The stem-like STAT3-responsive cells of zebrafish intestine are WNT/β-catenin dependent

Abstract

The transcription factor Stat3 is required for proliferation and pluripotency of embryonic stem cells; we have prepared and characterized fluorescent Stat3-reporter zebrafish based on repeats of minimal responsive elements. These transgenic lines mimic in vivo Stat3 expression patterns and are responsive to exogenous Stat3; notably, fluorescence is inhibited by both stat3 knockout and IL6/Jak/ STAT inhibitors. At larval stages, Stat3 reporter activity correlates with proliferating regions of the brain, haematopoietic tissue and intestine. In the adult gut, the reporter is active in sparse proliferating cells, located at the base of intestinal folds, expressing the stemness marker sox9b and having the morphology of mammalian crypt base columnar cells; noteworthy, zebrafish stat3 mutants show defects in intestinal folding. Stat3 reporter activity in the gut is abolished with mutation of T cell factor 4 (Tcf7l2), the intestinal mediator of Wnt/βcatenin-dependent transcription. The Wnt/β-catenin dependence of Stat3 activity in the gut is confirmed by abrupt expansion of Stat3positive cells in intestinal adenomas of apc heterozygotes. Our findings indicate that Jak/Stat3 signalling is needed for intestinal stem cell maintenance and possibly crucial in controlling Wnt/β-catenindependent colorectal cancer cell proliferation.

Y705 and S727 are required for mitochondrial import and transcriptional activities of STAT3 and regulate proliferation of embryonic and tissue stem cells

“…8A). The activity of Stat3 in the intestine of zebrafish is consistent with the facts that a) the proliferative and survival effects of IL-6 in murine IECs (intestinal epithelial cells) is largely mediated by STAT3 (Grivennikov et al, 2009), b) that STAT3 is needed for small-intestine crypt stem cell survival, as revealed by conditional mutant mice (Matthews et al, 2011), and c) that Stat3 positive cells in zebrafish intestine represent a population of intestinal Wnt responsive stem cells (Peron et al, 2020). Administration of 60 µM AG490 between 3 and 6 dpf was able to significantly reduce mitochondrial transcription in the intestine of treated larvae with respect to DMSO treated controls (Fig.…”

“…To overcome this issue and to determine whether genetic ablation of stat3 alters mitochondrial transcription and cell proliferation in 48-hpf larvae, we decided to analyse stat3 “CRISPants” generated after injection of Cas9 protein and sgRNAs which target the antisense strand of exons 14, 22 and 23 of stat3 gene: this approach is employed to target a gene and silence its RNA transcription, according to the principles of classic CRISPR/Cas9 (Strutt et al, 2018). We evaluated the efficiency of stat3 targeting by injection of the Tg(7xCRP-Hu.tk:EGFP) reporter line characterized in Peron et al (2020). Results show that Stat3-dependent fluorescence of reporters is significantly dampened in CRISPants when compared to control larvae (Fig.…”

“…To confirm data obtained by endogenous Stat3 chemical treatment with either MEK and Jak2 inhibitors, we used the stat3 ia23 mutant (from now on called stat3 -/-) (Peron et al, 2020), which is predicted to encode a premature stop codon at amino acid 456, thus lacking all functional domains including the dimerization domain and the transactivation domain, harboring Y705 and S727 phosphorylation sites, respectively. Consistently with our previous results, Stat3 genetic ablation caused a significant and clear reduction of both mt_nd2 and pcna transcripts, endorsing the link between Stat3 mitochondrial functions and its role in regulating cell proliferation in different tissues.…”

“…All experimental procedures complied with European Legislation for the Protection of Animals used for Scientific Purposes (Directive 2010/63/EU). stat3 ia23 mutants are described in Peron et al (2020). Tg(CoxVIII-mls:EGFP) transgenic zebrafish line is described in Martorano et al (2019).…”

“…Notably, zebrafish mutants lacking maternal and zygotic Stat3 expression display transient axis elongation defects due to reduced cell proliferation during embryogenesis (Liu et al , 2017). Additionally, it has been demonstrated that in zebrafish Stat3 is transcriptionally active in stem cells of highly proliferative tissues like Tectum Opticum (TeO), hematopoietic tissue and intestine (Peron et al , 2020).…”