In toto imaging of early enteric nervous system development reveals that gut colonization is tied to proliferation downstream of Ret

Abstract

The enteric nervous system (ENS) is a vast intrinsic network of neurons and glia within the gastrointestinal tract and is largely derived from enteric neural crest cells (ENCCs) that emigrate into the gut during vertebrate embryonic development. Study of ENCC migration dynamics and their genetic regulators provides great insights into fundamentals of collective cell migration and nervous system formation, and are a pertinent subject for study due to their relevance to the human congenital disease, Hirschsprung disease (HSCR). For the first time, we performed in toto gut imaging and single-cell generation tracing of ENCC migration in WT and a novel ret heterozygous background zebrafish (retwmr1/+) to gain insight into ENCC dynamics in vivo. We observed that retwmr1/+ zebrafish produced fewer ENCCs while localized along the gut, which failed to reach the hindgut, resulting in HSCR-like phenotypes. Specifically, we observed a proliferation dependent migration mechanism, where cell divisions were associated with inter-cell distances and migration speed. Lastly, we detected a premature neuronal differentiation gene expression signature in retwmr1/+ ENCCs, collectively suggesting that Ret signaling may function to regulate maintenance of a stem-state in ENCCs.

A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system

“…Of the twelve candidate genes we screened, phenotyped ( Figure 2 , Table 1 ) and validated ( Figure 3 ), two of them served as positive controls, having known knockout/knockdown zebrafish models. For ret wmr1 and ret hu2486 mutants, they present with HSCR-like phenotypes in larval fish (Baker et al, 2022; Heanue et al, 2016), and for gfra1α morphants, they had a reduction in the number of ENs, displaying hypoganglionosis (Shepherd et al, 2004). To our knowledge, for the additional ten genes there are no mutational models or phenotypes described in the ENS, highlighting the importance of our screen for illuminating these novel genes with different functions.…”

“…Pools of 30 to 50 embryos fertilized from in-crossing Tg(−8.3 phox2bb :Kaede) adults were injected at the one-cell stage in the yolk with a solution containing 100 picogram (pg) of gene specific sgRNA, 2 µM Cas9 NLS nuclease (Synthego) and Phenol red. A total of eight injected F0 larvae were dissociated, used in T7 endonuclease I activity assays (NEB E3321) as previously described (Baker et al, 2022), and the percentage of indels was determined. PCR pair of primers per gene used for theT7E1 activity (Supplementary table 1) targeted the specific sgRNA region for each gene and amplified regions between 200 to 300 bp.…”

“…Notably, various stages of zebrafish ENS development, genes, and signaling pathways have been elucidated, contributing to a deeper understanding of the molecular basis of ENS development (Ganz, 2018; Kuil et al, 2021). In zebrafish, ENCCs migrate into and along the developing gut between 32-72 hours post fertilization (hpf), and by 4 days post fertilization (dpf) an ENS network begins to form around the whole length of the GI tract (Baker et al, 2022; Bandla et al, 2022; Harrison et al, 2014; Kuil et al, 2021; Uyttebroek et al, 2010). Thus, its genetic conservation, and rapid, simple ENS development, make zebrafish an attractive animal model for elucidating ENS development.…”

BMP signaling pathway member expression is enriched in enteric neural progenitors and required for zebrafish enteric nervous system development

“…Zebrafish embryos obtained from in-crossing Tg(−8.3 phox2bb :Kaede) +/− adults were injected at the one-cell stage with a cocktail containing: phenol red dye .5 nanoliter (nl), 966 picogram (pg) New England Biolabs Cas9 (M0646T) and 120 pg of sgRNA per embryo targeting exon 1 of bmp5 (5’-CGGGCAAGCAGGCGTCGTCC -3’) (as described above). Injected F0 larvae were screened at 5 dpf for ENS defects in which regions of the intestine demonstrated reduced Kaede + cells, and a subset of F0 embryos were dissociated and used in T7 endonuclease activity assays (NEB E3321) in order to validate successful CRISPR-Cas9 activity, as previously described (Baker et al, 2022). Injected F0 larvae exhibiting WT phenotype were collected and raised in our in-house zebrafish facility, and when reaching sexual maturity were outcrossed to AB WT.…”

“…Ret responds to the binding of TGF-β signaling factor, glial cell-derived neurotrophic factor (GDNF), and promotes the migration of enteric progenitors (Natarajan et al, 2022). Recent work in zebrafish and mouse demonstrates more complex roles of traditionally studied ENS factors, such as Ret (Baker et al, 2022; Natarajan et al, 2022; Vincent et al, 2023), where it was recently found to be necessary for driving not only ENCC migration but the proliferative capacity and population density of enteric neurons along the gut. While these recent efforts have extended our understanding of how certain previously known pathways, such as Ret, mechanistically regulate ENCC ontogenesis, delineating how other known factors may be involved in specific phases ENS formation have not been as well studied.…”