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1. Sox10 is required for systemic initiation of bone mineralization

   https://doi.org/10.1101/2024.07.24.604990  

   Gjorcheska, Stefani; Paudel, Sandhya; McLeod, Sarah; Snape, Louisa; Sosa, Karen Camargo; Duan, Cunming; Kelsh, Robert; Barske, Lindsey (July 2024, bioRxiv)

   NA (Ed.)

   Abstract Heterozygous variants in the gene encoding the SOX10 transcription factor cause congenital syndromes affecting pigmentation, digestion, hearing, and neural function. Most of these symptoms are attributable to failed differentiation and loss of neural crest cells. Extensive research on mouse and zebrafish models has confirmed that Sox10 is essential for most non-skeletal crest derivatives, but seemingly dispensable for skeletal development. We challenge that concept here by revealing a novel requirement for Sox10 in skeletal mineralization. Neither neural crest- nor mesoderm-derived bones initiate mineralization on time in zebrafishsox10mutants, despite normal osteoblast differentiation and matrix production. We show that mutants are deficient in the ionocyte subpopulation tasked with taking up calcium from the environment through the Trpv6 epithelial calcium channel, leading to a severe calcium deficit that explains the lack of mineralization. As these ionocytes do not derive from asox10+ lineage, we hypothesized that the primary defect instead resides in a separate organ that regulates ionocyte numbers or calcium uptake at a systemic level. Screening of the endocrine hormones known to regulate calcium homeostasis in adult vertebrates revealed significantly elevated levels of stanniocalcin (Stc1a), an anti-hypercalcemic hormone, in larvalsox10mutants. Previous studies demonstrated that Stc1a inhibits calcium uptake in fish by repressingtrpv6expression and blocking proliferation of Trpv6+ ionocytes. Our epistasis assays indicate that excess Stc1a is the proximate cause of the calcium deficit insox10mutants. Lineage tracing shows that the pronephros-derived glands that synthesize Stc1a interact withsox10+ neural crest-derived cells, and that the latter are missing in mutants. We conclude that a subpopulation of Sox10+ neural crest non-cell-autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary for the initiation of bone mineralization. 

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2. Stanniocalcin 1a regulates organismal calcium balance and survival by suppressing Trpv6 expression and inhibiting IGF signaling in zebrafish

   https://doi.org/10.3389/fendo.2023.1276348  

   Li, Shuang; Li, Helena; Wang, Zhengyi; Duan, Cunming (October 2023, Frontiers in Endocrinology)

   Stanniocalcin 1 (Stc1) is well known for its role in regulating calcium uptake in fish by acting on ionocytes or NaR cells. A hallmark of NaR cells is the expression of Trpv6, a constitutively open calcium channel. Recent studies in zebrafish suggest that genetical deletion of Stc1a and Trpv6 individually both increases IGF signaling and NaR cell proliferation. Whiletrpv6^-/-fish suffered from calcium deficiency and died prematurely,stc1a^-/-fish had elevated body calcium levels but also died prematurely. The relationship between Stc1a, Trpv6, and IGF signaling in regulating calcium homeostasis and organismal survival is unclear. Here we report that loss of Stc1a increases Trpv6 expression in NaR cells in an IGF signaling-dependent manner. Treatment with CdCl₂, a Trpv6 inhibitor, reduced NaR cell number instc1a^-/-fish to the sibling levels. Genetic and biochemical analysis results suggest that Stc1a and Trpv6 regulate NaR cell proliferation via the same IGF pathway. Alizarin red staining detected abnormal calcium deposits in the yolk sac region and kidney stone-like structures instc1a^-/-fish. Double knockout or pharmacological inhibition of Trpv6 alleviated these phenotypes, suggesting that Stc1a inhibit epithelial Ca²⁺uptake by regulating Trpv6 expression and activity.stc1a^-/-mutant fish developed cardiac edema, body swelling, and died prematurely. Treatment ofstc1a^-/-fish with CdCl₂or double knockout of Trpv6 alleviated these phenotypes. These results provide evidence that Stc1a regulates calcium homeostasis and organismal survival by suppressing Trpv6 expression and inhibiting IGF signaling in ionocytes. 

