The ELT-3 GATA factor specifies endoderm in Caenorhabditis angaria in an ancestral gene network
Posted on: 11 June 2022 , updated on: 19 December 2022
Preprint posted on 26 May 2022
Article now published in Development at http://dx.doi.org/10.1242/dev.200984
How many ways to make a worm gut? Extensive rewiring of the gene network underlying gut development in Caenorhabditis
Selected by Chee Kiang Ewe
Categories: developmental biology, evolutionary biology, genetics
Updated 28 October 2022 with a postLight by Swathi Arur
When I received and read this manuscript as an editor, I thought this manuscript may be a good fit for Development and sent it out for peer review since I was struck by what seemed like an example of evolution of a gene regulatory network that led to endoderm specification in two distant yet related species (in this case of the nematode Caenorhabhditis). Using a combination of gene expression reporters, single molecule FISH analysis, and either genetic mutants or RNA interference to deplete gene function, the authors argued that the gene regulatory network that led to specification of the endoderm was not the same between C. elegans and C. angaria. In fact C. angaria lacked some ‘critical’ gut specification genes, ‘critical’ as defined by the model nematode C. elegans, which led the group to identify a more basic gene regulatory network which drove endoderm specification in nematodes.
Updated 26 October 2022 with a postLight by Chee Kiang Ewe
This preprint was recently published in Development. In the peer-reviewed article, the authors included an updated high quality whole genome sequence of C. angaria, which will aid future comparative studies with C. elegans. The authors also expanded their smFISH analysis to C. monodelphis and showed that elt-3 is expressed in the embryonic endoderm, as observed in C. portoensis and C. angaria, further strengthening the conclusion that ELT-3 plays a conserved role in endoderm specification in nematodes outside of the Elegans supergroup. Moreover, the authors performed additional genetic analyses and could show that Can-ELT-3 (driven under the end-3 promotor) may rescue endoderm defects in the C. elegans end-1 end-3 double mutant. Strikingly, the authors were able to move C. angaria network into C. elegans by knocking out the entire GATA cascade in C. elegans and replacing it with Can-ELT-3 and Can-ELT-2, which strongly supports the conclusion that Can-ELT-3 and Can-ELT-2 drive endoderm specification and differentiation, respectively. Interestingly, the rescue appeared to be temperature sensitive as it occurred at lower efficiency at 20oC compared to 25oC, suggesting reduced robustness in nematodes with a simple network as was also pointed out in the preLights Q&A. Overall, I think that the published, peer-reviewed paper has significantly improved compared to the preprint. Congratulations to Broitman-Maduro et al.!
Background:
Development is driven by gene regulatory networks (GRNs) that are composed of complex interactions between many transcription factors. The nematode C. elegans has been a great system to study the principals underlying developmental GRN owing to its defined cell lineage and the extensive genetic tools available for manipulating the GRN. Furthermore, comparative studies involving different nematode species spanning the Caenorhabditis genus (Figure 1) have provided insights into the evolutionary variation in developmental mechanisms, and how GRNs might be modified during evolution (Ewe et al., 2020).
Figure 1: The phylogenetic relationships of the Caenorhabditis nematodes. MEDs, ENDs, and ELT-7 originate at the base of the Elegans Supergroup (adapted from Figure 1; Broitman-Maduro & Maduro, 2022).
In C. elegans, the endoderm comprises the mid-gut. In the early embryo, the endoderm GRN is initiated by SKN-1, which activates a gene cascade driven by six GATA transcription factors – two MEDs, two ENDs, and two ELTs. (Figure 2). Additionally, the Wnt effector POP-1 activates endoderm specification by acting on END-1 and -3. Interestingly, the orthologues of the MEDs and ENDs, as well as ELT-7, are missing in nematodes outside of the Elegans Supergroup, including C. angaria and C. portoensis (Figure 1) (Maduro, 2020), raising the question of how gut is specified in their absence.
In this preprint, the authors found that ELT-3, a GATA factor that is mostly expressed in the hypodermal cells in C. elegans, is present in early endodermal progenitors and drives gut specification in C. angaria. This study demonstrates an extensive rewiring of the endoderm GRN within the Caenorhabditis genus (Figure 2).
Figure 2: The evolution of the endoderm GRN in nematodes. POP-1 activates ELT-3, which in turns activates ELT-2 in C. angaria. In the derived GRN in C. elegans, MEDs and ENDs, instead of ELT-3, drive endoderm specification. This eventually leads to the activation of ELT-7 and -2, which lock down the differentiated endodermal cell fate.
