Embryological manipulation to probe early evo-devo in the fish Astyanax mexicanus
Preprint posted on 7 October 2020 https://www.biorxiv.org/content/10.1101/2020.10.06.328500v1
Article now published in Frontiers in Cell and Developmental Biology at http://dx.doi.org/10.3389/fcell.2021.667296
Overview of the research
The mexican cavefish (Astyanax mexicanus) is a freshwater fish living in the rivers and caves bordering the Mexican Gulf. Uniquely, members of this species exist in two very different forms (“eco-morphotypes”) (See Figure 1). The surface morph lives in rivers, is sighted and pigmented. The cave morph lives deep into dark caves, is blind, depigmented, but compensates for the loss of sight by a highly developed olfactory system. Clearly, each form of this species is highly adapted to its environment, but how is their same genetic identity executed into such different body plans during development? What are the developmental differences that turn an Astyanax embryo into a “surface” fish or a “cave” one?
Figure 1: the two Astyanax morphs. Morph picture is from wikimedia commons (public domain). Embryos and pescoids are reused with permission from preprint Fig.2.
Previous work in this species has identified that there are differences in the maternal determinants provided by each mother morph during egg formation, and deposited in the yolk. (Torres-Paz, et al; 2019, Ma, et al; 2018). Eggs from cave mothers are enriched in ventralising signals compared to surface eggs, suggesting that some of the morphological differences observed come maternally rather than from the embryo itself (maternal genetic effect). How to test the extent of such maternal contributions?
The authors turn to experimental and synthetic embryology, and describe the successful application, to the mexican cavefish, of two techniques that both probe the intrinsic developmental programmes of surface and cave embryonic cells. Accordingly, the authors describe the procedure to generate cave-surface chimeric embryos: i.e. unique embryos made from cells of both morph types. Secondly, the authors use cavefish embryonic cells to generate gastruloids, a novel in vitro system widely employed to dissect the role of embryonic versus extraembryonic cues (see the preList on the topic).
The authors overcome significant hurdles related to Astyanax reproductive biology, and describe a protocol to generate stage-matched chimeric embryos between the two morphs: that is, to take cells from a surface fish embryo and incorporate them into a cave morph (and vice versa). Preliminary results show that cells from different morphs are able to survive together and to contribute to embryonic tissues based on the time of transplantation (restriction of potential as time progresses). It would be interesting to follow up on whether these cells initiate independent developmental programmes typical of their morph of origin, or if either developmental programme prevails over the other.
Ecotype-specific pescoids (fish gastruloids)
By applying a recently described protocol to generate fish gastruloids (“pescoids”, Fulton et al; 2020) the authors generate “surfoids” (pescoids made from surface fish embryonic cells), and “caveoids” (pescoids made from cave fish embryonic cells) (See Figure 2). By showing that cells from Astyanax embryos also aggregate and elongate in vitro by self organisation, in the absence of yolk, the authors expand on the now growing collection of species whose embryonic cells have been shown to recapitulate features of normal development in vitro (human, mouse, zebrafish). The rationale of this approach is particularly intriguing: if you take Astyanax cells away from the yolk and maternal factors and make them assemble as pescoids, do they still “know” they should execute “surface” and “cave” programmes? For now, the authors do not report any notable difference between the behaviour of the two pescoid morph types, at least with respect to the readout analysed in this report. Incidentally, staining for the marker no tail (ntl, equivalent of the mouse Brachyury) shows that mesoderm tissue internalises, at the site equivalent to the blastopore in an intact embryo. An observation that enriches current knowledge about how pescoids develop.
Figure 2: Pescoid generation. Surfoids and Caveoids generated by in vitro culture of Astyanax embryonic cells away from the yolk. Purple: staining for the mesoderm marker no tail (ntl) (reused with permission from preprint Fig.1)
This preprint describes early steps towards the novel application of experimental embryology techniques to a very interesting case study: the mexican cavefish. The recourse to the gastruloid/pescoid approach is extremely interesting, because here is a system where cells with the same genetic identity give rise to embryos with drastically different morphology and adult structures. Gastruloids, where embryonic cells are left to their own means without any other developmental cue, are an excellent system to tease out the relative contribution of embryonic and maternal inputs to development. If features of cave and surface morphology are instructed by maternal cues, one would expect caveoids and surfoids not to be able to develop such features. We are left wondering what will come next from the application of the two techniques described in this paper.
Questions to the authors:
- One of the major differences of cave morphs is their degenerate eye and the loss of vision. In the preprint you indeed mention the availability of an Astyanax eye reporter that would allow to track the fate of these cells during development. Could you elaborate more on this project? Do you expect cave cells to be rescued in a cave-surface chimaera?
- On the same topic, many notable differences of cave morphs relate to facial anatomy and anterior neural structures. These are also the structures that have traditionally been missing in gastruloids. Do you think this will represent a significant obstacle to the pescoid approach?
- Pescoids have originally been developed with zebrafish cells. What is known about the evolutionary divergence between cavefish and zebrafish? Do you believe pescoids could be generated from any fish species?
- A technical question: why are cave morphs maintained at a 12:12 light-dark cycle? Do they not usually live in dark caves all day long? Would your observations be different if the fish were maintained in a longer dark phase?
- Torres-Paz, J., Leclercq, J., & Rétaux, S. 2019. Maternally-regulated gastrulation as a source of variation contributing to cavefish forebrain evolution. ELife, 8. https://doi.org/10.7554/eLife.50160
- Ma, Li, et al. “Maternal genetic effects in Astyanax cavefish development.” Developmental biology 441.2 (2018): 209-220.
- Fulton, Timothy, et al. “Axis Specification in Zebrafish Is Robust to Cell Mixing and Reveals a Regulation of Pattern Formation by Morphogenesis.” Current Biology 30.15 (2020): 2984-2994
Posted on: 26 November 2020 , updated on: 1 January 2021Read preprint
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