Elevated Hoxb5b Expands Vagal Neural Crest Pool and Blocks Enteric Neuronal Development in Zebrafish

Abstract

Neural crest cells (NCCs) are a migratory, transient, and multipotent stem cell population essential to vertebrate embryonic development, contributing to numerous cell lineages in the adult organism. While great strides have been made in elucidating molecular and cellular events that drive NCC specification, comprehensive knowledge of the genetic factors that orchestrate NCC developmental programs is still far from complete. We discovered that elevated Hoxb5b levels promoted an expansion of zebrafish NCCs, which persisted throughout multiple stages of development. Correspondingly, elevated Hoxb5b also specifically expanded expression domains of the vagal NCC markers foxd3 and phox2bb. Increases in NCCs were most apparent after pulsed ectopic Hoxb5b expression at early developmental stages, rather than later during differentiation stages, as determined using a novel transgenic zebrafish line. The increase in vagal NCCs early in development led to supernumerary Phox2b+ enteric neural progenitors, while leaving many other NCC-derived tissues without an overt phenotype. Surprisingly, these NCC-derived enteric progenitors failed to expand properly into sufficient quantities of enterically fated neurons and stalled in the gut tissue. These results suggest that while Hoxb5b participates in vagal NCC development as a driver of progenitor expansion, the supernumerary, ectopically localized NCC fail to initiate expansion programs in timely fashion in the gut. All together, these data point to a model in which Hoxb5b regulates NCCs both in a tissue specific and temporally restricted manner.

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

“…We performed wholemount immunohistochemistry at 6 dpf to detect if changes were present in the neurochemical coding of the ENS. To achieve this, we used antibodies against Phox2b [ 49 ] and different markers that are present in differentiated ENs, such as HuC/D (Elavl3/4), 5-HT (5-hydroxytryptamine), and Chat (acetylcholine) [ 19 ]. Control larvae displayed a complete ENS along the gut, and based on the markers we assayed, showed 5 main populations: Phox2b + /HuC/D + ; Phox2b + /HuC/D + /5-HT + ; HuC/D + /5-HT + /Chat + ; Phox2b + ; and HuC/D + ( Fig 5A ).…”

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

“…Our single-cell transcriptomic and HCR analyses (Fig.1,2) predict BMP signaling activity during development of the zebrafish ENS. To validate the predicted activity of the BMP pathway in NCC and enteric progenitors, we utilized immunohistochemical analysis of activated BMP-specific Smad 1/5/9 (Das and Crump, 2012) and Phox2b (Howard et al, 2022) (Fig.3A,B). Phox2b and phosphorylated R-Smad 1/5/9 (pSMAD) lacked co-expression at 24 hpf (Fig.3C-E’), though BMP signaling showed activation in regions of neural crest migratory paths along the vagal-level neural tube.…”

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

“…We therefore performed wholemount immunohistochemistry at 6 dpf to detect if changes were present in the neurochemical coding of the ENS. To achieve this, we used antibodies against Phox2b (Howard et al, 2022), and different markers that are present in differentiated ENs, such as HuC/D (Elavl3/4), 5-HT (5-hydroxytryptamine) and Chat (acetylcholine) (Uyttebroek et al, 2010). Control larvae displayed a complete ENS along the gut, and based on the markers we assayed, showed 5 main populations: Phox2b + /HuC/D + ; Phox2b + /HuC/D + /5-HT + ; HuC/D + /5-HT + /Chat + ; Phox2b + ; and HuC/D + ( Figure 5A ).…”