Chin Chiang

Sonic hedgehog is essential to foregut development

Congenital malformation of the foregut is common in humans, with an estimated incidence of 1 in 3000 live births, although its aetiology remains largely unknown. Mice with a targeted deletion of Sonic hedgehog ( Shh) have foregut defects that are apparent as early as embryonic day 9.5, when the tracheal diverticulum begins to outgrow. Homozygous Shh-null mutant mice show oesophageal atresia/stenosis, tracheo-oesophageal fistula and tracheal and lung anomalies, features similar to those observed in humans with foregut defects. The lung mesenchyme shows enhanced cell death, decreased cell proliferation and downregulation of Shh target genes. These results indicate that Shh is required for the growth and differentiation of the oesophagus, trachea and lung, and suggest that mutations in SHH and its signalling components may be involved in foregut defects in humans.

Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity

Most current models propose Sonic hedgehog (Shh) as the primary determinant of anteroposterior development of amniote limbs. Shh protein is said to be required to direct the formation of skeletal elements and to specify digit identity through dose-dependent activation of target gene expression. However, the identity of genes targeted by Shh, and the regulatory mechanisms controlling their expression, remain poorly understood. Gli3 (the gene implicated in human Greig cephalopolysyndactyly syndrome) is proposed to negatively regulate Shh by restricting its expression and influence to the posterior mesoderm. Here we report genetic analyses in mice showing that Shh and Gli3 are dispensable for formation of limb skeletal elements: Shh-/- Gli3-/- limbs are distally complete and polydactylous, but completely lack wild-type digit identities. We show that the effects of Shh signalling on skeletal patterning and ridge maintenance are necessarily mediated through Gli3. We propose that the function of Shh and Gli3 in limb skeletal patterning is limited to refining autopodial morphology, imposing pentadactyl constraint on the limbs polydactyl potential, and organizing digit identity specification, by regulating the relative balance of Gli3 transcriptional activator and repressor activities.

Induction of midbrain dopaminergic neurons by Sonic hedgehog

Midbrain dopaminergic neurons, whose loss in adults results in Parkinsons disease, can be specified during embryonic development by a contact-dependent signal from floor plate cells. Here we show that the amino-terminal product of Sonic hedgehog autoproteolysis (SHH-N), an inductive signal expressed by floor plate cells, can induce dopaminergic neurons in vitro. We show further that manipulations to increase the activity of cyclic AMP-dependent protein kinase A, which is known to antagonize hedgehog signaling, can block dopaminergic neuron induction by floor plate cells. Our results and those of other studies indicate that SHH-N can function in a dose-dependent manner to induce different cell types within the neural tube. Our results also provide the basis for a potential cell transplantation therapy for Parkinsons disease.

Specification of ventral neuron types is mediated by an antagonisticinteraction between Shh and Gli3

Specification of distinct neuron types in the ventral spinal cord is thought to be mediated by a graded concentration of Sonic hedgehog (Shh), a secreted signaling protein. Shh is made in the notochord, the most ventral part of the spinal cord, and in mice lacking Shh, ventral cell types are reduced or absent. The response to Shh depends on transcription factors of the Gli family, but the detailed mechanism is not understood. Here we show that Gli3 represses ventral fates in a dose-dependent manner. Whereas Shh−/− mutant mice show reductions in several classes of ventral interneurons and a complete absence of motor neurons, these cell types were rescued in Shh−/−;Gli3−/− double mutants. This rescue of the Shh null phenotype depended on the level of Gli3 function; a partial rescue was observed in Shh−/−;Gli3+/− embryos. We propose that Shh is required to antagonize Gli3, which would otherwise repress ventral fates. Differences between rostral and caudal regions suggest that other signaling molecules—in addition to Shh—may be involved in specifying ventral fates, particularly in the caudal region of the spinal cord.

A Sonic Hedgehog–Independent, Retinoid-Activated Pathway of Neurogenesis in the Ventral Spinal Cord

Sonic hedgehog (Shh) is thought to control the generation of motor neurons and interneurons in the ventral CNS. We show here that a Shh-independent pathway of interneuron generation also operates in the ventral spinal cord. Evidence for this parallel pathway emerged from an analysis of the induction of ventral progenitors that express the Dbx homeodomain proteins and of Evx1/2 (V0) and En1 (V1) neurons. Shh signaling is sufficient to induce Dbx cells and V0 and V1 neurons but is not required for their generation in vitro or in vivo. Retinoids appear to mediate this parallel pathway. These findings reveal an unanticipated Shh-independent signaling pathway that controls progenitor cell identity and interneuron diversity in the ventral spinal cord.

Coordinate regulation and synergistic actions of BMP4, SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles.

