GSE193281
Identification of an evolutionarily conserved domain in Neurod1 essential for triggering enteroendocrine cell differentiation
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 one is 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. This novel domain acts as a goblet cell fate repressor and 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 several EE subtypes, validating the importance of the UCE domain in enteroendocrine cell differentiation. Importantly, the neurod1 null mutant displays very similar defects supporting 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. Overall design: RNA-sequencing of 23 samples. Enteroendocrine cell (pax6b:GFP +) transcriptomic profiles of 4dpf (days post fertilization) wild type and neurod1 deleted from its conserved domain (neurod1DelUCE-/-)  zebrafish embryos were generated in in 3 replicates and 4 replicates, respectively.  Endodermal cells (sox17:dsRed +) transcriptomic profiles of 52hpf (hours post fertilization) WT or transgenic (hsp70:ascl1a, hsp70:neurod1, hsp70: neurod1DelUCE, hsp70: neurod1DelECD) zebrafish embryos were generated in triplicates or in 4 replicates (hsp70:neurod1) after 2 heat-shocks inducing transgene expression.