Caudal migration and proliferation of renal progenitors regulates early nephron segment size in zebrafish

Abstract

The nephron is the functional unit of the kidney and is divided into distinct proximal and distal segments. The factors determining nephron segment size are not fully understood. In zebrafish, the embryonic kidney has long been thought to differentiate in situ into two proximal tubule segments and two distal tubule segments (distal early; DE, and distal late; DL) with little involvement of cell movement. Here, we overturn this notion by performing lineage-labelling experiments that reveal extensive caudal movement of the proximal and DE segments and a concomitant compaction of the DL segment as it fuses with the cloaca. Laser-mediated severing of the tubule, such that the DE and DL are disconnected or that the DL and cloaca do not fuse, results in a reduction in tubule cell proliferation and significantly shortens the DE segment while the caudal movement of the DL is unaffected. These results suggest that the DL mechanically pulls the more proximal segments, thereby driving both their caudal extension and their proliferation. Together, these data provide new insights into early nephron morphogenesis and demonstrate the importance of cell movement and proliferation in determining initial nephron segment size.

Figure 1. Lineage labelling of the intermediate mesoderm.

(A) Panels show dorsal views of flat mounted embryos double stained for pax2a, pax8 and hnf1ba (purple) and myod1 (red) transcripts at the 0-2-somite and 5-6-somite stages. The caudalmost limit of the hnf1ba expression domain in the intermediate mesoderm is indicated with a black arrow. (B) Panels show the results of uncaging a lineage tracer in cells posterior to the end of the notochord (first panel) and the descendants of the labelled cells (purple) compared to the unlabelled cdh17-expressing kidney tubules (red; second and third panels, arrow in third panel labels cloaca cells). The fourth panel shows a cross-section, as indicated by the grey dotted line, of the embryo in panel two (red staining has been lost, but red dotted lines highlight the pronephric tubules). (C) Left panel shows lineage labelling of the ‘horseshoe’ domain at the 5-somite stage (fluorescence observed in the more rostral midline is background). Middle panel shows their contribution to non-renal cell types at 24 hpf by whole mount in situ hybridization. Labelled ventral fin cells are highlighted with black arrows and a population of mesenchyme lateral to the posterior pronephros is indicated with a white arrow, which is also highlighted in the cross-section view in the right panel. (D) Panels show lineage labelling of cloacal precursors (white arrow) located in a small cluster of cells near the end of the intermediate mesoderm. Images in (B,C,D) are shown in lateral view.

Figure 2. Expression analysis of segment formation in the pronephros.

Panels show dorsal views of embryos double stained for slc4a4 (red)/slc12a1 (purple) and slc4a4 (red)/slc12a3 (purple) at the stages indicated. The position of initial slc12a1 expression is highlighted with a black arrow and a schematic representation of the pronephros at 24 hpf is shown in the top left. Abbreviations; PCT, Proximal convoluted tubule; PST, Proximal Straight tubule; DE, Distal Early tubule; DL, Distal Late segment.

Figure 3. Lineage labelling of renal precursors.

(A) Dorsal views of flat-mounted embryos that are double stained for slc4a4 (purple) and myod1/smyhc1 (red) are shown at the indicated stages. (B) Panels show uncaging of a lineage tracer in the intermediate mesoderm lateral to somite 8 at the 12-somite stage and tracking of these cells posteriorly (white arrows) up to the 24 hpf stage (upper panels show live brightfield images, middle panels show live fluorescent images). Lower panels show lateral views of the trunk from the same fate-mapped embryo double stained for the lineage tracer (‘anti-flu’; purple) and slc12a1 (red).

Figure 4. Live morphology of the pronephric tubule.

(A,B) Top panels show a lateral view of the pronephric tubule in live Tg(cdh17:egfp) embryos at 24 hpf treated with either DMSO (A) or HUA (B). The two panels below show the same embryos with higher magnification views of the proximal (left) and distal (right) tubule. The bottom panels are single plane lateral views of Hnf1b stained pronephric nuclei in the proximal (left) and distal (right) tubule. (C) Histogram representing the inter-nuclear distances observed in the proximal and distal tubules of DMSO (control) and HUA treated embryos.

Figure 5. Effects of HUA treatment on tubulogenesis and caudal migration.

(A) Left panel shows the PST region and the right panel shows the DL region of an embryo labelled with EdU (red) and Hnf1b (green) at the 24 hpf stage. (B) Transverse sections of Tg(cdh17:egfp) embryos treated with HUA and DMSO and stained with Phalloidin to label F-actin and DAPI to label nuclei. (C) Panels show lateral views of HUA and DMSO treated embryos double stained for cdh17 and uncaged fluorescein (lineage tracer) that was labelled in the intermediate mesoderm lateral to somite 8 at the 12-somite stage. (D) Oblique lateral views of embryos treated with HUA and DMSO (control) and analysed for pax2a, slc4a4, slc12a1 and slc12a3 transcripts (black arrowheads indicate the anteriormost position of the DL segment).

Figure 6. Laser ablation of the DL segment reduces DE segment size.

(A–E) Dorsal views of the trunk of 24 hpf embryos that have undergone laser ablation of the intermediate mesoderm at the 12-somite stage at the indicated positions and double-stained for cdh17 (red) and slc12a1 (purple) transcripts. Manual cell counts of slc12a1⁺ cells are shown as dot-plots to the right.

Figure 7. DL caudal compaction regulates proximal tubule proliferation and segment size.

(A) Top panels show low magnification lateral views of the same embryo labelled with Hnf1b. Asterix denotes the site of ablation. Bottom panels show close-up views of the highlighted region of the ablated and contralateral control side of the embryo. (B) Histogram of the percentage of Hnf1b nuclei that are EdU⁺ in the control non-ablated side and ablated side of the tubule in 6 embryos. (C) Panels show lateral views of 24 hpf embryos treated with LY294002 or DMSO (control) and labelled with EdU from the 12-somite stage to 24 hpf. (D) Histogram of the percentage of Hnf1b nuclei that are EdU⁺ in control and LY294002 treated embryos.