Embryological manipulation to probe early evo-devo in the fish Astyanax mexicanus

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Jorge and Sylvie shared

• 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?

Adult cavefish lack completely their eyes, however as embryos they develop an eye primordium that is small in size and mis-patterned, and that later degenerates by apoptotic mechanisms. A former PhD student in the lab, Lucie Devos, generated transgenic reporter lines for the “eye gene” zic1 (zic1:GFP) in order to visualize and compare eye morphogenesis in the two morphs. By doing live imaging analyses she showed that cell behaviors are impaired in cavefish embryos compared to surface fish (Devos et al., 2019). In addition, our lab has shown that developmental differences between the two morphotypes start at the onset of gastrulation and that maternal information contained in the unfertilized oocytes is responsible for these changes (Torres-Paz et al., 2019).

We decided to implement the recently described method to generate pescoids in Astyanax mexicanus in order to study the influences of extra-embryonic (yolk) and embryonic signals in normal and defective eye development. To our knowledge, eye development has not yet been proven to occur in gastruloid systems. We will take advantage of the available transgenic lines as direct readout to study if early steps of eye development could be recapitulated in gastruloids. Combining gastruloid methods and eye-specific reporter lines will help us to uncover early developmental mechanisms leading to eye developmental defects. Note that we can also envisage producing mixed gastruloids, i.e., combining blastoderms from a cavefish and a surface fish embryo.

Regarding the experiments on chimeric embryos in the Astyanax model, we hope to reveal autonomous and non-autonomous mechanisms involved, first in the incorrect specification of the presumptive eye, and then in the degenerative process. We bet that the embryonic environment will have a strong influence in cell fate decisions made by the grafted cells and we aim to identify the signals involved and the timing during which these cell decisions are made.

• 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? 

Morphological changes in anterior brain structures observed in Astyanax morphs, including the eye loss and enlargement of olfactory organs, take their origin during gastrulation or earlier. We and others have demonstrated that changes in mature forebrain structures can be predicted precociously in embryos at the neural plate stages by looking at the expression of key regionalization markers. We think that even though development of structures such as the eyes, telencephalon or the olfactory organs do not occur in gastruloids, maybe due to failure in morphogenesis, some of the earlier developmental aspects, such as initial tissue specification could be advantageously studied. If this is true, Astyanax gastruloids will help us characterizing the signaling systems (and early embryonic structures) that are involved in neural plate regionalization.

• 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?

Zebrafish embryogenesis occurs by meroblastic discoidal early cleavages, were embryonic cells divide on top of a large extraembryonic yolk cell. Pescoids were developed in zebrafish by removing the yolk cell and culturing the embryonic cells under appropriate conditions.

Divergence between Astyanax (characid) and zebrafish (cyprinid) is estimated to have occurred around 150mya, and both species share a very similar pattern of early development, with meroblastic discoidal early cleavages. This developmental pattern is also observed in most teleost fish such as the medaka, which have diverged from the clade containing both Astyanax and zebrafish (ostariophysi) around 250 mya. We guess that pescoids could be obtained also from medaka and other teleost embryos.

In contrast, earlier-derived, non-teleost ray-finned fishes such as sturgeons, gars and bowfins develop through holoblastic cleavages, similarly to amphibians, where the early blastomeres are completely divided during early cell cycles. Thus, as there is no yolk cell in this type of embryos, pescoids in the proper sense cannot be obtained.

• 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?

In natural conditions Astyanax cavefish live in total and permanent darkness, while the river-dwelling surface fish live in a “normal” circadian rhythm. In our lab as in most cavefish labs around the world, both morphs are maintained at a 12:12 light-dark cycle, in order to keep them under the same experimental conditions- except for specific experimental designs and questions. Also, it is technically very complicated to keep fish under cave-like conditions throughout their life cycle. We have not studied the effect of the absence of light on early morphogenesis, but we believe there would be no effect. In fact, work from the lab has shown that raising fish in the absence of light does not have any impact in general morphology compared to fish raised in normal light-dark cycle – even though some behavioral plasticity has been observed in larvae, particularly an enhancement in olfactory responses in surface fish raised in the dark (Blin et al., 2018).

References

Blin, M., Tine, E., Meister, L., Elipot, Y., Bibliowicz, J., Espinasa, L., & Rétaux, S. (2018). Developmental evolution and developmental plasticity of the olfactory epithelium and olfactory skills in Mexican cavefish. Developmental Biology. https://doi.org/10.1016/J.YDBIO.2018.04.019

Devos, L., Klee, F., Edouard, J., Simon, V., Legendre, L., Khallouki, N. El, Blin, M., Barbachou, S., Sohm, F., & Rétaux, S. (2019). Morphogenetic and patterning defects explain the coloboma phenotype of the eye in the Mexican cavefish. BioRxiv, 698035. https://doi.org/10.1101/698035

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