Dataset Information

Iroquois transcription factor irx2a is required for multiciliated and transporter cell fate decisions during zebrafish pronephros development.

ABSTRACT: The genetic regulation of nephron patterning during kidney organogenesis remains poorly understood. Nephron tubules in zebrafish are composed of segment populations that have unique absorptive and secretory roles, as well as multiciliated cells (MCCs) that govern fluid flow. Here, we report that the transcription factor iroquois 2a (irx2a) is requisite for zebrafish nephrogenesis. irx2a transcripts localized to the developing pronephros and maturing MCCs, and loss of function altered formation of two segment populations and reduced MCC number. Interestingly, irx2a deficient embryos had reduced expression of an essential MCC gene ets variant 5a (etv5a), and were rescued by etv5a overexpression, supporting the conclusion that etv5a acts downstream of irx2a to control MCC ontogeny. Finally, we found that retinoic acid (RA) signaling affects the irx2a expression domain in renal progenitors, positioning irx2a downstream of RA. In sum, this work reveals new roles for irx2a during nephrogenesis, identifying irx2a as a crucial connection between RA signaling, segmentation, and the control of etv5a mediated MCC formation. Further investigation of the genetic players involved in these events will enhance our understanding of the molecular pathways that govern renal development, which can be used help create therapeutics to treat congenital and acquired kidney diseases.

SUBMITTER: Marra AN

PROVIDER: S-EPMC6478698 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Iroquois transcription factor irx2a is required for multiciliated and transporter cell fate decisions during zebrafish pronephros development.

Marra Amanda N AN Cheng Christina N CN Adeeb Basma B Addiego Amanda A Wesselman Hannah M HM Chambers Brooke E BE Chambers Joseph M JM Wingert Rebecca A RA

Scientific reports 20190423 1

The genetic regulation of nephron patterning during kidney organogenesis remains poorly understood. Nephron tubules in zebrafish are composed of segment populations that have unique absorptive and secretory roles, as well as multiciliated cells (MCCs) that govern fluid flow. Here, we report that the transcription factor iroquois 2a (irx2a) is requisite for zebrafish nephrogenesis. irx2a transcripts localized to the developing pronephros and maturing MCCs, and loss of function altered formation o ...[more]

PMID: 31015532

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Project description:The simplified and genetically conserved zebrafish pronephros is an excellent model to examine the cryptic processes of cell fate decisions during the development of nephron segments as well as the origins of associated endocrine cells that comprise the corpuscles of Stannius (CS). Using whole mount in situ hybridization, we found that transcripts of the zebrafish genes t-box 2a (tbx2a) and t-box 2b (tbx2b), which belong to the T-box family of transcription factors, were expressed in the caudal intermediate mesoderm progenitors that give rise to the distal pronephros and CS. Deficiency of tbx2a, tbx2b or both tbx2a/b reduced the size of the distal late (DL) segment, which was accompanied by a proximal convoluted segment (PCT) expansion. Further, tbx2a/b deficiency led to significantly larger CS clusters. These phenotypes were also observed in embryos with the from beyond (fby)c144 mutation, which encodes a premature stop codon in the tbx2b T-box sequence. Conversely, overexpression of tbx2a and tbx2b in wild-type embryos expanded the DL segment where cells were comingled with the adjacent DE, and also decreased CS cell number, but notably did not alter PCT development-providing independent evidence that tbx2a and tbx2b are each necessary and sufficient to promote DL fate and suppress CS genesis. Epistasis studies indicated that tbx2a acts upstream of tbx2b to regulate the DL and CS fates, and likely has other targets as well. Retinoic acid (RA) addition and inhibition studies revealed that tbx2a and tbx2b are negatively regulated by RA signaling. Interestingly, the CS cell expansion that typifies tbx2a/b deficiency also occurred when blocking Notch signaling with the chemical DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). Ectopic activation of Notch in Tg(hsp70::Gal4; UAS::NICD)(NICD) embryos led to a reduced CS post heat-shock induction. To further examine the link between the tbx2a/b genes and Notch during CS formation, DAPT treatment was used to block Notch activity in tbx2a/b deficient embryos, and tbx2a/b knockdown was performed in NICD transgenic embryos. Both manipulations caused similar CS expansions, indicating that Notch functions upstream of the tbx2a/b genes to suppress CS ontogeny. Taken together, these data reveal for the first time that tbx2a/b mitigate pronephros segmentation downstream of RA, and that interplay between Notch signaling and tbx2a/b regulate CS formation, thus providing several novel insights into the genetic regulatory networks that influence these lineages.

Project description:E2f5 is a member of the E2f family of transcription factors that play essential roles during many cellular processes. E2f5 was initially characterized as a transcriptional repressor in cell proliferation studies through its interaction with the Retinoblastoma (Rb) protein for inhibition of target gene transcription. However, the precise roles of E2f5 during embryonic and post-embryonic development remain incompletely investigated. Here, we report that zebrafish E2f5 plays critical roles during spermatogenesis and multiciliated cell (MCC) differentiation. Zebrafish e2f5 mutants develop exclusively as infertile males. In the mutants, spermatogenesis is arrested at the zygotene stage due to homologous recombination (HR) defects, which finally leads to germ cell apoptosis. Inhibition of cell apoptosis in e2f5;tp53 double mutants rescued ovarian development, although oocytes generated from the double mutants were still abnormal, characterized by aberrant distribution of nucleoli. Using transcriptome analysis, we identified dmc1, which encodes an essential meiotic recombination protein, as the major target gene of E2f5 during spermatogenesis. E2f5 can bind to the promoter of dmc1 to promote HR, and overexpression of dmc1 significantly increased the fertilization rate of e2f5 mutant males. Besides gametogenesis defects, e2f5 mutants failed to develop MCCs in the nose and pronephric ducts during early embryonic stages, but these cells recovered later due to redundancy with E2f4. Moreover, we demonstrate that ion transporting principal cells in the pronephric ducts, which remain intercalated with the MCCs, do not contain motile cilia in wild-type embryos, while they generate single motile cilia in the absence of E2f5 activity. In line with this, we further show that E2f5 activates the Notch pathway gene jagged2b (jag2b) to inhibit the acquisition of MCC fate as well as motile cilia differentiation by the neighboring principal cells. Taken together, our data suggest that E2f5 can function as a versatile transcriptional activator and identify novel roles of the protein in spermatogenesis as well as MCC differentiation during zebrafish development.

Dataset Information

Iroquois transcription factor irx2a is required for multiciliated and transporter cell fate decisions during zebrafish pronephros development.

Publications

Iroquois transcription factor irx2a is required for multiciliated and transporter cell fate decisions during zebrafish pronephros development.

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