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ABSTRACT Sea urchins and other echinoderms are important experimental models for studying developmental processes. The lack of approaches for conditional gene perturbation, however, has made it challenging to investigate the late developmental functions of genes that have essential roles during early embryogenesis and genes that have diverse functions in multiple tissues. The doxycycline-controlled Tet-On system is a widely used molecular tool for temporally and spatially regulated transgene expression. Here, we optimized the Tet-On system to conditionally induce gene expression in sea urchin embryos. Using this approach, we explored the roles the MAPK signaling plays in skeletogenesis by expressing genes that perturb the pathway specifically in primary mesenchyme cells during later stages of development. We demonstrated the wide utility of the Tet-On system by applying it to a second sea urchin species and in cell types other than the primary mesenchyme cells. Our work provides a robust and flexible platform for the spatiotemporal regulation of gene expression in sea urchins, which will considerably enhance the utility of this prominent model system. 

The gene regulatory network (GRN) that underlies echinoderm skeletogenesis is a prominent model of GRN architecture and evolution. KirrelL is an essential downstream effector gene in this network and encodes an Ig-superfamily protein required for the fusion of skeletogenic cells and the formation of the skeleton. In this study, we dissected the transcriptional control region of the kirrelL gene of the purple sea urchin, Strongylocentrotus purpuratus . Using plasmid- and bacterial artificial chromosome-based transgenic reporter assays, we identified key cis -regulatory elements (CREs) and transcription factor inputs that regulate Sp-kirrelL , including direct, positive inputs from two key transcription factors in the skeletogenic GRN, Alx1 and Ets1. We next identified kirrelL cis -regulatory regions from seven other echinoderm species that together represent all classes within the phylum. By introducing these heterologous regulatory regions into developing sea urchin embryos we provide evidence of their remarkable conservation across ~500 million years of evolution. We dissected in detail the kirrelL regulatory region of the sea star, Patiria miniata , and demonstrated that it also receives direct inputs from Alx1 and Ets1. Our findings identify kirrelL as a component of the ancestral echinoderm skeletogenic GRN. They support the view that GRN subcircuits, including specific transcription factor–CRE interactions, can remain stable over vast periods of evolutionary history. Lastly, our analysis of kirrelL establishes direct linkages between a developmental GRN and an effector gene that controls a key morphogenetic cell behavior, cell–cell fusion, providing a paradigm for extending the explanatory power of GRNs. 

We used capped analysis of gene expression with sequencing (CAGE-seq) to profile eRNA expression and enhancer activity during embryogenesis of a model echinoderm: the sea urchin, Strongylocentrotus purpuratus . We identified more than 18,000 enhancers that were active in mature oocytes and developing embryos and documented a burst of enhancer activation during cleavage and early blastula stages. We found that a large fraction (73.8%) of all enhancers active during the first 48 h of embryogenesis were hyperaccessible no later than the 128-cell stage and possibly even earlier. Most enhancers were located near gene bodies, and temporal patterns of eRNA expression tended to parallel those of nearby genes. Furthermore, enhancers near lineage-specific genes contained signatures of inputs from developmental gene regulatory networks deployed in those lineages. A large fraction (60%) of sea urchin enhancers previously shown to be active in transgenic reporter assays was associated with eRNA expression. Moreover, a large fraction (50%) of a representative subset of enhancers identified by eRNA profiling drove tissue-specific gene expression in isolation when tested by reporter assays. Our findings provide an atlas of developmental enhancers in a model sea urchin and support the utility of eRNA profiling as a tool for enhancer discovery and regulatory biology. The data generated in this study are available at Echinobase, the public database of information related to echinoderm genomics. 

The skeleton-forming cells of sea urchins and other echinoderms have been studied by developmental biologists as models of cell specification and morphogenesis for many decades. The gene regulatory network (GRN) deployed in the embryonic skeletogenic cells of euechinoid sea urchins is one of the best understood in any developing animal. Recent comparative studies have leveraged the information contained in this GRN, bringing renewed attention to the diverse patterns of skeletogenesis within the phylum and the evolutionary basis for this diversity. The homeodomain-containing transcription factor, Alx1, was originally shown to be a core component of the skeletogenic GRN of the sea urchin embryo. Alx1 has since been found to be key regulator of skeletal cell identity throughout the phylum. As such, Alx1 is currently serving as a lens through which multiple developmental processes are being investigated. These include not only GRN organization and evolution, but also cell reprogramming, cell type evolution, and the gene regulatory control of morphogenesis. This review summarizes our current state of knowledge concerning Alx1 and highlights the insights it is yielding into these important developmental and evolutionary processes. 

Members of the alx gene family encode transcription factors that contain a highly conserved Paired-class, DNA-binding homeodomain, and a C-terminal OAR/Aristaless domain. Phylogenetic and comparative genomic studies have revealed complex patterns of alx gene duplications during deuterostome evolution. Remarkably, alx genes have been implicated in skeletogenesis in both echinoderms and vertebrates. In this review, we provide an overview of current knowledge concerning alx genes in deuterostomes. We highlight their evolutionarily conserved role in skeletogenesis and draw parallels and distinctions between the skeletogenic gene regulatory circuitries of diverse groups within the superphylum.