Major findings:
1) Wnt effector POP-1 is required for embryonic gut development in C. angaria
To test the roles of SKN-1 and POP-1 orthologues in endoderm development in C. angaria, the authors knocked down Can-skn-1 and Can-pop-1 using RNAi. While knocking down Can-skn-1 did not cause obvious developmental defects, embryos lacking Can-pop-1 underwent developmental arrest and failed to development a gut.
2) GATA factor ELT-2 plays conserved role in endoderm differentiation
To examine the role of C. angaria ELT-2 in endoderm development, the authors cloned Can-elt-2 and introduced the transgene into C. elegans. They found that Can-elt-2, like Cel-elt-2, was expressed exclusively in the endodermal lineage. In C. elegans, eliminating both elt-2 and elt-7 causes larval arrest and a severe block to endoderm differentiation (Sommermann et al., 2010). The authors showed that the expression of Can-elt-2 transgene strongly rescued larval lethality in C. elegans double mutant lacking elt-2 and elt-7. Importantly, knocking down Can-elt-2 in C. angaria caused penetrant larval lethality and gut differentiation defects. Together, these results demonstrate the functional conservation of ELT-2 in endoderm differentiation.
3) ELT-3 drives endoderm specification in C. angaria
The authors showed that the expression of Can-elt-2 in C. elegans required END-1 and -3, the two endoderm-specifying GATA factors, as knocking out end-1/3 abrogated Can-elt-2 transgene expression; however, C. angaria does not contain END-1 and -3 orthologues. To identify the upstream activator(s) of Can-elt-2, the authors performed smFISH to examine the expression of other GATA-encoding genes (elt-1, elt-3, and elt-5) in C. angaria embryos. Interestingly, the authors found that the transcripts of elt-3, but not elt-1 and -5, were found in the early endodermal lineage, and the expression of Can-elt-3 depended on Can-POP-1, but not Can-SKN-1.
To examine the role of elt-3 in endoderm specification, the authors overexpressed Can-elt-3 (under the control of a heat-shock promotor) in C. elegans and found a widespread activation Can-elt-2 and ectopic gut development in the arrested embryos. Furthermore, knocked down elt-3 in C. angaria by RNAi resulted in development arrest and a severe block to endoderm development, showing the function of Can-ELT-3 as a potent driver of endoderm specification, akin to END-1 and -3 in C. elegans.
4) The evolution of the endoderm gene regulatory network in nematodes
Lastly, the authors found elt-3 and elt-2 to be expressed in the endodermal lineage of C. portoensis, another species outside of the Elegans Supergroup, suggesting that ELT-3 -> ELT-2 interaction is conserved in the ancestral endoderm GRN, prior to the gene duplication events that gave rise to meds, ends, and elt-7 (Figure 2). The intercalation of the redundant GATA factors in the endoderm GRN may ensure developmental robustness during rapid embryogenesis in C. elegans.
What I liked about this preprint: This work provides an important insight into the dynamics of developmental system drift in the endoderm GRN in nematodes – an excellent paradigm to study the molecular mechanisms of developmental robustness and evolutionary plasticity.
Question for the authors:
The genetic redundancy in C. elegans may indeed promote developmental robustness; the simplicity of the ancestral gene network may therefore result in increased transcriptional noise. Have you noted obvious variability in elt-2 onset timing and/or expression level in C. angaria?
References:
Ewe, C. K., Torres Cleuren, Y. N., & Rothman, J. H. (2020). Evolution and developmental system drift in the endoderm gene regulatory network of Caenorhabditis and other nematodes. Frontiers in Cell and Developmental Biology, 8, 170. https://doi.org/10.3389/FCELL.2020.00170
Maduro, M. F. (2020). Evolutionary dynamics of the Skn-1/Med/end-1,3 regulatory gene cascade in Caenorhabditis endoderm specification. G3: Genes, Genomes, Genetics, 10(1), 333–356. https://doi.org/10.1534/g3.119.400724
Sommermann, E. M., Strohmaier, K. R., Maduro, M. F., & Rothman, J. H. (2010). Endoderm development in Caenorhabditis elegans: The synergistic action of ELT-2 and -7 mediates the specification→differentiation transition. Developmental Biology, 347(1), 154–166. https://doi.org/10.1016/j.ydbio.2010.08.020
doi: https://doi.org/10.1242/prelights.32270
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2 years
Katherine Brown
This paper was recently selected by the editors of Development as a finalist of our 2022 Outstanding Paper prize (see this recent editorial for more details: https://journals.biologists.com/dev/article/150/7/dev201810/306247/)
It’s been great seeing the journey of this paper from preprint to published paper, and to read the comments from Chee Kiang and Swathi on why they both found it an exciting piece of research!