Shh signaling is not required for initiating hair follicle development. Shh signaling is essential, however, for controlling ingrowth and morphogenesis of the hair follicle.

The mammalian lung, like many other organs, develops by branching morphogenesis of an epithelium [1]. Development initiates with evagination of two ventral buds of foregut endoderm into the underlying splanchnic mesoderm. As the buds extend, they send out lateral branches at precise, invariant positions, establishing the primary airways and the lobes of each lung. Dichotomous branching leads to further extension of the airways. Grafting studies have demonstrated the importance of bronchial mesenchyme in inducing epithelial branching, but the significance of epithelial signaling has largely been unstudied. The morphogen Sonic hedgehog (Shh) is widely expressed in the foregut endoderm and is specifically upregulated in the distal epithelium of the lung where branching is occurring [2]. Ectopic expression of Shh disrupts branching and increases proliferation, suggesting that local Shh signaling regulates lung development [2]. We report here that Shh is essential for development of the respiratory system. In Shh null mutants, we found that the trachea and esophagus do not separate properly and the lungs form a rudimentary sac due to failure of branching and growth after formation of the primary lung buds. Interestingly, normal proximo-distal differentiation of the airway epithelium occurred, indicating that Shh is not needed for differentiation events. In addition, the transcription of several mesenchymally expressed downstream targets of Shh is abolished. These results highlight the importance of epithelially derived Shh in regulating branching morphogenesis of the lung.

We have generated a transgenic line that expresses the Cre gene product under the regulation of a 12.5 kb upstream regulatory sequence from the Sox2 gene. Using a R26R reporter line, we show that this transgenic line induces recombination in all epiblast cells by embryonic day (E) 6.5 but little or no activity in other extraembryonic cell types at this time. When crossed to a conditional allele of the Sonic hedgehog gene (Shhc), all Sox2Cre;Shhn/Shhc embryos displayed a phenotype indistinguishable from that of the Shh null mutant. Sox2Cre functioned more efficiently in epiblast-mediated recombination than the Mox2Cre (MORE) transgenic line, which has also been shown to drive Cre-mediated recombination exclusively in the embryonic component of the early mouse embryo. Although most MORE; shhh/shhc embryos have a shh hull phenotype, 33% displayed a milder skeletal phenotype, most likely result of incomplete recombination at egg cylinder stages. In agreement with these findings, Sox2Cre was active earlier and Sox2Cre-mediated recombination was more advanced than MORE-mediated recombination at early gastrulation stages. The Sox2Cre line is likely to be more effective in generating complete, epiblast-specific removal of gene activity, and the mosaic activity of the MORE line will be helpful in generating partial loss-of-function phenotypes in the embryo-proper.

Sonic hedgehog (Shh) signaling from the posterior zone of polarizing activity (ZPA) is the primary determinant of anterior-posterior polarity in the vertebrate limb field. An active signal is produced by an autoprocessing reaction that covalently links cholesterol to the N-terminal signaling moiety (N-Shh(p)), tethering N-Shh(p) to the cell membrane. We have addressed the role played by this lipophilic modification in Shh-mediated patterning of mouse digits. Both the distribution and activity of N-Shh(p) indicate that N-Shh(p) acts directly over a few hundred microns. In contrast, N-Shh, a form that lacks cholesterol, retains similar biological activity to N-Shh(p), but signaling is posteriorly restricted. Thus, cholesterol modification is essential for the normal range of signaling. It also appears to be necessary for appropriate modulation of signaling by the Shh receptor, Ptc1.

The signals that promote regional growth and development of the brain are not well understood. Sonic hedgehog (Shh) is produced by Purkinje cells of the cerebellum and is a potent inducer of granule cell proliferation. Here, we demonstrate that Shh protein is present in the murine cerebellum during late stages of embryogenesis and is associated with Purkinje cell bodies and their processes. To better determine the role of Shh during cerebellar development, we genetically removed Shh activity specifically from Purkinje cells and the cerebellar anlage of the mouse embryo. We show that Shh is required for expansion of the granule neuron precursor population, but not for the subsequent differentiation of these cells. In addition, the loss of Shh activity influences Purkinje cell development and the formation of folia in the cerebellum. A role for Shh in compartmentalization of the cerebellum is also suggested by the more severe rostral defects observed in the absence of Hedgehog signaling. Together, these findings provide additional evidence for Shh's key regulatory role in controlling growth of the cerebellar primordium.

