Endothelial versus pronephron fate decision is modulated by the transcription factors Cloche/Npas4l, Tal1, and Lmo2

Abstract

Endothelial specification is a key event during embryogenesis; however, when, and how, endothelial cells separate from other lineages is poorly understood. In zebrafish, Npas4l is indispensable for endothelial specification by inducing the expression of the transcription factor genes etsrp , tal1 , and lmo2 . We generated a knock-in reporter in zebrafish npas4l to visualize endothelial progenitors and their derivatives in wild-type and mutant embryos. Unexpectedly, we find that in npas4l mutants, npas4l reporter–expressing cells contribute to the pronephron tubules. Single-cell transcriptomics and live imaging of the early lateral plate mesoderm in wild-type embryos indeed reveals coexpression of endothelial and pronephron markers, a finding confirmed by creERT2-based lineage tracing. Increased contribution of npas4l reporter–expressing cells to pronephron tubules is also observed in tal1 and lmo2 mutants and is reversed in npas4l mutants injected with tal1 mRNA. Together, these data reveal that Npas4l/Tal1/Lmo2 regulate the fate decision between the endothelial and pronephron lineages.

Drivers of Vessel Progenitor Fate Define Intermediate Mesoderm Dimensions by Inhibiting Kidney Progenitor Specification

“…In npas4l mutants, we observed expanded glomerular precursors and pronephron tubules (Fig. 4C), similar to defects seen in hand2 mutants (Perens et al, 2016) and consistent with previously characterized npas4l mutant pronephron phenotypes (Majumdar & Drummond, 1999; Mattonet et al, 2022). Remarkably, in npas4l ; osr1 double mutants, glomerular precursors and pronephron tubules appeared most similar to wild-type embryos (Fig.…”

“…Last, hand2 and osr1 expression mark the lateral territory. Some progenitors within this lateral territory continue to express hand2 and migrate medially (Yin et al, 2010), giving rise to derivatives that include the mesothelium (Prummel et al, 2022). Notably, hand2 is an essential and potent inhibitor of IM fate within this territory; we have only observed ectopic Pax2a expression in this territory in hand2 mutants, but not in npas4l loss-of-function or osr1 overexpression scenarios.…”

“…Interestingly, recent single cell profiling of endothelial progenitors in zebrafish identified a small population that expresses IM markers such as pax2a and osr1 (Mattonet et al, 2022; Sahai-Hernandez et al, 2023), further raising the possibility of a shared progenitor. Additionally, although not direct evidence of a bipotential progenitor, it is intriguing that genetic fate mapping of pax2a -expressing descendants in zebrafish and Osr1 -expressing descendants in mouse labeled both kidney and vasculature (Mattonet et al, 2022; Mugford et al, 2008). Ultimately, high-resolution fate mapping will be necessary to determine the existence of a bipotential progenitor, as well as to determine the fates of the different posterior mesoderm territories and how they are altered in different genetic backgrounds.…”

“…More recently, Mattonet and colleagues found additional factors that promote vessel progenitor formation and also inhibit kidney cell fate (Mattonet et al, 2022). Specifically, they found that npas4l ( cloche ), an early essential regulator of vessel and blood progenitor formation (Liao et al, 1998; Liao et al, 1997; Reischauer et al, 2016; Stainier et al, 1995; Sumanas et al, 2005), along with downstream vessel-promoting factors tal1 and lmo2 , inhibits kidney formation.…”

Hemato-vascular specification requiresarnt1andarnt2genes in zebrafish embryos

“…We asked whether the expression of npas4l , arnt1 and arnt2 was the same in the anterior LPM (ALPM) and posterior LPM (PLPM). npas4l and its associated downstream targets exhibit more expression in the PLPM compared to the ALPM in embryos throughout segmentation (Liao et al, 1997; Liao et al, 2000; Marass et al, 2019; Mattonet et al, 2022; Qian et al, 2005; Stainier et al, 1995). At 4- 6 somite stage embryos, we found that expression of npas4l and arnt2 was greater in the PLPM than in the ALPM, but the expression of arnt1 was similar in both regions ( Figure S3C, S3E, npas4l Welsh’s t- test, p<0.0001; arnt2 Welsh’s t-test, p<0.005 ).…”