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

ABSTRACT: 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 ?-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.

SUBMITTER: Li Y

PROVIDER: S-EPMC3920184 | biostudies-other | 2014 Feb

REPOSITORIES: biostudies-other

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

Li Yue Y Cheng Christina N CN Verdun Valerie A VA Wingert Rebecca A RA

Developmental biology 20131203 1

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 ...[more]

PMID: 24309209

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Project description:Retinoic acid (RA) is known to regulate definitive myelopoiesis but its role in vertebrate primitive myelopoiesis remains unclear. Here we report that zebrafish primitive myelopoiesis is restricted by RA in a dose dependent manner mainly before 11 hpf (hours post fertilization) when anterior hemangioblasts are initiated to form. RA treatment significantly reduces expressions of anterior hemangioblast markers scl, lmo2, gata2 and etsrp in the rostral end of ALPM (anterior lateral plate mesoderm) of the embryos. The result indicates that RA restricts primitive myelopoiesis by suppressing formation of anterior hemangioblasts. Analyses of ALPM formation suggest that the defective primitive myelopoiesis resulting from RA treatment before late gastrulation may be secondary to global loss of cells for ALPM fate whereas the developmental defect resulting from RA treatment during 10-11 hpf should be due to ALPM patterning shift. Overexpressions of scl and lmo2 partially rescue the block of primitive myelopoiesis in the embryos treated with 250 nM RA during 10-11 hpf, suggesting RA acts upstream of scl to control primitive myelopoiesis. However, the RA treatment blocks the increased primitive myelopoiesis caused by overexpressing gata4/6 whereas the abolished primitive myelopoiesis in gata4/5/6 depleted embryos is well rescued by 4-diethylamino-benzaldehyde, a retinal dehydrogenase inhibitor, or partially rescued by knocking down aldh1a2, the major retinal dehydrogenase gene that is responsible for RA synthesis during early development. Consistently, overexpressing gata4/6 inhibits aldh1a2 expression whereas depleting gata4/5/6 increases aldh1a2 expression. The results reveal that RA signaling acts downstream of gata4/5/6 to control primitive myelopoiesis. But, 4-diethylamino-benzaldehyde fails to rescue the defective primitive myelopoiesis in either cloche embryos or lycat morphants. Taken together, our results demonstrate that RA signaling restricts zebrafish primitive myelopoiesis through acting downstream of gata4/5/6, upstream of, or parallel to, cloche, and upstream of scl.

Project description:All-trans-retinoic acid (ATRA) and its synthetic analogues EC23 and EC19 direct cellular differentiation by interacting as ligands for the retinoic acid receptor (RAR?, ? and ?) family of nuclear receptor proteins. To date, a number of crystal structures of natural and synthetic ligands complexed to their target proteins have been solved, providing molecular level snap-shots of ligand binding. However, a deeper understanding of receptor and ligand flexibility and conformational freedom is required to develop stable and effective ATRA analogues for clinical use. Therefore, we have used molecular modelling techniques to define RAR interactions with ATRA and two synthetic analogues, EC19 and EC23, and compared their predicted biochemical activities to experimental measurements of relative ligand affinity and recruitment of coactivator proteins. A comprehensive molecular docking approach that explored the conformational space of the ligands indicated that ATRA is able to bind the three RAR proteins in a number of conformations with one extended structure being favoured. In contrast the biologically-distinct isomer, 9-cis-retinoic acid (; 9CRA), showed significantly less conformational flexibility in the RAR binding pockets. These findings were used to inform docking studies of the synthetic retinoids EC23 and EC19, and their respective methyl esters. EC23 was found to be an excellent mimic for ATRA, and occupied similar binding modes to ATRA in all three target RAR proteins. In comparison, EC19 exhibited an alternative binding mode which reduces the strength of key polar interactions in RAR?/? but is well-suited to the larger RAR? binding pocket. In contrast, docking of the corresponding esters revealed the loss of key polar interactions which may explain the much reduced biological activity. Our computational results were complemented using an in vitro binding assay based on FRET measurements, which showed that EC23 was a strongly binding, pan-agonist of the RARs, while EC19 exhibited specificity for RAR?, as predicted by the docking studies. These findings can account for the distinct behaviour of EC23 and EC19 in cellular differentiation assays, and additionally, the methods described herein can be further applied to the understanding of the molecular basis for the selectivity of different retinoids to RAR?, ? and ?.

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

Publications

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

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