9348 Colloquium Paper : Marigo and Tabin PTC Induction by Sonic Hedgehog Misexpression in the Neural Tube and Paraxial

Ventral cell fates in the central nervous system are induced by Sonic hedgehog, a homolog ofhedgehog, a secreted Drosophila protein. In the central nervous system, Sonic hedgehog has been identified as the signal inducing floor plate, motor neurons, and dopaminergic neurons. Sonic hedgehog is also involved in the induction of ventral cell type in the developing somites. ptc is a key gene in the Drosophila hedgehog signaling pathway where it is involved in transducing the hedgehog signal and is also a transcriptional target of the signal. PTC, a vertebrate homolog of this Drosophila gene, is genetically qownstream of Sonic hedgehog (Shh) in the limb bud. We analyze PTC expression during chicken neural and somite development and find it expressed in all regions of these tissues known to be responsive to Sonic hedgehog signal. As in the limb bud, ectopic expression of Sonic hedgehog leads to ectopic induction of PTC in the neural tube and paraxial mesoderm. This conservation of regulation allows us to use PTC as a marker for Sonic hedgehog response. The pattern of PTC expression suggests that Sonic hedgehog may play an inductive role in more dorsal regions of the neural tube than have been previously demonstrated. Examination of the pattern of PTC expression also suggests that PTC may act in a negative feedback loop to attenuate hedgehog signaling. During early vertebrate development, signaling centers from the midline direct the dorsal-ventral fate of cells in neural tube and somites. Recent studies (1) have identified Sonic hedgehog as a signaling molecule that is responsible for establishing aspects of dorsal-ventral polarity in the vertebrate central nervous system and somites. In the developing neural tube, contact-mediated signaling from the underlying notochord is responsible for inducing the formation of the floor plate along the ventral midline (2, 3). In addition the floor plate and the notochord induce the formation of motor neurons lateral and dorsal to the floor plate in a contact independent manner (2, 4). These two signaling centers, the floor plate and notochord, also produce signals responsible for inducing ventral fates in the somites (5, 6). Sonic hedgehog is expressed in both the notochord and the floor plate (7-11). Ectopic expression of Sonic hedgehog in vivo as well as application of Sonic hedgehog protein to explants in vitro result in induction of both floor plate and motor neuron marker genes (7, 8, 11-14). This raises an apparent paradox: a single signal is capable of generating both contact-dependent and -independent responses. A possible resolution is that different thresholds of the protein can trigger induction of different cell fates. In fact, low concentrations of Sonic hedgehog induce motor neurons but not floor plate markers, whereas high concentrations induce floor plate cells (12, 13). Hence the experimental demonstration of contact dependence likely reflects the requirement for extremely high concentrations of Sonic hedgehog present on the surface of cells producing it rather than a need for direct contact per se. In addition to acting at a distance in motor neuron induction, several lines of evidence have implicated Sonic hedgehog as the long-range factor from the floor plate and the notochord mediating ventral sclerotomal cell fate in the somites (15, 16). Finally, Sonic hedgehog has also been identified as the signal inducing dopaminergic neurons, a ventral cell type in the midbrain (17, 18). How Sonic hedgehog acts to regulate embryonic patterning remains unknown and needs not be direct. In fact, the assays described above have not definitively established whether these inductive activities require secondary signals. If Sonic hedgehog is directly responsible for induction of motor neurons and sclerotome, it would need to diffuse over many cell diameters in vivo. However, no evidence for diffusion of the protein was observed by immunohistochemistry, perhaps due to limited sensitivity of the technique (12, 19). In Drosophila, hedgehog (hh), the fly homolog of Sonic hedgehog, has been implicated in short-range (e.g., in the wing imaginal discs and in establishing early segment borders) and long-range inductions (e.g., cellular patterning of the dorsal epidermis) (20-24). Supporting the idea that hh is a diffusible factor, in the fly the protein can be detected a few cell diameters beyond cells transcribing the hh gene (24-26). The regulation of gene expression by hh is mediated by the gene patched (ptc) (27, 28). ptc constitutively represses downstream targets, including its own transcription (29). The hh signal relieves ptc repression, thus inducing transcription of target genes (27, 29). Low levels of ptc transcription are therefore indicative of cells capable of responding to hh, since without ptc hh has no effect on transcription. High levels of ptc transcription, on the other hand, serve as a marker for cells that are directly responding to hh, since ptc is strongly derepressed in all cells receiving the hh signal. We previously reported the cloning of a chicken homolog (PTC) of the Drosophila patched gene and demonstrated it to be in the Sonic hedgehog signaling pathway during vertebrate limb development (30). Here we report the expression pattern of PTC during chicken neural and somitic development. PTC is highly expressed in chicken tissues responsive to Sonic hedgehog signal, including the ventral neural tube and the ventral somites. We also demonstrate that PTC is genetically downstream of Sonic hedgehog in these tissues being induced in the neural tissue and paraxial mesoderm by ectopic expres*To whom reprint requests should be addressed. e-mail: Tabin@ rascal.med.harvard.edu. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.