A 3D molecular map of the cavefish neural plate illuminates eyefield organization and its borders in vertebrates

The vertebrate retinas originate from a specific anlage in the anterior neural plate called the eyefield. The eyefield shares its anterior border with the prospective telencephalon and is in contact ventrally and posteriorly with hypothalamic and diencephalic precursors. Eyefield identity is conferred by a set of "eye transcription factors", whose combinatorial expression has not been precisely characterized. Here, we use the dimorphic teleost species Astyanax mexicanus, which develops proper eyes in the wild type and smaller colobomatous eyes in the blind cavefish embryo, to unravel the molecular anatomy of the eyefield and its micro-evolutionary variations in the two Astyanax morphs. Using a series of markers (Rx3, Pax6, CxCr4b, Zic1, Lhx2, Emx3, Nkx2.1), we draw a comparative 3D expression map at the end of gastrulation/onset of neurulation, which highlights hyper-regionalization of the eyefield into sub-territories of distinct sizes, shapes, cell identities and putative fates along the three body axes. We also discover sub-domains within the prospective telencephalon, and we characterize cell identities at the frontiers of the eyefield. Analyses at the tissue scale and single cell level show variations in volumes and shapes of eyefield subdivisions as well as cellular gene expression levels and identity changes in cavefish. The ventro-medial border and the anterior border of the eyefield contain cells co-expressing hypothalamic and telencephalic marker, respectively, in cavefish embryos. Altogether, we provide a new model of eyefield patterning in 3D and suggest a developmental origin for the emergence of the coloboma phenotype in the natural mutant cavefish embryo.