Sonic Hedgehog Is a Member of the Hh/DD-Peptidase Family That Spans the Eukaryotic and Bacterial Domains of Life.

Sonic Hedgehog (Shh) coordinates Zn2+ in a manner that resembles that of peptidases. The ability of Shh to undergo autoproteolytic processing is impaired in mutants that affect the Zn2+ coordination, while mutating residues essential for catalytic activity results in more stable forms of Shh. The residues involved in Zn2+ coordination in Shh are found to be mutated in some individuals with the congenital birth defect holoprosencephaly, demonstrating their importance in development. Highly conserved Shh domains are found in parts of some bacterial proteins that are members of the larger family of DD-peptidases, supporting the notion that Shh acts as a peptidase. Whereas this Hh/DD-peptidase motif is present in Hedgehog (Hh) proteins of nearly all animals, it is not present in Drosophila Hh, indicating that Hh signaling in fruit flies is derived, and perhaps not a good model for vertebrate Shh signaling. A sequence analysis of Hh proteins and their possible evolutionary precursors suggests that the evolution of modern Hh might have involved horizontal transfer of a bacterial gene coding of a Hh/DD-peptidase into a Cnidarian ancestor, recombining to give rise to modern Hh

Sonic hedgehog promotes rod photoreceptor differentiation in mammalian retinal cells in vitro.

Journal ArticleThe hedgehog gene family encodes secreted proteins important in many developmental patterning events in both vertebrates and invertebrates. In the Drosophila eye disk, hedgehog controls the progression of photoreceptor differentiation in the morphogenetic furrow. To investigate whether hedgehog proteins are also involved in the development of the vertebrate retina at stages of photoreceptor differentiation, we analyzed expression of the three known vertebrate hedgehog genes. We found that Sonic hedgehog and Desert hedgehog are expressed in the developing retina, albeit at very low levels, whereas Indian hedgehog (Ihh) is expressed in the developing and mature retinal pigmented epithelium, beginning at embryonic day 13. To determine whether hedgehog proteins have activities on developing retinal cells, we used an in vitro system in which much of retinal histogenesis is recapitulated. N-terminal recombinant Sonic Hedgehog protein (SHH-N) was added to rat retinal cultures for 3-12 d, and the numbers of retinal cells of various phenotypes were analyzed by immunohistochemistry. We found that SHH-N caused a transient increase in the number of retinal progenitor cells, and a 2- to 10-fold increase in the number of photoreceptors differentiating in the cultures when analyzed with three different photoreceptor-specific antigens. In contrast, the numbers of retinal ganglion cells and amacrine cells were similar to those in control cultures. These results show that Hedgehog proteins can regulate mitogenesis and photoreceptor differentiation in the vertebrate retina, and Ihh is a candidate factor from the pigmented epithelium to promote retinal progenitor proliferation and photoreceptor differentiation

Induction of motor neurons by Sonic hedgehog is independent of floor plate differentiation

AbstractBackground: The differentiation of floor plate cells and motor neurons in the vertebrate neural tube appears to be induced by signals from the notochord. The secreted protein encoded by the Sonic hedgehog (Shh) gene is expressed by axial midline cells and can induce floor plate cells in vivo and in vitro. Motor neurons can also be induced in vitro by cells that synthesize Sonic hedgehog protein (Shh). It remains unclear, however, if the motor-neuron-inducing activity of Shh depends on the synthesis of a distinct signaling molecule by floor plate cells. To resolve this issue, we have developed an in vitro assay which uncouples the notochord-mediated induction of motor neurons from floor plate differentiation, and have used this assay to examine whether Shh induces motor neurons in the absence of floor plate differentiation.Results Floor plate cells and motor neurons were induced in neural plate explants grown in contact with the notochord, but only motor neurons were induced when explants were separated from the notochord. COS cells transfected with Shh induced both floor plate cells and motor neurons when grown in contact with neural plate explants, whereas only motor neurons were induced when the explants were grown at a distance from Shh-transfected COS cells. Direct transfection of neural plate cells with an Shh-expression construct induced both floor plate cells and motor neurons, with motor neuron differentiation occurring prior to, or coincidentally with, floor plate differentiation. The induction of motor neurons appears, therefore, not to depend on floor plate differentiation.Conclusion The induction of motor neurons by Shh does not depend on distinct floor-plate-derived signaling molecules. Shh can, therefore, initiate the differentiation of two cell types that are generated in the ventral region of the neural tube. These results show that the early development of motor neurons involves the inductive action of Shh, whereas the survival of motor neurons at later stages of embryonic development requires neurotrophic factors