Sonic Hedgehog (Shh) and Indian Hedgehog (Ihh) are members of the Hedgehog (Hh) family of signaling molecules known to be involved in embryonic patterning and morphogenesis. The Hh proteins undergo an autocatalytic cleavage to yield an N-terminal and a C-terminal peptide, with the signaling capacities confined to the N peptide. Drosophila Hh-N has been shown to act via both short- and long-range signaling. In vertebrates, however, attempts to directly demonstrate Shh (SHH) or Ihh (IHH) proteins at a distance from producing cells have been largely unsuccessful. Furthermore, the fact that the Hh N peptides occur in a cholesterol-modified, membrane-tethered form is not easily reconciled with long-range signaling. This study used optimized immunohistochemistry combined with tissue separation and biochemical analyses in vivo and in vitro to determine the range of action of SHH and IHH in the mouse embryo. In all embryonic structures studied, we detect signaling peptides in producing cells, but we also find that ligands move over considerable distances depending on the tissue. These data provide direct evidence for the presence of Hedgehog signaling peptides in target compartments, suggesting a direct long-range action without a need for secondary mediators. Visualization of Hedgehog proteins in target tissues was achieved only under conditions that allowed proteoglycan/glycosaminoglycan (PG/GAG) preservation. Furthermore, we show that induced changes of the composition of PG/GAG in the tooth alter SHH signaling. These data suggest a crucial role for PG/GAGs in Hedgehog movement.

The adult retina is organized into three cellular layers--an outer photoreceptor, a middle interneuron and an inner retinal ganglion cell (RGC) layer. Although the retinal pigment epithelium (RPE) and Müller cells are important in the establishment and maintenance of this organization, the signals involved are unknown. Here we show that Sonic hedgehog signaling from RGCs is required for the normal laminar organization in the vertebrate retina.

During mammalian tooth development, the oral ectoderm and mesenchyme coordinate their growth and differentiation to give rise to organs with precise shapes, sizes and functions. The initial ingrowth of the dental epithelium and its associated dental mesenchyme gives rise to the tooth bud. Next, the epithelial component folds to give the tooth its shape. Coincident with this process, adjacent epithelial and mesenchymal cells differentiate into enamel-secreting ameloblasts and dentin-secreting odontoblasts, respectively. Growth, morphogenesis and differentiation of the epithelium and mesenchyme are coordinated by secreted signaling proteins. Sonic hedgehog (Shh) encodes a signaling peptide which is present in the oral epithelium prior to invagination and in the tooth epithelium throughout its development. We have addressed the role of Shh in the developing tooth in mouse by using a conditional allele to remove Shh activity shortly after ingrowth of the dental epithelium. Reduction and then loss of Shh function results in a cap stage tooth rudiment in which the morphology is severely disrupted. The overall size of the tooth is reduced and both the lingual epithelial invagination and the dental cord are absent. However, the enamel knot, a putative organizer of crown formation, is present and expresses Fgf4, Wnt10b, Bmp2 and Lef1, as in the wild type. At birth, the size and the shape of the teeth are severely affected and the polarity and organization of the ameloblast and odontoblast layers is disrupted. However, both dentin- and enamel-specific markers are expressed and a large amount of tooth-specific extracellular matrix is produced. This observation was confirmed by grafting studies in which tooth rudiments were cultured for several days under kidney capsules. Under these conditions, both enamel and dentin were deposited even though the enamel and dentin layers remained disorganized. These studies demonstrate that Shh regulates growth and determines the shape of the tooth. However, Shh signaling is not essential for differentiation of ameloblasts or odontoblasts.