Vertebrate cdx genes are members of the parahox gene cluster and are homologs of Drosophila Caudal. They are expressed in the posterior tissues of every major group of bilaterians, where they control growth and development of the posterior embryo. In zebrafish, and likely all vertebrates, Cdx homologs have acquired an additional role, namely, specification of the spinal cord: cdx genes are among the earliest expressed in the spinal cord territory during development and are necessary and sufficient for spinal cord fate within the zebrafish posterior neural plate. Additionally, unlike the brain and anterior spinal cord, cells of the posterior spinal cord are not specified by the end of gastrulation and instead must be continually generated and patterned from precursor cells within the tailbud as the embryo elongates. Work from our lab and others indicates that Cdx plays a key role in specifying/committing posterior cells to the posterior spinal cord fate, as well as maintaining the posterior growth zone itself from which the caudal tissues of the embryo emerge.
In this work, we ask: how does zebrafish Cdx4 specify/commit posterior cells to the spinal cord fate? Currently, we do not know at what step or steps, from precursor specification to neural differentiation, Cdx acts in this process. To understand the role of Cdx in posterior neural cell fate, we developed a scalable loss-of-function assay that takes advantage of the CRISPR/Cas9 system to generate F0, genetically mosaic loss-of-function zebrafish embryos. We use this assay, a powerful and efficient alternative to morpholino knockdown and akin to RNAi-based screens in other systems, to query a candidate gene list of Cdx4 direct and indirect effectors compiled from multiple high-throughput experiments. We have confirmed several candidates as expressed in the spinal cord, responsive to Cdx4 levels, and whose loss-of-function causes ectopic expression of hindbrain marker genes within the spinal cord territory as loss of Cdx4 does. Some of these candidates, like the helix-loop-helix protein Id1, are known regulators of neural differentiation; others, like the metalloproteinase Adamts18, are poorly characterized. In particular, we have found that mosaic knockdown of multiple components of the BMP signaling pathway also produce ectopic marker gene expression in our assay.
Creating mutant lines for some of these Cdx4 effector loci, we are further characterizing their role in posterior neural development. This work should shed light on the generation of posterior neural tissue from the multipotent stem cell-like niche within the vertebrate tailbud.