Loss of ascl1a prevents secretory cell differentiation within the zebrafish intestinal epithelium resulting in a loss of distal intestinal motility

Abstract

The vertebrate intestinal epithelium is renewed continuously from stem cells at the base of the crypt in mammals or base of the fold in fish over the life of the organism. As stem cells divide, newly formed epithelial cells make an initial choice between a secretory or enterocyte fate. This choice has previously been demonstrated to involve Notch signaling as well as Atonal and Her transcription factors in both embryogenesis and adults. Here, we demonstrate that in contrast to the atoh1 in mammals, ascl1a is responsible for formation of secretory cells in zebrafish. ascl1a−/− embryos lack all intestinal epithelial secretory cells and instead differentiate into enterocytes. ascl1a−/− embryos also fail to induce intestinal epithelial expression of deltaD suggesting that ascl1a plays a role in initiation of Notch signaling. Inhibition of Notch signaling increases the number of ascl1a and deltaD expressing intestinal epithelial cells as well as the number of developing secretory cells during two specific time periods: between 30 and 34 hpf and again between 64 and 74 hpf. Loss of enteroendocrine products results in loss of anterograde motility in ascl1a−/− embryos. 5HT produced by enterochromaffin cells is critical in motility and secretion within the intestine. We find that addition of exogenous 5HT to ascl1a−/− embryos at near physiological levels (measured by differential pulse voltammetry) induce anterograde motility at similar levels to wild type velocity, distance, and frequency. Removal or doubling the concentration of 5HT in WT embryos does not significantly affect anterograde motility, suggesting that the loss of additional enteroendocrine products in ascl1a−/− embryos also contributes to intestinal motility. Thus, zebrafish intestinal epithelial cells appear to have a common secretory progenitor from which all subtypes form. Loss of enteroendocrine cells reveals the critical need for enteroendocrine products in maintenance of normal intestinal motility.

Injury Induces Endogenous Reprogramming and Dedifferentiation of Neuronal Progenitors to Multipotency

“…1C, Uninjured). Ascl1 has also been associated with both neuroendocrine and secretory cell development (Kokubu et al, 2008; Roach and Wallace, 2013), which is concordant with the generation of the non-neuronal progeny here. More surprising, however, were the cell types generated by Ascl1+ GBCs after injury.…”

bHLH proneural genes as cell fate determinants of entero-endocrine cells, an evolutionarily conserved lineage sharing a common root with sensory neurons

“…In mammals, Math1, a member of the atonal family of bHLH genes, is expressed in all intestinal progenitors destined to become secretory (including gland and endocrine) cells (Yang et al, 2001). In zebrafish, Ascl1, a achaete-scute-like bHLH factor, appears to be the first gene signaling a secretory fate (Roach et al, 2013; Flasse et al, 2013). Downstream of Math1, a bHLH factor of the neurogenin family, Ngn3, is expressed in a smaller subset of progenitors which embark on the endocrine fate (Jenny et al, 2002).…”

“…In zebrafish, atoh1 does not appear to play a role at an early stage of intestinal development to specify secretory lineages. Instead, the achaete-scute family member, ascl1 , carries out this function (Roach et al, 2013; Flasse et al, 2013). Ngn3 is expressed in subsets of entero-endocrine cells in the zebrafish gut, at later stages than ascl1 , but does not to have a similarly widespread role as in mammals (K.Wallace, pers comm).…”

“…This pathway controls an early developmental choice between continued proliferation (high N activity) and differentiation as a secretory (gland/endocrine) cell (low N activity; Fig.3A3; May and Kaestner, 2010; Schonhoff et al, 2004a). As a result of low N activity levels, the bHLH transcription factor Math1 (=Atoh1, in mammals) or Ascl1 (zebrafish) is expressed in progenitors destined to become secretory cells (Yang et al, 2001; Li et al, 2011; Roach et al, 2013; Flasse et al, 2013; Fig.3A3). This lineage splits up further when (as a result of so far unknown signaling interactions) additional cell fate determinants become active.…”

Can the ‘neuron theory’ be complemented by a universal mechanism for generic neuronal differentiation

“…Here, a Math1/Atoh1>Neurog3/Ngn3>NeuroD1/NeuroD cascade governs the generation of enteroendocrine cells (see Li et al 2011 for review). Different from the mouse, in the zebrafish intestine, ascl1a/zash1a is crucial for differentiation of all secretory cell types including enteroendocrine cells (Flasse et al 2013;Roach et al 2013) that in mutant animals appear to differentiate into enterocytes.…”