Depletion of Tissue-Specific Ion Transporters Causes Differential Expression of PRL Targets in Response to Increased Levels of Endogenous PRL.

Prolactin (PRL) has been considered a key regulator of ion uptake in zebrafish. The genes slc12a10.2 and slc12a3, which are Na+ and chloride Cl- co-transporters, have been reported to be regulated by PRL in freshwater fish. The integrative network of PRL signaling dissected from the knockout of tissue-specific downstream PRL ion transporters remains poor. In the present study, zebrafish models with increased endogenous levels of PRL were generated through the knockout of slc12a10.2 or slc12a3, and the developmental consequences were analyzed. The increased levels of pituitary PRL were observed in both slc12a10.2- and slc12a3-deficient fish. Unlike the slc12a3-deficient fish, which could survive to adulthood, the slc12a10.2-deficient fish began to die at 9 days post-fertilization (dpf) and did not survive beyond 17 dpf. This survival defect is a result of defective Cl- uptake in this mutant, indicating that Slc12a10.2 plays an essential role in Cl- uptake. Intriguingly, compared to the levels in control fish, no significant differences in the levels of Na+ in the body were observed in slc12a10.2- or slc12a3-deficient zebrafish. The upregulations of the PRL downstream transporters, slc9a3.2, slc12a10.2, and atp1a1a.5 were observed in slc12a3-deficient fish in both the gills/skin and the pronephric duct. However, this type of response was not observed in the pronephric duct of slc12a10.2-deficient fish, except under Na+-deprived conditions. Our results show that PRL is susceptible to deficiencies in downstream ion transporters. Moreover, both the gills/skin and pronephric duct show differential expression of downstream PRL targets in response to increased levels of pituitary PRL caused by the depletion of tissue-specific ion transporters.

Expression patterns of dnmt3aa, dnmt3ab, and dnmt4 during development and fin regeneration in zebrafish.

Epigenetic modifications such as DNA methylation and chromatin modifications are critical for regulation of spatiotemporal gene expression during development. In mammals, the de novo-type DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. In addition to developmental processes, we recently showed that DNA methylation levels are dynamically changed during zebrafish fin regeneration, suggesting that the de novo-type Dnmts might play roles in the regulation of gene expression during regeneration processes. Here, we showed the detailed expression profiles of three zebrafish dnmt genes (dnmt3aa, dnmt3ab, and dnmt4), which were identified as the orthologues of mammalian dnmt3a and dnmt3b, during embryonic and larval development, as well as fin regeneration processes. dnmt3aa and dnmt3ab are expressed in the brain, pharyngeal arches, pectoral fin buds, intestine, and swim bladder; the specific expression of dnmt3aa is observed in the pronephric duct during larval development. dnmt4 expression is observed in the zona limitans intrathalamica, midbrain-hindbrain boundary, ciliary marginal zone, pharyngeal arches, auditory capsule, pectoral fin buds, intestine, pancreas, liver, and hematopoietic cells in the aorta-gonad-mesonephros and caudal hematopoietic tissue from 48 to 72 h post-fertilization. Furthermore, during fin regeneration, strong dnmt3aa expression, and faint dnmt3ab and dnmt4 expression are detected in blastema cells at 72 h post-amputation. Taken together, our results suggest that zebrafish Dnmt3aa, Dnmt3ab, and Dnmt4 may play roles in the formation of various organs, such as the brain, kidney, digestive organs, and/or hematopoietic cells, as well as in the differentiation of blastema cells.

Zebrafish nephrogenesis is regulated by interactions between retinoic acid, mecom, and Notch signaling.

The zebrafish pronephros provides a conserved model to study kidney development, in particular to delineate the poorly understood processes of how nephron segment pattern and cell type choice are established. Zebrafish nephrons are divided into distinct epithelial regions that include a series of proximal and distal tubule segments, which are comprised of intercalated transporting epithelial cells and multiciliated cells (MCC). Previous studies have shown that retinoic acid (RA) regionalizes the renal progenitor field into proximal and distal domains and that Notch signaling later represses MCC differentiation, but further understanding of these pathways has remained unknown. The transcription factor mecom (mds1/evi1 complex) is broadly expressed in renal progenitors, and then subsequently marks the distal tubule. Here, we show that mecom is necessary to form the distal tubule and to restrict both proximal tubule formation and MCC fate choice. We found that mecom and RA have opposing roles in patterning discrete proximal and distal segments. Further, we discovered that RA is required for MCC formation, and that one mechanism by which RA promotes MCC fate choice is to inhibit mecom. Next, we determined the epistatic relationship between mecom and Notch signaling, which limits MCC fate choice by lateral inhibition. Abrogation of Notch signaling with the γ-secretase inhibitor DAPT revealed that Notch and mecom did not have additive effects in blocking MCC formation, suggesting that they function in the same pathway. Ectopic expression of the Notch signaling effector, Notch intracellular domain (NICD), rescued the expansion of MCCs in mecom morphants, indicating that mecom acts upstream to induce Notch signaling. These findings suggest a model in which mecom and RA arbitrate proximodistal segment domains, while MCC fate is modulated by a complex interplay in which RA inhibition of mecom, and mecom promotion of Notch, titrates MCC number. Taken together, our studies have revealed several essential and novel mechanisms that control pronephros development in the zebrafish.