Caenorhabditis elegans PTR/PTCHD PTR-18 promotes the clearance of extracellular hedgehog-related protein via endocytosis.

Spatiotemporal restriction of signaling plays a critical role in animal development and tissue homeostasis. All stem and progenitor cells in newly hatched C. elegans larvae are quiescent and capable of suspending their development until sufficient food is supplied. Here, we show that ptr-18, which encodes the evolutionarily conserved patched-related (PTR)/patched domain-containing (PTCHD) protein, temporally restricts the availability of extracellular hedgehog-related protein to establish the capacity of progenitor cells to maintain quiescence. We found that neural progenitor cells exit from quiescence in ptr-18 mutant larvae even when hatched under starved conditions. This unwanted reactivation depended on the activity of a specific set of hedgehog-related grl genes including grl-7. Unexpectedly, neither PTR-18 nor GRL-7 were expressed in newly hatched wild-type larvae. Instead, at the late embryonic stage, both PTR-18 and GRL-7 proteins were first localized around the apical membrane of hypodermal and neural progenitor cells and subsequently targeted for lysosomal degradation before hatching. Loss of ptr-18 caused a significant delay in GRL-7 clearance, causing this protein to be retained in the extracellular space in newly hatched ptr-18 mutant larvae. Furthermore, the putative transporter activity of PTR-18 was shown to be required for the appropriate function of the protein. These findings not only uncover a previously undescribed role of PTR/PTCHD in the clearance of extracellular hedgehog-related proteins via endocytosis-mediated degradation but also illustrate that failure to temporally restrict intercellular signaling during embryogenesis can subsequently compromise post-embryonic progenitor cell function.

Hedgehog-related genes regulate reactivation of quiescent neural progenitors in Caenorhabditis elegans.

The animal body contains various types of stem and progenitor cells. These undifferentiated cells coordinate the balance between quiescence and proliferation with dynamics of various physiological conditions such as the developmental stage, food availability, and injury. Although regulation of such coordination plays a critical role in maintaining tissue homeostasis, controlling the growth rate and regeneration, much of its mechanism remains elusive. Newly hatched Caenorhabditis elegans larvae possess quiescent stem and progenitor cells in several tissues, and these cells are reactivated by the insulin/insulin-like growth factor (IGF) signaling (IIS) pathway only when sufficient food is supplied. Maintenance of the quiescence of neuronal and mesodermal progenitor cells requires microRNA (miRNA), miR-235, which is upregulated under the starvation. On the other hand, feeding ample food downregulates the miRNA via the activity of the IIS pathway. As miR-235 in the hypodermis can non-autonomously regulate quiescence of neuronal and mesodermal progenitor cells, a cell-cell signaling pathway has been hypothesized to act downstream of the miRNA. Here, we provide evidence that two hedgehog-related (hh-r) genes, grl-5 and grl-7, are targets of miR-235 that promote reactivation of quiescent neuroblasts. These grl genes possess an miR-235 binding site on 3'UTRs of their transcripts, and are upregulated in starved mir-235 mutant larvae. grl-5 and grl-7 promoters can continuously drive the expression of GFP-pest reporter protein in the hypodermis under the fed condition. However, expression of these reporters is strikingly downregulated under the starvation condition after hatching. We found that miR-235 can repress expression of reporter genes via the predicted miR-235 binding sites on the grl-5 and grl-7 3'UTRs. Furthermore, activity of grl-5 and grl-7 genes are required for reactivation of neural progenitor cells in starved mir-235 mutant larvae. These findings suggest that the IIS pathway-miR-235 signaling in the hypodermis non-autonomously regulates quiescence of neural progenitor cells, partly via grl-5 and grl-7.

KCNJ8/ABCC9-containing K-ATP channel modulates brain vascular smooth muscle development and neurovascular coupling.

Loss- or gain-of-function mutations in ATP-sensitive potassium channel (K-ATP)-encoding genes, KCNJ8 and ABCC9, cause human central nervous system disorders with unknown pathogenesis. Here, using mice, zebrafish, and cell culture models, we investigated cellular and molecular causes of brain dysfunctions derived from altered K-ATP channel function. We show that genetic/chemical inhibition or activation of KCNJ8/ABCC9-containing K-ATP channel function leads to brain-selective suppression or promotion of arterial/arteriolar vascular smooth muscle cell (VSMC) differentiation, respectively. We further show that brain VSMCs develop from KCNJ8/ABCC9-containing K-ATP channel-expressing mural cell progenitor and that K-ATP channel cell autonomously regulates VSMC differentiation through modulation of intracellular Ca2+ oscillation via voltage-dependent calcium channels. Consistent with defective VSMC development, Kcnj8 knockout mice showed deficiency in vasoconstrictive capacity and neuronal-evoked vasodilation leading to local hyperemia. Our results demonstrate a role for KCNJ8/ABCC9-containing K-ATP channels in the differentiation of brain VSMC, which in turn is necessary for fine-tuning of cerebral blood flow.

Patched regulates lipid homeostasis by controlling cellular cholesterol levels.

Hedgehog (Hh) signaling is essential during development and in organ physiology. In the canonical pathway, Hh binding to Patched (PTCH) relieves the inhibition of Smoothened (SMO). Yet, PTCH may also perform SMO-independent functions. While the PTCH homolog PTC-3 is essential in C. elegans, worms lack SMO, providing an excellent model to probe non-canonical PTCH function. Here, we show that PTC-3 is a cholesterol transporter. ptc-3(RNAi) leads to accumulation of intracellular cholesterol and defects in ER structure and lipid droplet formation. These phenotypes were accompanied by a reduction in acyl chain (FA) length and desaturation. ptc-3(RNAi)-induced lethality, fat content and ER morphology defects were rescued by reducing dietary cholesterol. We provide evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as of the PPARα homolog NHR-49 and NHR-181, and affects FA composition. Our data uncover a role for PTCH in organelle structure maintenance and fat metabolism.