Mutant mice bearing a targeted disruption of the heparan sulfate (HS) modifying enzyme GlcNAc N-deacetylase/N-sulfotransferase 1 (Ndst1) exhibit severe developmental defects of the forebrain and forebrain-derived structures, including cerebral hypoplasia, lack of olfactory bulbs, eye defects and axon guidance errors. Neural crest-derived facial structures are also severely affected. We show that properly synthesized heparan sulfate is required for the normal development of the brain and face, and that Ndst1 is a modifier of heparan sulfate-dependent growth factor/morphogen signalling in those tissues. Among the multiple heparan sulfate-binding factors potentially affected in Ndst1 mutant embryos, the facial phenotypes are consistent with impaired sonic hedgehog (Shh) and fibroblast growth factor (Fgf) interaction with mutant heparan sulfate. Most importantly, the data suggest the possibility that defects in heparan sulfate synthesis could give rise to or contribute to a number of developmental brain and facial defects in humans.

The ectodomains of numerous proteins are released from cells by matrix metalloproteases to yield soluble intercellular regulators. A disintegrin and metalloprotease (ADAM) family members have often been found to be the responsible "sheddases," ADAM17/tumor necrosis factor-alpha-converting enzyme being its best characterized member. In this work, we show that ShhNp (lipidated and membrane-tethered Sonic hedgehog) is released from Bosc23 cells by metalloprotease-mediated ectodomain shedding, resulting in a soluble and biologically active morphogen. ShhNp shedding is increased by ADAM17 coexpression and cholesterol depletion of cells with methyl-beta-cyclodextrin and is reduced by metalloprotease inhibitors as well as ADAM17 RNA interference. We also show that the amount of shed ShhNp is modulated by extracellular heparan sulfate (HS) and that ShhNp shedding depends on specific HS sulfations. Based on those data, we suggest new roles for metalloproteases, including but not restricted to ADAM17, and for HS-proteoglycans in Hedgehog signaling.

Although hundreds of cytosolic or transmembrane molecules form the primary cilium, few secreted molecules are known to contribute to ciliogenesis. Here, homologous secreted metalloproteases ADAMTS9 and ADAMTS20 are identified as ciliogenesis regulators that act intracellularly. Secreted and furin-processed ADAMTS9 bound heparan sulfate and was internalized by LRP1, LRP2 and clathrin-mediated endocytosis to be gathered in Rab11 vesicles with a unique periciliary localization defined by super-resolution microscopy. CRISPR-Cas9 inactivation of ADAMTS9 impaired ciliogenesis in RPE-1 cells, which was restored by catalytically active ADAMTS9 or ADAMTS20 acting in trans, but not by their proteolytically inactive mutants. Their mutagenesis in mice impaired neural and yolk sac ciliogenesis, leading to morphogenetic anomalies resulting from impaired hedgehog signaling, which is transduced by primary cilia. In addition to their cognate extracellular proteolytic activity, ADAMTS9 and ADAMTS20 thus have an additional proteolytic role intracellularly, revealing an unexpected regulatory dimension in ciliogenesis.

We report the generation and analysis of mutant mice bearing a targeted disruption of the heparan sulfate (HS)-modifying enzyme GlcNAc N-deacetylase/N-sulfotransferase 3 (NDST3). NDST3^-/- mice develop normally, are fertile, and show only subtle hematological and behavioral abnormalities in agreement with only moderate HS undersulfation. Compound mutant mice made deficient in NDST2;NDST3 activities also develop normally, showing that both isoforms are not essential for development. In contrast, NDST1^-/-;NDST3^-/- compound mutant embryos display developmental defects caused by severe HS undersulfation, demonstrating NDST3 contribution to HS synthesis in the absence of NDST1. Moreover, analysis of HS composition in dissected NDST3 mutant adult brain revealed regional changes in HS sulfation, indicating restricted NDST3 activity on nascent HS in defined wild-type tissues. Taken together, we show that NDST3 function is not essential for development or adult homeostasis despite contributing to HS synthesis in a region-specific manner and that the loss of NDST3 function is compensated for by the other NDST isoforms to a varying degree.

Major developmental morphogens of the Hedgehog (Hh) family act at short range and long range to direct cell fate decisions in vertebrate and invertebrate tissues. To this end, Hhs are released from local sources and act at a distance on target cells that express the Hh receptor Patched. However, morphogen secretion and spreading are not passive processes because all Hhs are synthesized as dually (N- and C-terminal) lipidated proteins that firmly tether to the surface of producing cells. On the cell surface, Hhs associate with each other and with heparan sulfate (HS) proteoglycans. This raises the question of how Hh solubilization and spreading is achieved. We recently discovered that Sonic hedgehog (Shh) is solubilized by proteolytic processing (shedding) of lipidated peptide termini in vitro. Because unprocessed N termini block Patched receptor binding sites in the cluster, we further suggested that their proteolytic removal is required for simultaneous Shh activation. In this work we confirm inactivity of unprocessed protein clusters and demonstrate restored biological Shh function upon distortion or removal of N-terminal amino acids and peptides. We further show that N-terminal Shh processing targets and inactivates the HS binding Cardin-Weintraub (CW) motif, resulting in soluble Shh clusters with their HS binding capacities strongly reduced. This may explain the ability of Shh to diffuse through the HS-containing extracellular matrix, whereas other HS-binding proteins are quickly immobilized. Our in vitro findings are supported by the presence of CW-processed Shh in murine brain samples, providing the first in vivo evidence for Shh shedding and subsequent solubilization of N-terminal-truncated proteins.

Decision making in cellular ensembles requires the dynamic release of signaling molecules from the producing cells into the extracellular compartment. One important example of molecules that require regulated release in order to signal over several cell diameters is the Hedgehog (Hh) family, because all Hhs are synthesized as dual-lipidated proteins that firmly tether to the outer membrane leaflet of the cell that produces them. Factors for the release of the vertebrate Hh family member Sonic Hedgehog (Shh) include cell-surface sheddases that remove the lipidated terminal peptides, as well as the soluble glycoprotein Scube2 that cell-nonautonomously enhances this process. This raises the question of how soluble Scube2 is recruited to cell-bound Shh substrates to regulate their turnover. We hypothesized that heparan sulfate (HS) proteoglycans (HSPGs) on the producing cell surface may play this role. In this work, we confirm that HSPGs enrich Scube2 at the surface of Shh-producing cells and that Scube2-regulated proteolytic Shh processing and release depends on specific HS. This finding indicates that HSPGs act as cell-surface assembly and storage platforms for Shh substrates and for protein factors required for their release, making HSPGs critical decision makers for Scube2-dependent Shh signaling from the surface of producing cells.