Identification of an evolutionarily conserved domain in Neurod1 favouring enteroendocrine versus goblet cell fate

Abstract

ARP/ASCL transcription factors are key determinants of cell fate specification in a wide variety of tissues, coordinating the acquisition of generic cell fates and of specific subtype identities. How these factors, recognizing highly similar DNA motifs, display specific activities, is not yet fully understood. To address this issue, we overexpressed different ARP/ASCL factors in zebrafish ascl1a-/- mutant embryos to determine which ones are able to rescue the intestinal secretory lineage. We found that Ascl1a/b, Atoh1a/b and Neurod1 factors are all able to trigger the first step of the secretory regulatory cascade but distinct secretory cells are induced by these factors. Indeed, Neurod1 rescues the enteroendocrine lineage while Ascl1a/b and Atoh1a/b rescue the goblet cells. Gain-of-function experiments with Ascl1a/Neurod1 chimeric proteins revealed that the functional divergence is encoded by a 19-aa ultra-conserved element (UCE), present in all Neurod members but absent in the other ARP/ASCL proteins. Importantly, inserting the UCE into the Ascl1a protein reverse the rescuing capacity of this Ascl1a chimeric protein that cannot rescue the goblet cells anymore but efficiently rescue the enteroendocrine cells. This novel domain acts indeed as a goblet cell fate repressor that inhibits gfi1aa expression, known to be important for goblet cell differentiation. Deleting the UCE domain of the endogenous Neurod1 protein leads to an increase in the number of goblet cells concomitant with a reduction of the enteroendocrine cells, phenotype also observed in the neurod1 null mutant. This highlight the crucial function of the UCE domain for NeuroD1 activity in the intestine. As Gfi1 acts as a binary cell fate switch in several tissues where Neurod1 is also expressed, we can envision a similar role of the UCE in other tissues, allowing Neurod1 to repress Gfi1 to influence the balance between cell fates.

M1 and M2 macrophages differentially regulate colonic crypt renewal

“…Within the secretory cell lineage, Cro-A+ enteroendocrine cell numbers were maintained throughout the epithelium in our study. The differentiation of enteroendocrine cells from crypt progenitor cells requires the expression of Atoh1 and Neurogenin3 and as studies have also shown that enteroendocrine cells are a highly conserved population within the intestinal epithelium, our findings suggests that it is unlikely that macrophages are able to influence the enteroendocrine cell fate (Hartenstein et al, 2011; Reuters et al, 2022) Instead, it is likely that enteroendocrine cell-derived hormones and peptides may alter macrophage function and further studies should investigate the potential impact these crosstalk mechanism may have in gastrointestinal tract disorders (Worthington et al, 2018).…”

Interleukin-10 regulates goblet cell numbers through Notch signaling in the developing zebrafish intestine

“…To gain insights into the potential mechanisms by which il10 modulates goblet cell numbers in zebrafish larvae, we analyzed the activity of signaling pathways involved in goblet cell differentiation, such as Notch, 18 RAR, 28 and ARP/ASCL factors. 43 , 44 For this purpose, we dissected intestines from 5dpf WT and il10 -Mut larvae (Fig. 5a ) and we analyzed the expression of Goblet cell markers and key target genes for the above-mentioned signaling pathways by qRT-PCR.…”

microRNA‐124 regulates Notch and NeuroD1 to mediate transition states of neuronal development

“…Although we do not understand the regulatory mechanism of NeuroD1 on Delta expression, the number of Delta ‐expressing cells correlated with the level of qPCR, if the number of Delta transcripts expressed by each cell is similar (Figures 1c and S1). Recently, it has been shown that an OE of NeuroD1 in zebrafish embryos leads to increased expression of Delta ligands (deltaB, deltaC, and delta‐like4) during the hatching stage prior to the larval stage (Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995; Reuter et al., 2022). We do not know if zebrafish NeuroD1 temporally regulates Delta differently during various developmental timepoints.…”