesendoderm and express Fgf10. Fgf10+ progenitors undergo anisotropic growth: those displaced rostrally differentiate into anterior cells, then those displaced caudally differentiate into mammillary cells. A stable population of Fgf10+ progenitors is retained within the tuberal domain; a subset of these gives rise to the tuberal infundibulum – the precursor of the posterior pituitary. Pharmacological approaches reveal that Shh signalling promotes the growth and differentiation of anterior progenitors, and also orchestrates the development of the infundibulum and Rathke's pouch – the precursor of the anterior pituitary. Together, our studies identify a hypothalamic progenitor population defined by Fgf10 and highlight a role for Shh signalling in the integrated development of the hypothalamus and pituitary., Summary: Anterior and then mammillary hypothalamic cells differentiate from Fgf10+ progenitors that are retained as central tuberal hypothalamic cells. Shh is required for anterior regionalisation and also for integrated hypothalamic/pituitary development.

In the developing brain, growth and differentiation are intimately linked. Here, we show that in the zebrafish embryo, the homeodomain transcription factor Rx3 coordinates these processes to build the tuberal/anterior hypothalamus. Analysis of rx3 chk mutant/rx3 morphant fish and EdU pulse-chase studies reveal that rx3 is required to select tuberal/anterior hypothalamic progenitors and to orchestrate their anisotropic growth. In the absence of Rx3 function, progenitors accumulate in the third ventricular wall, die or are inappropriately specified, the shh+ anterior recess does not form, and its resident pomc+, ff1b+ and otpb+ Th1+ cells fail to differentiate. Manipulation of Shh signalling shows that Shh coordinates progenitor cell selection and behaviour by acting as an on-off switch for rx3. Together, our studies show that Shh and Rx3 govern formation of a distinct progenitor domain that elaborates patterning through its anisotropic growth and differentiation., Summary: During zebrafish tuberal/anterior hypothalamus development, Shh and Rx3 govern the formation of a distinct progenitor domain that elaborates pattern through its anisotropic growth and differentiation.

Hypothalamic neurons play a key role in homeostasis, yet little is known about their differentiation. Here, we demonstrate that Shh and Bmp7 from the adjacent prechordal mesoderm govern hypothalamic neural fate, their sequential action controlling hypothalamic dopaminergic neuron generation in a Six3-dependent manner. Our data suggest a temporal distinction in the requirement for the two signals. Shh acts early to specify dopaminergic neurotransmitter phenotype. Subsequently, Bmp7 acts on cells that are ventralised by Shh, establishing aspects of hypothalamic regional identity in late-differentiating/postmitotic cells. The concerted actions of Shh and Bmp7 can direct mouse embryonic stem cell-derived neural progenitor cells to a hypothalamic dopaminergic fate ex vivo.

To begin to reconcile models of floor plate formation in the vertebrate neural tube, we have performed experiments aimed at understanding the development of the early floor plate in the chick embryo. Using real-time analyses of cell behaviour, we provide evidence that the principal contributor to the early neural midline, the future anterior floor plate, exists as a separate population of floor plate precursor cells in the epiblast of the gastrula stage embryo, and does not share a lineage with axial mesoderm. Analysis of the tissue interactions associated with differentiation of these cells to a floor plate fate reveals a role for the nascent prechordal mesoderm, indicating that more than one inductive event is associated with floor plate formation along the length of the neuraxis. We show that Nr1, a chick nodal homologue, is expressed in the nascent prechordal mesoderm and we provide evidence that Nodal signalling can cooperate with Shh to induce the epiblast precursors to a floor-plate fate. These results indicate that a shared lineage with axial mesoderm cells is not a pre-requisite for floor plate differentiation and suggest parallels between the development of the floor plate in amniote and anamniote embryos.

Two populations of axial mesoderm cells can be recognised in the chick embryo, posterior notochord and anterior prechordal mesoderm. We have examined the cellular and molecular events that govern the specification of prechordal mesoderm. We report that notochord and prechordal mesoderm cells are intermingled and share expression of many markers as they initially extend out of Hensen’s node. In vitro culture studies, together with in vivo grafting experiments, reveal that early extending axial mesoderm cells are labile and that their character may be defined subsequently through signals that derive from anterior endodermal tissues. Anterior endoderm elicits aspects of prechordal mesoderm identity in extending axial mesoderm by repressing notochord characteristics, briefly maintaining gsc expression and inducing BMP7 expression. Together these experiments suggest that, in vivo, signalling by anterior endoderm may determine the extent of prechordal mesoderm. The transforming growth factor β (TGFββ superfamily members BMP2, BMP4, BMP7 and activin, all of which are transiently expressed in anterior endoderm mimic distinct aspects of its patterning actions. Together our results suggest that anterior endoderm-derived TGFβs may specify prechordal mesoderm character in chick axial mesoderm.

Ventral midline cells in the neural tube have distinct properties at different rostrocaudal levels, apparently in response to differential signalling by axial mesoderm. Floor plate cells are induced by sonic hedgehog (SHH) secreted from the notochord whereas ventral midline cells of the rostral diencephalon (RDVM cells) appear to be induced by the dual actions of SHH and bone morphogenetic protein 7 (BMP7) from prechordal mesoderm. We have examined the cellular and molecular events that govern the program of differentiation of RDVM cells under the influence of the axial mesoderm. By fate mapping, we show that prospective RDVM cells migrate rostrally within the neural plate, passing over rostral notochord before establishing register with prechordal mesoderm at stage 7. Despite the co-expression of SHH and BMP7 by rostral notochord, prospective RDVM cells appear to be specified initially as caudal ventral midline neurectodermal cells and to acquire RDVM properties only at stage 7. We provide evidence that the signalling properties of axial mesoderm over this period are regulated by the BMP antagonist, chordin. Chordin is expressed throughout the axial mesoderm as it extends, but is downregulated in prechordal mesoderm coincident with the onset of RDVM cell differentiation. Addition of chordin to conjugate explant cultures of prechordal mesoderm and neural tissue prevents the rostralization of ventral midline cells by prechordal mesoderm. Chordin may thus act to refine the patterning of the ventral midline along the rostrocaudal axis.

Developing axons are guided to their targets by molecular cues in their local environment. Some cues are shortrange, deriving from cells along axonal pathways. There is also increasing evidence for longer-range guidance cues, in the form of gradients of diffusible chemoattractant molecules, which originate from restricted populations of target cells. The guidance of developing commissural axons within the spinal cord depends on one of their intermediate cellular targets, the floor plate. We have shown previously that floor plate cells secrete a diffusible factor(s) that can alter the direction of commissural axon growth in vitro. Here we show that the factor is an effective chemoattractant for commissural axons. It can diffuse considerable distances through a collagen gel matrix and through dorsal and ventral neural epithelium in vitro to reorient the growth of virtually all commissural axons. The orientation of axons occurs in the absence of detectable effects on the survival of commissural neurons or on the rate of commissural axon extension. The regionally restricted expression of the factor suggests that it is present in the embryonic spinal cord in a gradient with its high point at the floor plate. These observations support the idea that the guidance of commissural axons to the ventral midline of the spinal cord results in part from the secretion of a chemoattractant by the floor plate.