Targeted Gene Disruption in Zebrafish Reveals Noncanonical Functions of LH Signaling in Reproduction

The pivotal role of gonadotropin signaling in regulating gonadal development and functions has attracted much research attention in the past 2 decades. However, the precise physiological role of gonadotropin signaling is still largely unknown in fish. In this study, we have established both LH beta-subunit (lhb) and LH receptor (lhr) knockout zebrafish lines by transcription activator-like effector nucleases. Intriguingly, both homozygous lhb and lhr mutant male fish are fertile. The fertilization rate, sperm motility, and histological structure of the testis were not affected in either lhb or lhr mutant males. On the contrary, homozygous lhb mutant females are infertile, whereas homozygous lhr mutant females are fertile. Folliculogenesis was not affected in either lhb or lhr mutants, but oocyte maturation and ovulation were disrupted in lhb mutant, whereas only ovulation was affected in lhr mutant. Differential expression of genes in the ovary involved in steroidogenesis, oocyte maturation, and ovulation was found between the lhb and lhr mutants. These data demonstrate the essential role of LH signaling in oocyte maturation and ovulation, and support the notion that LH acts through the FSH receptor in the absence of LH receptor. Moreover, the defects of lhb mutant could be partially restored by administration of human chorionic gonadotropin. This in vivo evidence in the present study demonstrates, for the first time in any vertebrate species, that LH signaling is indispensable in female reproduction but not in male reproduction. LH signaling is demonstrated to control oocyte maturation and ovulation in the ovary

In vivo drug discovery for increasing incretin-expressing cells identifies DYRK inhibitors that reinforce the enteroendocrine system

Analogs of the incretin hormones Gip and Glp-1 are used to treat type 2 diabetes and obesity. Findings in experimental models suggest that manipulating several hormones simultaneously may be more effective. To identify small molecules that increase the number of incretin-expressing cells, we established a high-throughput in vivo chemical screen by using the gip promoter to drive the expression of luciferase in zebrafish. All hits increased the numbers of neurogenin 3-expressing enteroendocrine progenitors, Gip-expressing K-cells, and Glp-1-expressing L-cells. One of the hits, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor, additionally decreased glucose levels in both larval and juvenile fish. Knock-down experiments indicated that nfatc4, a downstream mediator of DYRKs, regulates incretin+ cell number in zebrafish, and that Dyrk1b regulates Glp-1 expression in an enteroendocrine cell line. DYRK inhibition also increased the number of incretin-expressing cells in diabetic mice, suggesting a conserved reinforcement of the enteroendocrine system, with possible implications for diabetes

In vivo screen identifies a SIK inhibitor that induces beta cell proliferation through a transient UPR

It is known that beta cell proliferation expands the beta cell mass during development and under certain hyperglycemic conditions in the adult, a process that may be used for beta cell regeneration in diabetes. Here, through a new high-throughput screen using a luminescence ubiquitination-based cell cycle indicator (LUCCI) in zebrafish, we identify HG-9-91-01 as a driver of proliferation and confirm this effect in mouse and human beta cells. HG-9-91-01 is an inhibitor of salt-inducible kinases (SIKs), and overexpression of Sik1 specifically in beta cells blocks the effect of HG-9-91-01 on beta cell proliferation. Single-cell transcriptomic analyses of mouse beta cells demonstrate that HG-9-91-01 induces a wave of activating transcription factor (ATF)6-dependent unfolded protein response (UPR) before cell cycle entry. Importantly, the UPR wave is not associated with an increase in insulin expression. Additional mechanistic studies indicate that HG-9-91-01 induces multiple signalling effectors downstream of SIK inhibition, including CRTC1, CRTC2, ATF6, IRE1 and mTOR, which integrate to collectively drive beta cell proliferation. A high-throughput chemical screen identifies the salt-inducible kinase inhibitor HG-9-91-01 as a driver of beta cell proliferation, acting through an ATF6-dependent unfolded protein response

Nitrate-NRT1.1B-SPX4 cascade integrates nitrogen and phosphorus signalling networks in plants

To ensure high crop yields in a sustainable manner, a comprehensive understanding of the control of nutrient acquisition is required. In particular, the signalling networks controlling the coordinated utilization of the two most highly demanded mineral nutrients, nitrogen and phosphorus, are of utmost importance. Here, we reveal a mechanism by which nitrate activates both phosphate and nitrate utilization in rice (Oryza sativa L.). We show that the nitrate sensor NRT1.1B interacts with a phosphate signalling repressor SPX4. Nitrate perception strengthens the NRT1.1B-SPX4 interaction and promotes the ubiquitination and degradation of SPX4 by recruiting NRT1.1B interacting protein 1 (NBIP1), an E3 ubiquitin ligase. This in turn allows the key transcription factor of phosphate signalling, PHR2, to translocate to the nucleus and initiate the transcription of phosphorus utilization genes. Interestingly, the central transcription factor of nitrate signalling, NLP3, is also under the control of SPX4. Thus, nitrate-triggered degradation of SPX4 activates both phosphate- and nitrate-responsive genes, implementing the coordinated utilization of nitrogen and phosphorus