HNF1<em>β</em> Is Essential for Nephron Segmentation during Nephrogenesis

Richard Naylor

Aneta Przepiorski

Qun Ren

Jing Yu

Alan Davidson

Supplementary Material

Supplemental Data:

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^{Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand; and}

^{Department of Cell Biology, University of Virginia, Charlottesville, Virginia}

^{Corresponding author.}

Correspondence: Dr. Alan J. Davidson, Department of Molecular Medicine and Pathology, University of Auckland, Auckland 1142, New Zealand. Email: zn.ca.dnalkcua@nosdivad.a

Received 2012 Jul 31; Accepted 2012 Oct 9.

Abstract

Nephrons comprise a blood filter and an epithelial tubule that is subdivided into proximal and distal segments, but what directs this patterning during kidney organogenesis is not well understood. Using zebrafish, we found that the HNF1β paralogues hnf1ba and hnf1bb, which encode homeodomain transcription factors, are essential for normal segmentation of nephrons. Embryos deficient in hnf1ba and hnf1bb did not express proximal and distal segment markers, yet still developed an epithelial tubule. Initiating hnf1ba/b expression required Pax2a and Pax8, but hnf1ba/b-deficient embryos did not exhibit the expected downregulation of pax2a and pax8 at later stages of development, suggesting complex regulatory loops involving these molecules. Embryos deficient in hnf1ba/b also did not express the irx3b transcription factor, which is responsible for differentiation of the first distal tubule segment. Reciprocally, embryos deficient in irx3b exhibited downregulation of hnf1ba/b transcripts in the distal early segment, suggesting a segment-specific regulatory circuit. Deficiency of hnf1ba/b also led to ectopic expansion of podocytes into the proximal tubule domain. Epistasis experiments showed that the formation of podocytes required wt1a, which encodes the Wilms’ tumor suppressor-1 transcription factor, and rbpj, which encodes a mediator of canonical Notch signaling, downstream or parallel to hnf1ba/b. Taken together, these results suggest that Hnf1β factors are essential for normal segmentation of nephrons during kidney organogenesis.

Abstract

How renal progenitors differentiate into the different nephron segments is poorly understood. The zebrafish pronephric kidney has emerged as a useful model to study nephrogenesis due to the high degree of cellular and genetic conservation with the mammalian nephron but without a dependency on prior renal structures.¹ The zebrafish pronephros comprises a single midline renal corpuscle attached to two tubules that are segmented into proximal and distal segments, including the proximal convoluted tubule (PCT), proximal straight tubule (PST), distal early tubule (DE), and distal late tubule (DL).²

The zebrafish pronephros arises from the intermediate mesoderm (IM) during development and initially expresses the transcription factor genes pax2a, pax8, and lhx1a.³5 The anteriormost cells adopt a podocyte fate and express genes such as nephrin and podocin, encoding critical components of the glomerular blood filter.⁶ The formation of podocytes is dependent on the activity of the Wilms’ tumor suppressor-1a (Wt1a) transcription factor and Notch signaling, similar to observations in mammals.⁷9 The remaining renal progenitors differentiate into a tubule with distinct proximal and distal segments. This process involves a considerable refinement in the expression domains of early expressed renal genes such as pax2a, and the activation of later-acting genes such as irx3b that is essential for the expression of DE segment-specific genes.¹⁰

In this study, we identified paralogues of hepatocyte nuclear factor-1 beta (hnf1ba and hnf1bb), encoding homeodomain transcription factors, as essential regulators of nephron formation. Zebrafish embryos deficient in hnf1ba/hnf1bb ectopically express podocyte markers in the region where the proximal tubule normally forms. Although an epithelial tubule arises from the remaining IM, it fails to express segment-specific solute transporter genes. Epistasis experiments revealed that hnf1ba/b acts upstream or in parallel to wt1a and Notch signaling to restrict podocyte fate. Taken together, these results implicate Hnf1β factors as key determinants of nephron segmentation that act to restrict podocyte formation and activate segment-specific gene expression programs.

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Acknowledgments

We thank Renee Vivien for assistance with zebrafish and Hilary Holloway for assistance with electron microscopy.

This work was funded by National Institutes of Health R01 grant DK069403-06 and Marsden fund 3626456 to A.J.D.

Acknowledgments

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2012070756/-/DCSupplemental.

Footnotes

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