Sonic Hedgehog Induces the Segregation of Patched and Smoothened in Endosomes

AbstractBackground: Sonic hedgehog (Shh) signal transduction involves the ligand binding Patched1 (Ptc1) protein and a signaling component, Smoothened (Smo). A select group of compounds inhibits both Shh signaling, regulated by Ptc1, and late endosomal lipid sorting, regulated by the Ptc-related Niemann-Pick C1 (NPC1) protein. This suggests that Ptc1 regulates Smo activity through a common late endosomal sorting pathway also utilized by NPC1. During signaling, Ptc accumulates in endosomal compartments, but it is unclear if Smo follows Ptc into the endocytic pathway.Results: We characterized the dynamic subcellular distributions of Ptc1, Smo, and activated Smo mutants individually and in combination. Ptc1 and Smo colocalize extensively in the absence of ligand and are internalized together after ligand binding, but Smo becomes segregated from Ptc1/Shh complexes destined for lysosomal degradation. In contrast, activated Smo mutants do not colocalize with nor are cotransported with Ptc1. Agents that block late endosomal transport and protein sorting inhibit the ligand-induced segregation of Ptc1 and Smo. We show that, like NPC1-regulated lipid sorting, Shh signal transduction is blocked by antibodies that specifically disrupt the internal membranes of late endosomes, which provide a platform for protein and lipid sorting.Conclusions: These data support a model in which Ptc1 inhibits Smo only when in the same compartment. Ligand-induced segregation allows Smo to signal independently of Ptc1 after becoming sorted from Ptc1/Shh complexes in the late endocytic pathway

Threshold-Dependent BMP-Mediated Repression: A Model for a Conserved Mechanism That Patterns the Neuroectoderm

Subdivision of the neuroectoderm into three rows of cells along the dorsal-ventral axis by neural identity genes is a highly conserved developmental process. While neural identity genes are expressed in remarkably similar patterns in vertebrates and invertebrates, previous work suggests that these patterns may be regulated by distinct upstream genetic pathways. Here we ask whether a potential conserved source of positional information provided by the BMP signaling contributes to patterning the neuroectoderm. We have addressed this question in two ways: First, we asked whether BMPs can act as bona fide morphogens to pattern the Drosophila neuroectoderm in a dose-dependent fashion, and second, we examined whether BMPs might act in a similar fashion in patterning the vertebrate neuroectoderm. In this study, we show that graded BMP signaling participates in organizing the neural axis in Drosophila by repressing expression of neural identity genes in a threshold-dependent fashion. We also provide evidence for a similar organizing activity of BMP signaling in chick neural plate explants, which may operate by the same double negative mechanism that acts earlier during neural induction. We propose that BMPs played an ancestral role in patterning the metazoan neuroectoderm by threshold-dependent repression of neural identity genes

Sonic Hedgehog Is a Member of the Hh/DD-Peptidase Family That Spans the Eukaryotic and Bacterial Domains of Life

