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The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros.

ABSTRACT: Kidney function depends on the nephron, which comprises a blood filter, a tubule that is subdivided into functionally distinct segments, and a collecting duct. How these regions arise during development is poorly understood. The zebrafish pronephros consists of two linear nephrons that develop from the intermediate mesoderm along the length of the trunk. Here we show that, contrary to current dogma, these nephrons possess multiple proximal and distal tubule domains that resemble the organization of the mammalian nephron. We examined whether pronephric segmentation is mediated by retinoic acid (RA) and the caudal (cdx) transcription factors, which are known regulators of segmental identity during development. Inhibition of RA signaling resulted in a loss of the proximal segments and an expansion of the distal segments, while exogenous RA treatment induced proximal segment fates at the expense of distal fates. Loss of cdx function caused abrogation of distal segments, a posterior shift in the position of the pronephros, and alterations in the expression boundaries of raldh2 and cyp26a1, which encode enzymes that synthesize and degrade RA, respectively. These results suggest that the cdx genes act to localize the activity of RA along the axis, thereby determining where the pronephros forms. Consistent with this, the pronephric-positioning defect and the loss of distal tubule fate were rescued in embryos doubly-deficient for cdx and RA. These findings reveal a novel link between the RA and cdx pathways and provide a model for how pronephric nephrons are segmented and positioned along the embryonic axis.

SUBMITTER: Wingert RA

PROVIDER: S-EPMC2042002 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that cyp26a1 in zebrafish and its paralog Cyp26b1 in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish.

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The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros.

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