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Hnf4a-mediated regulation of proximal tubule progenitors in the mouse kidney

Sierra S. Marable, Eunah Chung, Joo-Seop Park

Preprint posted on February 17, 2020 https://www.biorxiv.org/content/10.1101/2020.02.16.951731v1

Tracking down proximal tubule progenitors: Marable et al develop lineage tracing and a tissue-specific Hnf4a mutant to elucidate downstream mechanisms at play during kidney development

Selected by Brooke Chambers

Background:

The kidneys are a pair of homeostatic organs that participate in blood filtration, toxin removal, fluid regulation, electrolyte balance, and acid-base control. To complete these tasks, this organ employs thousands of microscopic functional units called nephrons. Each nephron is comprised of a glomerulus (blood filter), a segmented tubule, and a collecting duct. The nephron tubule encompasses a sequence of proximal, intermediate and distal segments, each which execute distinct physiological functions via ion channel proteins. In particular, the proximal tubules are the major site of reabsorption in the nephron and therefore exhibit high metabolic activity (Zhuo & Li, 2013). Despite the functional importance of proximal tubules, the genetic signals necessary for the development of these epithelial cells remain poorly understood. To this end, current single-cell RNA sequencing initiatives of developing kidney tissue have shed light on segment-specific nephron regulators (Lindström et al., 2018; Menon et al., 2018; Combes et al., 2019). These comprehensive transcriptomic profiles provide a great launching point for functional validation studies of top genetic candidates in a vertebrate model system.

Experimental System:

The authors developed a new Hnf4a knockout mouse model using Osr2Cre to conditionally delete Hnf4a in developing proximal-intermediate nephron progenitors. Mutant kidney sections were phenotyped by immunofluorescence for proximal tubule markers: LTL (Lotus Tetragonolobus Lectin), Lrp2 (low-density lipoprotein-related protein 2), and Cdh6 (Cadherin-6). Lineage tracing experiments were conducted by a tamoxifen-inducible Cre recombinase system driven by the Cdh6 promoter (Cdh6CreER) paired with the Cre-inducible Rosa26Ai3 reporter. Chromatin immunoprecipitation followed by high-throughput sequencing (CHIP-seq) and RNA-sequencing of postnatal day 0 (P0) mouse kidneys were completed to identify downstream proximal tubule genetic regulatory network candidates.

Key Findings:

Hnf4a is necessary for proximal tubule differentiation and postnatal survival

Analysis of Hnf4a mutant kidneys revealed decreased expression of mature proximal tubule markers LTL and Lrp2. Significantly, these mutant cells lacked an apical brush border, which is a post-mitotic feature required for proximal tubule function, specifically reabsorption of molecules and solutes. Further, histological examination of mutant kidneys indicated severely damaged tissue, cortical cysts, and hydronephrosis. These renal phenotypes caused significant postnatal lethality, where no Hnf4a mutant pups survived to weaning age. The authors’ findings corroborate the importance of proximal tubule formation for survival. The Hnf4a mutant mice in this study phenocopy many of the key clinical manifestations of human Fanconi renotubular syndrome (FRTS), which encompasses generalized proximal tubule dysfunction. To this point, a previous study found a heterozygous mutation in the DNA-binding domain of human HNF4A triggers atypical dominant FRTS (Hamilton et al., 2014). Therefore, the Hnf4a;Osr2Cre mouse system provides the opportunity to glean insight into mechanisms that contribute to human FRTS.

Nephron progenitors expressing high levels of Cdh6 and low levels of LTL are assigned to a proximal tubule lineage

Previous studies have suggested that Cdh6-expressing cells in the developing mouse kidney are proximal tubule precursors (Cho et al., 1998), however, this idea had not been conclusively tested until this study. The authors identified two discrete types of Cdh6+ cells in developing wild type kidneys: Cdh6high and Cdh6low. Interestingly, the majority of Cdh6high cells coexpressed Hnf4a but exhibited scarce LTL expression indicating this signature likely correlates to a progenitor cell state. Consistent with these findings, Hnf4a mutant kidneys contained elevated Cdh6highLTLlow (immature) cells and significantly decreased Cdh6lowLTLhigh (differentiated) cells. To determine if Cdh6high cells are in fact proximal tubule precursors, the researchers performed lineage tracing experiments utilizing their Rosa26Ai3/+;Cdh6CreER/+ line. After tamoxifen treatment, the labeled Cdh6high progeny expressed both Hnf4a and LTL, confirming for the first time that Cdh6-expressing cells are bona-fide proximal tubule progenitors. Furthermore, Cdh6high cells were found to have a 10-fold higher proliferative rate than their Cdh6low counterparts. In sum, this data fortifies Cdh6high cells comprise the proximal tubule progenitor pool and undergo a wave of proliferation before terminally differentiating.

