Stefan C. Materna

High accuracy, high-resolution prevalence measurement for the majority of locally expressed regulatory genes in early sea urchin development

Accurate measurements of transcript abundance are a prerequisite to understand gene activity in development. Using the NanoString nCounter, an RNA counting device, we measured the prevalence of 172 transcription factors and signaling molecules in early sea urchin development. These measurements show high fidelity over more than five orders of magnitude down to a few transcripts per embryo. Most of the genes included are locally restricted in their spatial expression, and contribute to the divergent regulatory states of cells in the developing embryo. In order to obtain high-resolution expression profiles from fertilization to late gastrulation samples were collected at hourly intervals. The measured time courses agree well with, and substantially extend, prior relative abundance measurements obtained by quantitative PCR. High temporal resolution permits sequences of successively activated genes to be precisely delineated providing an ancillary tool for assembling maps of gene regulatory networks. The data are available via an interactive website for quick plotting of selected time courses.

A comprehensive analysis of Delta signaling in pre-gastrular sea urchin embryos.

In sea urchin embryos Delta signaling specifies non-skeletogenic mesoderm (NSM). Despite the identification of some direct targets, several aspects of Delta Notch (D/N) signaling remain supported only by circumstantial evidence. To obtain a detailed and more complete image of Delta function we followed a systems biology approach and evaluated the effects of D/N perturbation on expression levels of 205 genes up to gastrulation. This gene set includes virtually all transcription factors that are expressed in a localized fashion by mid-gastrulation, and which thus provide spatial regulatory information to the embryo. Also included are signaling factors and some pigment cell differentiation genes. We show that the number of pregastrular D/N signaling targets among these regulatory genes is small and is almost exclusively restricted to non-skeletogenic mesoderm genes. However, Delta signaling also activates foxY in the small micromeres. As is the early NSM, the small micromeres are in direct contact with Delta expressing skeletogenic mesoderm. In contrast, no endoderm regulatory genes are activated by Delta signaling even during the second phase of delta expression, when this gene is transcribed in NSM cells adjacent to the endoderm. During this phase Delta provides an ongoing input which continues to activate foxY expression in small micromere progeny. Disruption of the second phase of Delta expression specifically abolishes specification of late mesodermal derivatives such as the coelomic pouches to which the small micromeres contribute.

Diversification of oral and aboral mesodermal regulatory states in pregastrular sea urchin embryos.

Specification of the non-skeletogenic mesoderm (NSM) in sea urchin embryos depends on Delta signaling. Signal reception leads to expression of regulatory genes that later contribute to the aboral NSM regulatory state. In oral NSM, this is replaced by a distinct oral regulatory state in consequence of Nodal signaling. Through regulome wide analysis we identify the homeobox gene not as an immediate Nodal target. not expression in NSM causes extinction of the aboral regulatory state in the oral NSM, and expression of a new suite of regulatory genes. All NSM specific regulatory genes are henceforth expressed exclusively, in oral or aboral domains, presaging the mesodermal cell types that will emerge. We have analyzed the regulatory linkages within the aboral NSM gene regulatory network. A linchpin of this network is gataE which as we show is a direct Gcm target and part of a feedback loop locking down the aboral regulatory state.

A protocol for unraveling gene regulatory networks

Regulatory genes form large networks that are fundamental to the developmental program. The protocol presented here describes a general approach to assemble maps of gene regulatory networks (GRNs). It combines high-resolution spatio-temporal profiling of regulatory genes, strategies to perturb gene expression and quantification of perturbation effects on other genes of the network. The map of the GRN emerges by integration of these data sources and explains developmental events in terms of functional linkages between regulatory genes. This protocol has been successfully applied to regulatory processes in the sea urchin embryo, but it is generally applicable to any developmental process that relies primarily on transcriptional regulation. Unraveling the GRN for a whole tissue or organ is a challenging undertaking and, depending on the complexity, may take anywhere from months to years to complete.

Notch and Nodal control forkhead factor expression in the specification of multipotent progenitors in sea urchin

Indirect development, in which embryogenesis gives rise to a larval form, requires that some cells retain developmental potency until they contribute to the different tissues in the adult, including the germ line, in a later, post-embryonic phase. In sea urchins, the coelomic pouches are the major contributor to the adult, but how coelomic pouch cells (CPCs) are specified during embryogenesis is unknown. Here we identify the key signaling inputs into the CPC specification network and show that the forkhead factor foxY is the first transcription factor specifically expressed in CPC progenitors. Through dissection of its cis-regulatory apparatus we determine that the foxY expression pattern is the result of two signaling inputs: first, Delta/Notch signaling activates foxY in CPC progenitors; second, Nodal signaling restricts its expression to the left side, where the adult rudiment will form, through direct repression by the Nodal target pitx2. A third signal, Hedgehog, is required for coelomic pouch morphogenesis and institution of laterality, but does not directly affect foxY transcription. Knockdown of foxY results in a failure to form coelomic pouches and disrupts the expression of virtually all transcription factors known to be expressed in this cell type. Our experiments place foxY at the top of the regulatory hierarchy underlying the specification of a cell type that maintains developmental potency.

The Sea Urchin Genome as a Window on Function

The emphasis on the sequencing of genomes seems to make this task an end in itself. However, genome sequences and the genes that are predicted from them are really an opportunity to examine the biological function of the organism constructed by that genome. This point is illustrated here by examples in which the newly annotated gene complement reveals surprises about the way Strongylocentrotus purpuratus, the purple sea urchin, goes about its business. The three topics considered here are the nature of the innate immune system; the unexpected complexity of sensory function implied by genes encoding sensory proteins; and the remarkable intricacy of the regulatory gene complement in embryogenesis.