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3. Regulation of cell quiescence–proliferation balance by Ca2+–CaMKK–Akt signaling

   https://doi.org/10.1242/jcs.253807  

   Xin, Yi; Guan, Jian; Li, Yingxiang; Duan, Cunming (October 2021, Journal of Cell Science)

   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. 

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4. The metalloproteinase Papp-aa controls epithelial cell quiescence-proliferation transition

   https://doi.org/10.7554/eLife.52322  

   Liu, Chengdong; Li, Shuang; Noer, Pernille Rimmer; Kjaer-Sorensen, Kasper; Juhl, Anna Karina; Goldstein, Allison; Ke, Caihuan; Oxvig, Claus; Duan, Cunming (April 2020, eLife)

   Ackert-Bicknell, Cheryl (Ed.)

   Human patients carrying PAPP‐A2 inactivating mutations have low bone mineral density. The underlying mechanisms for this reduced calcification are poorly understood. Using a zebrafish model, we report that Papp-aa regulates bone calcification by promoting Ca2+-transporting epithelial cell (ionocyte) quiescence-proliferation transition. Ionocytes, which are normally quiescent, re-enter the cell cycle under low [Ca2+] stress. Genetic deletion of Papp-aa, but not the closely related Papp-ab, abolished ionocyte proliferation and reduced calcified bone mass. Loss of Papp-aa expression or activity resulted in diminished IGF1 receptor-Akt-Tor signaling in ionocytes. Under low Ca2+ stress, Papp-aa cleaved Igfbp5a. Under normal conditions, however, Papp-aa proteinase activity was suppressed and IGFs were sequestered in the IGF/Igfbp complex. Pharmacological disruption of the IGF/Igfbp complex or adding free IGF1 activated IGF signaling and promoted ionocyte proliferation. These findings suggest that Papp-aa-mediated local Igfbp5a cleavage functions as a [Ca2+]-regulated molecular switch linking IGF signaling to bone calcification by stimulating epithelial cell quiescence-proliferation transition under low Ca2+ stress. 

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5. Calcium State-Dependent Regulation of Epithelial Cell Quiescence by Stanniocalcin 1a

   https://doi.org/10.3389/fcell.2021.662915  

   Li, Shuang; Liu, Chengdong; Goldstein, Allison; Xin, Yi; Ke, Caihuan; Duan, Cunming (April 2021, Frontiers in Cell and Developmental Biology)

   Yao, Guang (Ed.)

   The molecular mechanisms regulating cell quiescence-proliferation balance are not well defined. Using a zebrafish model, we report that Stc1a, a secreted glycoprotein, plays a key role in regulating the quiescence-proliferation balance of Ca²⁺transporting epithelial cells (ionocytes). Zebrafishstc1a, but not the otherstcgenes, is expressed in a Ca²⁺state-dependent manner. Genetic deletion ofstc1a, but notstc2b, increased ionocyte proliferation, leading to elevated body Ca²⁺levels, cardiac edema, body swelling, and premature death. The increased ionocyte proliferation was accompanied by an increase in the IGF1 receptor-mediated PI3 kinase-Akt-Tor signaling activity in ionocytes. Inhibition of the IGF1 receptor, PI3 kinase, Akt, and Tor signaling reduced ionocyte proliferation and rescued the edema and premature death instc1a^–/–fish, suggesting that Stc1a promotes ionocyte quiescence by suppressing local IGF signaling activity. Mechanistically, Stc1 acts by inhibiting Papp-aa, a zinc metalloproteinase degrading Igfbp5a. Inhibition of Papp-aa proteinase activity restored ionocyte quiescence-proliferation balance. Genetic deletion ofpapp-aaor its substrateigfbp5ain thestc1a^–/–background reduced ionocyte proliferation and rescued the edema and premature death. These findings uncover a novel and Ca²⁺state-dependent pathway regulating cell quiescence. Our findings also provide new insights into the importance of ionocyte quiescent-proliferation balance in organismal Ca²⁺homeostasis and survival. 

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