Hnf4a controls a genetic regulatory network that programs proximal tubule development

To identify downstream targets of Hnf4a, the researchers harvested P0 mouse kidney tissue and performed ChIP-seq experiments followed by high-throughput sequencing analysis. Hnf4a binding peaks were identified adjacent to promoters controlling proximal tubule genes, such as Slc34a1 and Ehhadh, which have been linked to human FRTS. Interestingly, the authors’ motif analysis suggested other nuclear receptors (ESRRA, RXR, PPAR, and HNF1B) likely bind similar renal targets as Hnf4a. RNA-sequencing of Hnf4a mutant kidneys revealed an inventory of downregulated genes associated with proximal tubule function. The authors cross-referenced differentially expressed genes from their RNA-seq experiment with the previously identified ChIP-seq binding regions. Overall, their integration of genomic and transcriptomic data substantiate Hnf4a regulates proximal tubule differentiation by controlling a repertoire of genes involved in transmembrane transport and cellular metabolism.

What I like about this paper:

I wanted to highlight this study because I believe it advances the field’s knowledge, not only about the role of a particular transcription factor (Hnf4a), but it also employs a new experimental mouse system to track proximal tubule progenitors. Previous studies proposed that Cdh6+ cells are putative proximal tubule precursors in the developing kidney. Importantly, this group tested this circulating idea and performed compelling lineage analysis experiments that unveiled the molecular signature of this progenitor population. I also admired how the researchers integrated their ChIP-seq and RNA-seq data sets to pinpoint promising Hnf4a targets. This paper introduces exciting directions for functional studies by providing a new collection of proximal tubule differentiation genes.

Open Questions:

  1. Based on your motif analysis, you provide exciting evidence that nuclear receptor transcription factors (ie. ESRRA, RXR, PPAR) may participate in cooperative binding with Hnf4a in developing proximal tubules. What are your thoughts on potentially conducting in vitro competition assays or other experiments to determine if this is the case?
  2. You mention that the Osr2 Cre system also deletes Hnf4a from the developing loops of Henle. From your ChIP-seq and transcriptomic analyses is there any indication that Hnf4a regulates genes specific to the loop of Henle program?

References:

Zhuo, J. L., & Li, X. C. (2013). Proximal nephron. Comprehensive Physiology, 3(3), 1079-1123.

Combes, A. N., Phipson, B., Lawlor, K. T., Dorison, A., Patrick, R., Zappia, L., … & Little, M. H. (2019). Single cell analysis of the developing mouse kidney provides deeper insight into marker gene expression and ligand-receptor crosstalk. Development, 146(12), dev178673.

Lindström, N. O., Brandine, G. D. S., Tran, T., Ransick, A., Suh, G., Guo, J., … & Thornton, M. E. (2018). Progressive recruitment of mesenchymal progenitors reveals a time-dependent process of cell fate acquisition in mouse and human nephrogenesis. Developmental cell, 45(5), 651-660.

Menon, R., Otto, E. A., Kokoruda, A., Zhou, J., Zhang, Z., Yoon, E., … & Cebrián, C. (2018). Single-cell analysis of progenitor cell dynamics and lineage specification in the human fetal kidney. Development, 145(16), dev164038.

Hamilton, A. J., Bingham, C., McDonald, T. J., Cook, P. R., Caswell, R. C., Weedon, M. N., … & Hamilton-Shield, J. P. (2014). The HNF4A R76W mutation causes atypical dominant Fanconi syndrome in addition to a β cell phenotype. Journal of medical genetics, 51(3), 165-169.

Cho, E. A., Patterson, L. T., Brookhiser, W. T., Mah, S., Kintner, C., & Dressler, G. R. (1998). Differential expression and function of cadherin-6 during renal epithelium development. Development, 125(5), 803-812.

Tags: developmental biology, genetics, kidney, nephron

Posted on: 10th March 2020

doi: Pending

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