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Abstract: TH-PO392

Transcriptional Control of Early Nephrogenesis in Xenopus tropicalis

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 600 Development, Stem Cells, and Regenerative Medicine

Authors

  • Ogar, Paulina, Universitat Zurich, Zurich, Zurich, Switzerland
  • Getwan, Maike, Universitat Zurich, Zurich, Zurich, Switzerland
  • Sugano, Yuya, Universitat Zurich, Zurich, Zurich, Switzerland
  • Naert, Thomas, Universiteit Gent, Gent, Belgium
  • Lienkamp, Soeren S., Universitat Zurich, Zurich, Zurich, Switzerland
Background

The transcription factors Pax8, Hnf1b, and Sall1 underpin embryonic kidney development, and are linked to Congenital Anomalies of the Kidney and Urinary Tract (CAKUT). They are localised in distinct regions within the ureteric bud epithelium, and metanephric mesenchyme, which interact to orchestrate kidney formation. Intriguingly, however, these factors are roughly co-expressed within the same developing pronephric region in Xenopus. Further understanding of their functions could unveil insights into the transcriptome underlying nephrogenesis.

Methods

We visualised cells in the developing pronephros, by confocal microscopy. Injections were combined with CRISPR/Cas9 knock-out (KO) of pax8, hnf1b, or sall1 to analyse effects on kidney morphology. Termed single embryo transcriptomics (seRNA-seq), we performed CRISPR/Cas9 KO of pax8, hnf1b and sall1 in individual embryos and bulk RNA-seq at an early stage. To filter for renal genes, seRNA-seq was intersected with a kidney-enriched X. tropicalis dataset. Localisation of seRNA-seq targets was performed using in situ hybridisation (ISH).

Results

We characterised pronephros development in molecular detail, highlighting key stages, from mesenchyme condensation to epithelialisation. KO of pax8, hnf1b, or sall1 disrupted size, cell organisation and the basement membrane, underscoring their importance in proper pronephros formation. Considering this, we explored their roles in the transcriptional network using CRISPR/Cas9 and seRNA-seq. Renal and morphogenetic genes were identified (e.g. slc12a1 up- and downregulated in pax8 and hnf1b KO respectively, twist1 upregulated in pax8 and hnf1b KO). Extracellular matrix genes (lama1, col4a6) were downregulated in the sall1 KO. ISH validation of seRNA-seq revealed expression of several targets within the early and developed pronephros (e.g. agpat3, traf4, and epha4).

Conclusion

Pax8, Hnf1b and Sall1 are key regulators of nephrogenesis, and influence a network of renal and morphogenetic targets in X. tropicalis. Clarifying their roles will improve understanding of inherited kidney malformations and disease.