A cellular atlas of Astyanax mexicanus embryogenesis reveals principles of developmental microevolution

The evolution of development is the primary mechanism by which morphological and functional diversity is generated. Comparing the developmental trajectories of single cells in entire embryos across species can reveal how gene expression is conserved and varies, as well as the molecular mechanisms underlying how cell- and tissue-specific changes in developmental regulation generate phenotypic differences. However, this perspective has not addressed the mechanisms of small-scale developmental microevolution, i.e, in closely-related species or even within a single species. The fish Astyanax mexicanus, which comes in two forms adapted to distinct environments - the eyed, river-dwelling morph and the eyeless, cave-adapted morph - is a remarkable model with which to investigate the evolution of gene regulation in a single species. Here, we generated and analyzed a single-nucleus RNA-seq atlas of the development of the two Astyanax morphs, from gastrula to swimming larva. Despite extensive changes in gene expression observed in the eight examined stages, for both ubiquitously-expressed and tissue-specific genes, and in both embryonic and extra-embryonic tissues, the temporal progression of cavefish and surface fish cells was similar in ordinal regression-based pseudotime models using 20-100 core genes. However, transcriptome-wide comparisons of gene expression waves revealed massive heterochronic shifts. In cavefish, some tissues had a majority of genes lagging behind in their transcriptional dynamics (e.g. the optic region), while in others, a majority of genes had accelerated dynamics (e.g. the mesoderm), or else, an equivalent number of genes were found in either category (e.g. the endoderm). Furthermore, cell fate acquisition and differentiation were variably affected in different cavefish lineages, with delays of ~6 hours in the retina, ~2 hours in the axial mesoderm and ~none in the extra-embryonic enveloping layer. Thus, cavefish embryos progress as a mosaic of different tissues, or modules, each following its own transcriptional tempo. Because gene expression differential regulation and time shifts were more prevalent in the early stages and gradually diminished throughout development, we propose that cavefish embryos initially undergo strong decanalisation before becoming re-canalized in line with the shared Astyanax developmental program.