Sonic Hedgehog (Shh) coordinates Zn2+ in a manner that resembles that of peptidases. The ability of Shh to undergo autoproteolytic processing is impaired in mutants that affect the Zn2+ coordination, while mutating residues essential for catalytic activity results in more stable forms of Shh. The residues involved in Zn2+ coordination in Shh are found to be mutated in some individuals with the congenital birth defect holoprosencephaly, demonstrating their importance in development. Highly conserved Shh domains are found in parts of some bacterial proteins that are members of the larger family of DD-peptidases, supporting the notion that Shh acts as a peptidase. Whereas this Hh/DD-peptidase motif is present in Hedgehog (Hh) proteins of nearly all animals, it is not present in Drosophila Hh, indicating that Hh signaling in fruit flies is derived, and perhaps not a good model for vertebrate Shh signaling. A sequence analysis of Hh proteins and their possible evolutionary precursors suggests that the evolution of modern Hh might have involved horizontal transfer of a bacterial gene coding of a Hh/DD-peptidase into a Cnidarian ancestor, recombining to give rise to modern Hh

Sonic Hedgehog Is a Member of the Hh/DD-Peptidase Family That Spans the Eukaryotic and Bacterial Domains of Life.

Sonic Hedgehog (Shh) coordinates Zn2+ in a manner that resembles that of peptidases. The ability of Shh to undergo autoproteolytic processing is impaired in mutants that affect the Zn2+ coordination, while mutating residues essential for catalytic activity results in more stable forms of Shh. The residues involved in Zn2+ coordination in Shh are found to be mutated in some individuals with the congenital birth defect holoprosencephaly, demonstrating their importance in development. Highly conserved Shh domains are found in parts of some bacterial proteins that are members of the larger family of DD-peptidases, supporting the notion that Shh acts as a peptidase. Whereas this Hh/DD-peptidase motif is present in Hedgehog (Hh) proteins of nearly all animals, it is not present in Drosophila Hh, indicating that Hh signaling in fruit flies is derived, and perhaps not a good model for vertebrate Shh signaling. A sequence analysis of Hh proteins and their possible evolutionary precursors suggests that the evolution of modern Hh might have involved horizontal transfer of a bacterial gene coding of a Hh/DD-peptidase into a Cnidarian ancestor, recombining to give rise to modern Hh

Gain-of-function Shh mutants activate Smo cell-autonomously independent of Ptch1/2 function

Sonic Hedgehog (Shh) signaling is characterized by non-cell autonomy; cells expressing Shh do not respond to the ligand. Here, we identify several Shh mutations that can activate the Hedgehog (Hh) pathway cell-autonomously. Cell-autonomous pathway activation requires the extracellular cysteine rich domain of Smoothened, but is otherwise independent of the Shh receptors Patched1 and -2. Many of the Shh mutants that gain activity fail to undergo auto processing resulting in the perdurance of the Shh pro-peptide, a form of Shh that is sufficient to activate the Hh response cell-autonomously. Our results demonstrate that Shh is capable of activating the Hh pathway via Smoothened, independently of Patched1/2, and that it harbors an intrinsic mechanism that prevents cell-autonomous activation of the Shh response

Loss of the Heparan Sulfate Proteoglycan Glypican5 Facilitates Long‐Range Sonic Hedgehog Signaling

As a morphogen, Sonic Hedgehog (Shh) mediates signaling at a distance from its sites of synthesis. After secretion, Shh must traverse a distance through the extracellular matrix (ECM) to reach the target cells and activate the Hh response. ECM proteins, in particular, the heparan sulfate proteoglycans (HSPGs) of the glypican family, have both negative and positive effects on Shh signaling, all attributed to their ability to bind Shh. Using mouse embryonic stem cell-derived mosaic tissues with compartments that lack the glycosyltransferases Exostosin1 and Exostosin2, or the HSPG core protein Glypican5, we show that Shh accumulates around its source cells when they are surrounded by cells that have a mutated ECM. This accumulation of Shh is correlated with an increased noncell autonomous Shh response. Our results support a model in which Shh presented on the cell surface accumulates at or near ECM that lacks HSPGs, possibly due to the absence of these Shh sequestering molecules. Stem Cells 2019;37:899-909