The long isoform of the C. elegans ELT-3 GATA factor can specify endoderm when overexpressed

Abstract

The C. elegans elt-3 gene encodes a GATA transcription factor that is expressed in the hypodermis and has roles in hypodermal specification and regulation of collagen and stress response genes. The gene encodes short and long isoforms, ELT-3A and ELT-3B respectively, that differ upstream of their DNA-binding domains. Previous work showed that ELT-3A can specify hypodermal cell fates when forcibly overexpressed throughout early embryos. We recently showed that the ELT-3B orthologue from the distantly related species C. angaria can specify endodermal fates when forcibly overexpressed in C. elegans. Here, we show that C. elegans ELT-3B can also specify endoderm.

Figure 1. Gut specification network, END-3 and ELT-3 structures, and evidence that ELT-3B is sufficient to drive gut specification in C. elegans

A. Simplified representations of the gene networks that specify gut in C. angaria and C. elegans , after a prior work (Broitman-Maduro et al., 2022). Factors below the horizontal dotted line act specifically in the E lineage. Factors upstream of ELT-2/-7 are transiently expressed. The common ancestor to both species is predicted to have used ELT-3 to specify the gut, while an expanded network of factors shown in blue acts in C. elegans . B. Comparison of ELT-3A/B from C. angaria and the ELT-3A/B and END-3 proteins from C. elegans . Percentages show identical amino acids (similar amino acids in brackets) across the regions identified by protein-protein BLAST (Altschul et al., 1990). The smaller ELT-3A isoforms start at amino acid positions 92 of Cel-ELT-3B and position 136 of Can-ELT-3B for C. elegans and C. angaria , respectively. Abbreviations: DBD, DNA-binding domain; EGD, Endodermal GATA Domain found in END-1 and END-3 orthologues (Maduro, 2020). C. Protein alignments of amino and carboxyl ends of Cel-END-3, Cel-ELT-3, and Can-ELT-3. Extended homology exists between the amino ends of the two ELT-3B orthologues, however Cel-END-3 shares only a similar Poly-S region with these. In the carboxyl ends which contain the DNA-binding domains (DBDs), those of Can-ELT-3 and Cel-ELT-3 are essentially identical, while Cel-END-3 shares only 52% identity (55% similarity) with Cel-ELT-3. Alignments were performed manually, assisted by BLASTP results (Altschul et al., 1990). D. Forced overexpression of Cel-ELT-3B, but not Cel-ELT-3A, promotes ectopic specification of gut. Left column: Representative DIC images of arrested transgenic embryos following heat shock (hs). Middle column: Polarized light showing presence of gut granules (white dots) in the same embryos as in the first column. In the case of hs-ELT-3B, granules are widely dispersed. Right column: Expression of integrated elt-2 reporter transgene in the same embryos as in the first column. In the middle and right-hand columns, the eggshells have been outlined in a dotted yellow line, and groups of gut cells, shown by both gut granules and elt-2 expression, have been outlined with dotted white lines. All images are at the same scale. Bottom: Table of results of heat shocked embryos. Gut was scored as ectopic if there were more than 40 elt-2- expressing nuclei present. Embryos of hs-Cel-ELT-3A generally had 20 or fewer elt-2 -expressing nuclei. E. Rescue of the embryonic/larval lethality and gut specification defects of end-1(ok558) end-3(ok1448) double mutants. Images show end-1,3(-) without or with rescue by extrachromosomal arrays carrying either end-3(+) (second column) or an end-3promoter::ELT-3B::CFP transgene (third column). Top row: DIC with GFP overlay showing expression of integrated elt-2promoter::NLS::GFP::lacZ reporter wIs84 in rescued comma-stage embryos. Bottom row: Representative arrested L1 larvae (arrows) or rescued transgenic adults on agar. Images across each row are at the same scale as the leftmost image. Bottom: Table showing rescue of end-1,3(-) lethality by various transgenes. Rescue was scored as positive if transgenic animals survived past the L3 stage.