The estrogen receptor signaling pathway plays an important role in vertebrate embryonic development and sexual differentiation. There are four major estrogen receptors in zebrafish: esr1, esr2a, esr2b and gper. However, the specific role of different estrogen receptors in zebrafish is not clear. To investigate the role of esr2b in zebrafish development and reproduction, this study utilized TALENs technology to generate an esr2b knockout homozygous zebrafish line. The number of eggs laid by esr2b knockout female zebrafish did not differ significantly from that of wild zebrafish. The embryonic development process of wild-type and esr2b knockout zebrafish was observed, revealing a significant developmental delay in the esr2b knockout zebrafish. Additionally, mortality rates were significantly higher in esr2b knockout zebrafish than in their wild-type counterparts at 24 hpf. The reciprocal cross experiment between esr2b knockout zebrafish and wild-type zebrafish revealed that the absence of esr2b resulted in a decline in the quality of zebrafish oocytes, while having no impact on sperm cells. The knockout of esr2b also led to an abnormal sex ratio in the adult zebrafish population, with a female-to-male ratio of approximately 1:7. The quantitative PCR (qPCR) and in situ hybridization results demonstrated a significant downregulation of cyp19ab1b expression in esr2b knockout embryos compared to wild-type embryos throughout development (at 2 dpf, 3 dpf and 4 dpf). Additionally, the estrogen-mediated induction expression of cyp19ab1b was attenuated, while the estradiol-induced upregulated expression of vtg1 was disrupted. These results suggest that esr2b is involved in regulating zebrafish oocyte development and sex differentiation.

Despite significant advances in understanding nephron segment patterning, many questions remain about the underlying genes and signaling pathways that orchestrate renal progenitor cell fate choices and regulate differentiation. In an effort to identify elusive regulators of nephron segmentation, our lab conducted a high-throughput drug screen using a bioactive chemical library and developing zebrafish, which are a conserved vertebrate model and particularly conducive to large-scale screening approaches. 17β-estradiol (E2), which is the dominant form of estrogen in vertebrates, was a particularly interesting hit from this screen. E2 has been extensively studied in the context of gonad development, but roles for E2 in nephron development were unknown. Here, we report that exogenous estrogen treatments affect distal tubule composition, namely, causing an increase in the distal early segment and a decrease in the neighboring distal late. These changes were noted early in development but were not due to changes in cell dynamics. Interestingly, exposure to the xenoestrogens ethinylestradiol and genistein yielded the same changes in distal segments. Further, upon treatment with an estrogen receptor 2 (Esr2) antagonist, PHTPP, we observed the opposite phenotypes. Similarly, genetic deficiency of the Esr2 analog, esr2b, revealed phenotypes consistent with that of PHTPP treatment. Inhibition of E2 signaling also resulted in decreased expression of essential distal transcription factors, irx3b and its target irx1a. These data suggest that estrogenic compounds are essential for distal segment fate during nephrogenesis in the zebrafish pronephros and expand our fundamental understanding of hormone function during kidney organogenesis.

Estradiol (E2) stimulates luteinizing hormone receptor (lhcgr) expression via nuclear estrogen receptors (nERs) in the zebrafish ovary. We have demonstrated that endocrine hormones such as gonadotropin (hCG) and paracrine factors such as epidermal growth factor (EGF) and pituitary adenylate cyclase-activating peptide (PACAP) can modulate E2-induced lhcgr expression in vitro. These observations raised a question on whether these hormones and factors exert their effects via regulating the expression of nERs. In this study, we first characterized the spatiotemporal expression profiles of three nER subtypes in the zebrafish ovary, including esr1 (ERα), esr2a (ERβ2) and esr2b (ERβ1). All three nERs increased their expression at the pre-vitellogenic stage and peaked at mid- (esr1 and esr2a) or late vitellogenic (esr2b) stage, followed by a significant decline at the full-grown stage. RT-PCR analysis showed that esr1 and esr2b were exclusively expressed in the follicle layer while esr2a was expressed in both compartments. We then examined how E2, hCG, PACAP and EGF regulated the expression of nERs in cultured zebrafish follicle cells. E2 quickly increased esr1 but reduced esr2a and esr2b expression from 1.5 to 12h of treatment. Similarly, EGF down-regulated esr2a significantly at 1.5h and this effect was further intensified at 24h. hCG decreased the expression of all three nER subtypes with similar potency throughout the 24-h time-course. Interestingly, PACAP exerted a biphasic regulation on esr2a. Our present study suggests that nERs, especially esr2a, provide potential target points for other hormones and factors to modulate E2 activity during folliculogenesis in the zebrafish.