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The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discover TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 causes mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrate that TMEM251 localizes to the Golgi and is required for the cleavage and activity of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 asGNPTABcleavage andactivityfactor (GCAF) and its related disease as Mucolipidosis Type V.

 

Many types of differentiated cells can reenter the cell cycle upon injury or stress. The underlying mechanisms are still poorly understood. Here, we investigated how quiescent cells are reactivated using a zebrafish model, in which a population of differentiated epithelial cells are reactivated under a physiological context. A robust and sustained increase in mitochondrial membrane potential was observed in the reactivated cells. Genetic and pharmacological perturbations show that elevated mitochondrial metabolism and ATP synthesis are critical for cell reactivation. Further analyses showed that elevated mitochondrial metabolism increases mitochondrial ROS levels, which induces Sgk1 expression in the mitochondria. Genetic deletion and inhibition of Sgk1 in zebrafish abolished epithelial cell reactivation. Similarly, ROS-dependent mitochondrial expression of SGK1 promotes S phase entry in human breast cancer cells. Mechanistically, SGK1 coordinates mitochondrial activity with ATP synthesis by phosphorylating F 1 F o -ATP synthase. These findings suggest a conserved intramitochondrial signaling loop regulating epithelial cell renewal. 

Using a zebrafish ionocyte model, transcriptomics and genetic analyses were performed to identify pathways and genes involved in cell quiescence‐proliferation regulation. Gene ontology and Kyoto encyclopedia of genes and genomes pathway analyses revealed that genes involved in transcription regulation, cell cycle, Foxo signalling and Wnt signalling pathway are enriched among the up‐regulated genes while those involved in ion transport, cell adhesion and oxidation–reduction are enriched among the down‐regulated genes. Among the top up‐regulated genes is FK506‐binding protein 5 (Fkbp5). Genetic deletion and pharmacological inhibition of Fkbp5 abolished ionocyte reactivation and impaired Akt signalling. Forced expression of a constitutively active form of Akt rescued the defects caused by Fkbp5 inhibition. These results uncover a key role of Fbkp5 in regulating the quiescence‐proliferation decision via Akt signalling.

 

ABSTRACT Compared with our extensive understanding of the cell cycle, we have limited knowledge of how the cell quiescence–proliferation decision is regulated. Using a zebrafish epithelial model, we report a novel signaling mechanism governing the cell quiescence–proliferation decision. Zebrafish Ca2+-transporting epithelial cells, or ionocytes, maintain high cytoplasmic Ca2+ concentration ([Ca2+]c) due to the expression of Trpv6. Genetic deletion or pharmacological inhibition of Trpv6, or reduction of external Ca2+ concentration, lowered the [Ca2+]c and reactivated these cells. The ionocyte reactivation was attenuated by chelating intracellular Ca2+ and inhibiting calmodulin (CaM), suggesting involvement of a Ca2+ and CaM-dependent mechanism. Long-term imaging studies showed that after an initial decrease, [Ca2+]c gradually returned to the basal levels. There was a concomitant decease in endoplasmic reticulum (ER) Ca2+ levels. Lowering the ER Ca2+ store content or inhibiting ryanodine receptors impaired ionocyte reactivation. Further analyses suggest that CaM-dependent protein kinase kinase (CaMKK) is a key molecular link between Ca2+ and Akt signaling. Genetic deletion or inhibition of CaMKK abolished cell reactivation, which could be rescued by expression of a constitutively active Akt. These results suggest that the quiescence–proliferation decision in zebrafish ionocytes is regulated by Trpv6-mediated Ca2+ and CaMKK–Akt signaling. 

Gonadotropin-releasing hormone (GnRH) neurons are master regulators of the reproductive axis in vertebrates. During early mammalian embryogenesis, GnRH1 neurons emerge in the nasal/olfactory placode. These neurons undertake a long-distance migration, moving from the nose to the preoptic area and hypothalamus. While significant advances have been made in understanding the functional importance of the GnRH1 neurons in reproduction, where GnRH1 neurons come from and how are they specified during early development is still under debate. In addition to the GnRH1 gene, most vertebrate species including humans have one or two additional GnRH genes. Compared to the GnRH1 neurons, much less is known about the development and regulation of GnRH2 neuron and GnRH3 neurons. The objective of this article is to review what is currently known about GnRH neuron development. We will survey various cell autonomous and non-autonomous factors implicated in the regulation of GnRH neuron development. Finally, we will discuss emerging tools and new approaches to resolve open questions pertaining to GnRH neuron development. 

Epithelial homeostasis and regeneration require a pool of quiescent cells. How the quiescent cells are established and maintained is poorly understood. Here, we report that Trpv6, a cation channel responsible for epithelial Ca2+ absorption, functions as a key regulator of cellular quiescence. Genetic deletion and pharmacological blockade of Trpv6 promoted zebrafish epithelial cells to exit from quiescence and re-enter the cell cycle. Reintroducing Trpv6, but not its channel dead mutant, restored the quiescent state. Ca2+ imaging showed that Trpv6 is constitutively open in vivo. Mechanistically, Trpv6-mediated Ca2+ influx maintained the quiescent state by suppressing insulin-like growth factor (IGF)-mediated Akt-Tor and Erk signaling. In zebrafish epithelia and human colon carcinoma cells, Trpv6/TRPV6 elevated intracellular Ca2+ levels and activated PP2A, which down-regulated IGF signaling and promoted the quiescent state. Our findings suggest that Trpv6 mediates constitutive Ca2+ influx into epithelial cells to continuously suppress growth factor signaling and maintain the quiescent state.