We investigated the roles of bare morphogenetic protein (BMP), sonic hedgehog (SHH) and fibroblast growth factor (FGF)-expressing signaling centers in regulating the patterned outgrowth of the telencephalic and optic vesicles. Implantation of BMP4 beads in the anterior neuropore of stage 10 chicken embryos repressed FGF8 and SHH expression. Similarly, loss of SHH expression in Shh mutant mice leads to increased BMP signaling and loss of Fgf8 expression in the prosencephalon. Increased BMP signaling and loss of FGF and SHH expression was correlated with decreased proliferation, increased cell death, and hypoplasia of the telencephalic and optic vesicles. However, decreased BMP signaling, through ectopic expression of Noggin, a BMP-binding protein, also caused decreased proliferation and hypoplasia of the telencephalic and optic vesicles, but with maintenance of Fgf8 and Shh expression, and no detectable increase in cell death. These results suggest that optimal growth requires a balance of BMP, FGF8 and SHH signaling. We suggest that the juxtaposition of Fgf8, Bmp4 and Shh expression domains generate patterning centers that coordinate the growth of the telencephalic and optic vesicles, similar to how Fgf8, Bmp4 and Shh regulate growth of the limb bud. Furthermore, these patterning centers regulate regional specification within the forebrain and eye, as exemplified by the regulation of Emx2 expression by different levels of BMP signaling. In summary, we present evidence that there is cross-regulation between BMP-, FGF- and SHH-expressing signaling centers in the prosencephalon which regulate morphogenesis of, and regional specification within, the telencephalic and optic vesicles.

Anorectal malformations caused by defects in sonic hedgehog signaling.

Anorectal malformations are a common clinical problem affecting the development of the distal hindgut in infants. The spectrum of anorectal malformations ranges from the mildly stenotic anus to imperforate anus with a fistula between the urinary and intestinal tracts to the most severe form, persistent cloaca. The etiology, embryology, and pathogenesis of anorectal malformations are poorly understood and controversial. Sonic hedgehog (Shh) is an endoderm-derived signaling molecule that induces mesodermal gene expression in the chick hindgut. However, the role of Shh signaling in mammalian hindgut development is unknown. Here, we show that mutant mice with various defects in the Shh signaling pathway exhibit a spectrum of distal hindgut defects mimicking human anorectal malformations. Shh null-mutant mice display persistent cloaca. Mutant mice lacking Gli2 or Gli3, two zinc finger transcription factors involved in Shh signaling, respectively, exhibit imperforate anus with recto-urethral fistula and anal stenosis. Furthermore, persistent cloaca is also observed in Gli2(-/-);Gli3(+/-), Gli2(+/-);Gli3(-/-), and Gli2(-/-);Gli3(-/-) mice demonstrating a gene dose-dependent effect. Therefore, Shh signaling is essential for normal development of the distal hindgut in mice and mutations affecting Shh signaling produce a spectrum of anorectal malformations that may reveal new insights into their human disease equivalents.

Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage

BACKGROUND Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH. RESULTS We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud. CONCLUSIONS Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.

MYCN haploinsufficiency is associated with reduced brain size and intestinal atresias in Feingold syndrome.

Feingold syndrome is characterized by variable combinations of esophageal and duodenal atresias, microcephaly, learning disability, syndactyly and cardiac defect. We show here that heterozygous mutations in the gene MYCN are present in Feingold syndrome. All mutations are predicted to disrupt both the full-length protein and a new shortened MYCN isoform, suggesting that multiple aspects of early embryogenesis and postnatal brain growth in humans are tightly regulated by MYCN dosage.

COUP-TFII is essential for radial and anteroposterior patterning of the stomach

COUP-TFII, an orphan member of the steroid receptor superfamily, has been implicated in mesenchymal-epithelial interaction during organogenesis. The generation of a lacZ knock-in allele in the COUP-TFII locus in mice allows us to use X-gal staining to follow the expression of COUP-TFII in the developing stomach. We found COUP-TFII is expressed in the mesenchyme and the epithelium of the developing stomach. Conditional ablation of floxed COUP-TFII by Nkx3-2Cre recombinase in the gastric mesenchyme results in dysmorphogenesis of the developing stomach manifested by major patterning defects in posteriorization and radial patterning. The epithelial outgrowth, the expansion of the circular smooth muscle layer and enteric neurons as well as the posteriorization of the stomach resemble phenotypes exhibited by inhibition of hedgehog signaling pathways. Using organ cultures and cyclopamine treatment, we showed downregulation of COUP-TFII level in the stomach, suggesting COUP-TFII as a target of hedgehog signaling in the stomach. Our results are consistent with a functional link between hedgehog proteins and COUP-TFII, factors that are vital for epithelial-mesenchymal interactions.