We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus , a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

We have independently carried out gene expression profiling of three types of stem or immature progenitor cells: ESCs, NPCs, and retinal progenitor/stem cells, or RPCs (Fig. 1) (4, 5). The intersection of ESC-, NPC- and RPC-enriched genes defined a list of 385 genes that are collectively expressed by all three stem cells (6). It can be inferred that these genes may represent or include putative “stemness” genes. For the approach taken here to be able to define and support the notion of “stemness” genes, however, would also require that very similar sets of genes can be identified regardless of the type of stem cells used. To test the validity of this notion, we have collectively analyzed our results along with those from the studies of Ramalho-Santos et al. (1) and Ivanova et al. (2) (Figs. 2 and 3). To our surprise, a comparison of the three independently derived lists of “stemness” genes showed only one gene (integrin alpha-6) commonly identified in the three studies (Figs. 2A and 3A) (6). This finding raised serious concerns about the conclusions reported in (1) and (2), as was also critically highlighted by Burns and Zon (7).

Previously, we have shown that Onecut1 (Oc1) and Onecut2 (Oc2) are expressed in retinal progenitor cells, developing retinal ganglion cells (RGCs), and horizontal cells (HCs). However, in Oc1-null mice, we only observed an 80% reduction in HCs, but no defects in other cell types. We postulated that the lack of defects in other cell types in Oc1-null retinas was a result of redundancy with Oc2. To test this theory, we have generated Oc2-null mice and now show that their retinas also only have defects in HCs, with a 50% reduction in their numbers. However, when both Oc1 and Oc2 are knocked out, the retinas exhibit more profound defects in the development of all early retinal cell types, including completely failed genesis of HCs, compromised generation of cones, reduced production (by 30%) of RGCs, and absence of starburst amacrine cells. Cone subtype diversification and RGC subtype composition also were affected in the double-null retina. Using RNA-Seq expression profiling, we have identified downstream genes of Oc1 and Oc2, which not only confirms the redundancy between the two factors and renders a molecular explanation for the defects in the double-null retinas, but also shows that the onecut factors suppress the production of the late cell type, rods, indicating that the two factors contribute to the competence of retinal progenitor cells for the early retinal cell fates. Our results provide insight into how onecut factors regulate the creation of cellular diversity in the retina and, by extension, in the central nervous system in general. Significance We show that the onecut transcription factors, Onecut1 and Onecut2, redundantly regulate the formation of all four early-born retinal cell types, namely horizontal cells, ganglion cells, cones, and amacrine cells, and prevent precocious formation of the late retinal cell type, rods. Expression profiling suggests these two factors regulate a shared set of downstream genes to maintain competence for the early retinal cell types and to regulate generation of various subtypes of retinal ganglion cells. This study lays the foundation for further examination of how onecut factors regulate cell differentiation in the retina, as well as in the central nervous system in general.

Understanding gene regulatory networks (GRNs) that control neuronal differentiation will provide systems-level perspectives on neurogenesis. We have previously constructed a model for a GRN in retinal ganglion cell (RGC) differentiation in which four hierarchical tiers of transcription factors ultimately control the expression of downstream terminal genes. Math5 occupies a central node in the hierarchy because it is essential for the formation of RGCs and the expression of the immediate downstream factor Pou4f2. Based on its expression, we also proposed that Isl1, a LIM-homeodomain factor, functions in parallel with Pou4f2 and downstream of Math5 in the RGC GRN. To determine whether this was the case, a conditional Isl1 allele was generated and deleted specifically in the developing retina. Although RGCs formed in Isl1 -deleted retinas, most underwent apoptosis, and few remained at later stages. By microarray analysis, we identified a distinct set of genes whose expression depended on Isl1. These genes are all downstream of Math5, and some of them, but not all, also depend on Pou4f2. Additionally, Isl1 was required for the sustained expression of Pou4f2 , suggesting that Isl1 positively regulates Pou4f2 after Math5 levels are diminished. The results demonstrate an essential role for Isl1 in RGC development and reveal two distinct but intersecting branches of the RGC GRN downstream of Math5, one directed by Pou4f2 and the other by Isl1. They also reveal that identical RGC expression patterns are achieved by different combinations of divergent inputs from upstream transcription factors.