Distinct regulators ofShhtranscription in the floor plate and notochord indicate separate origins for these tissues in the mouse node

Abstract

The establishment of the floor plate at the ventral midline of the CNS is dependent on an inductive signaling process mediated by the secreted protein Sonic hedgehog (Shh). To understand molecularly how floor plate induction proceeds we identified a Shh-responsive regulatory element that directs transgene reporter expression to the ventral midline of the CNS and notochord in a Shh-like manner and characterized critical cis-acting sequences regulating this element. Cross-species comparisons narrowed the activity of the Shh floor plate enhancer to an 88-bp sequence within intron 2 of Shh that included highly conserved binding sites matching the consensus for homeodomain, Tbx and Foxa transcription factors. Mutational analysis revealed that the homeodomain and Foxa binding sites are each required for activation of the Shh floor plate enhancer, whereas the Tbx site was required for repression in regions of the CNS where Shhis not normally expressed. We further show that Shh enhancer activity was detected in the mouse node from where the floor plate and notochord precursors derive. Shh reporter expression was restricted to the ventral (mesodermal)layer of the node in a pattern similar to endogenous Shh. X-gal-positive cells emerging from the node were only detected in the notochord lineage, suggesting that the floor plate and notochord arise from distinct precursors in the mouse node.

Directed differentiation of neural cells to hypothalamic dopaminergic neurons

“…As our data shows that Shh expression in hypothalamic explants precedes that of Nkx2.1, these results together support a cell-autonomous role for Nkx2.1 in the maintenance of Shh expression. Further support for a role for HD factors in regulating Shh expression derives through a recent dissection of the Shh enhancer element, SFPE2, which reveals that a HD site is required for Shh transcription in the posterior spinal cord (Jeong and Epstein, 2003). Thus, our studies suggest that Bmp signalling may play a dual role in the hypothalamus, inducing regional identity and maintaining Shh expression.…”

Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh

“…Otic explants were cultured according to the protocol described in Jeong and Epstein (2003) with slight modifications. Embryos generated from matings between Topgal males and CD-1 females were harvested in ice-cold L15 medium (GIBCO-BRL) at 9.25 dpc.…”

The zebrafish tailbud contains two independent populations of midline progenitor cells that maintain long-term germ layer plasticity and differentiate based on local signaling cues

“…During embryo elongation, MPCs continuously join growing tissues of the midline. In chicken embryos it is clear that different germ layer derivatives form from a single pool of MPCs, whereas in mouse embryos the notochord and floor plate progenitors are spatially distinct (Catala et al, 1996;Jeong and Epstein, 2003;Teillet et al, 1998). Whether MPC fate decisions are made solely during gastrulation or continuously during embryo elongation after gastrulation is unknown.…”

“…The timing and location of floor plate induction vary across vertebrate species. Cell lineage analyses in chick embryos show that notochord and medial floor plate arise from a single pool of progenitors, whereas in mouse embryos sonic hedgehog-expressing notochord-fated cells are separated from future floor plate cells in the node after gastrulation (Catala et al, 1996;Jeong and Epstein, 2003;Schoenwolf and Sheard, 1990;Selleck and Stern, 1991). In the mouse, Hedgehog signaling from the notochord induces both the medial and lateral floor plate, whereas in zebrafish medial floor plate formation is independent of Hedgehog signaling (Chen et al, 2001;Chiang et al, 1996;Ding et al, 1998;Matise et al, 1998).…”