SUMMARYThe gene regulatory network (GRN) that underlies the development of the embryonic skeleton in sea urchins is an important model for understanding the architecture and evolution of developmental GRNs. The initial deployment of the network is thought to be regulated by a derepression mechanism, which is mediated by the products of the pmar1 and hesC genes. Here, we show that the activation of the skeletogenic network occurs by a mechanism that is distinct from the transcriptional repression of hesC. By means of quantitative, fluorescent whole-mount in situ hybridization, we find that two pivotal early genes in the network, alx1 and delta, are activated in prospective skeletogenic cells prior to the downregulation of hesC expression. An analysis of the upstream regulation of alx1 shows that this gene is regulated by MAPK signaling and by the transcription factor Ets1; however, these inputs influence only the maintenance of alx1 expression and not its activation, which occurs by a distinct mechanism. By altering normal cleavage patterns, we show that the zygotic activation of alx1 and delta, but not that of pmar1, is dependent upon the unequal division of vegetal blastomeres. Based on these findings, we conclude that the widely accepted double-repression model is insufficient to account for the localized activation of the skeletogenic GRN. We postulate the existence of additional, unidentified repressors that are controlled by pmar1, and propose that the ability of pmar1 to derepress alx1 and delta is regulated by the unequal division of vegetal blastomeres.

Cell fates in the sea urchin embryo are remarkably labile, despite the fact that maternal polarity and zygotic programs of differential gene expression pattern the embryo from the earliest stages. Recent work has focused on transcriptional gene regulatory networks (GRNs) deployed in specific embryonic territories during early development. The micromere-primary mesenchyme cell (PMC) GRN drives the development of the embryonic skeleton. Although normally deployed only by presumptive PMCs, every lineage of the early embryo has the potential to activate this pathway. Here, we focus on one striking example of regulative activation of the skeletogenic GRN; the transfating of non-skeletogenic mesoderm (NSM) cells to a PMC fate during gastrulation. We show that transfating is accompanied by the de novo expression of terminal, biomineralization-related genes in the PMC GRN, as well as genes encoding two upstream transcription factors, Lvalx1 and Lvtbr. We report that Lvalx1, a key component of the skeletogenic GRN in the PMC lineage, plays an essential role in the regulative pathway both in NSM cells and in animal blastomeres. MAPK signaling is required for the expression of Lvalx1 and downstream skeletogenic genes in NSM cells, mirroring its role in the PMC lineage. We also demonstrate that Lvalx1 regulates the signal from PMCs that normally suppresses NSM transfating. Significantly, misexpression of Lvalx1 in macromeres (the progenitors of NSM cells) is sufficient to activate the skeletogenic GRN. We suggest that NSM cells normally deploy a basal mesodermal pathway and require only an Lvalx1-mediated sub-program to express a PMC fate. Finally, we provide evidence that, in contrast to the normal pathway, activation of the skeletogenic GRN in NSM cells is independent of Lvpmar1. Our studies reveal that, although most features of the micromere-PMC GRN are recapitulated in transfating NSM cells, different inputs activate this GRN during normal and regulative development.

SUMMARYThe well-known regulative properties of the sea urchin embryo, coupled with the recent elucidation of gene regulatory networks (GRNs) that underlie cell specification, make this a valuable experimental model for analyzing developmental plasticity. In the sea urchin, the primary mesenchyme cell (PMC) GRN controls the development of the embryonic skeleton. Remarkably, experimental manipulations reveal that this GRN can be activated in almost any cell of the embryo. Here, we focus on the activation of the PMC GRN during gastrulation by non-skeletogenic mesoderm (NSM) cells and by endoderm cells. We show that most transfating NSM cells are prospective blastocoelar cells, not prospective pigment cells, as was previously believed. Earlier work showed that the regulative deployment of the GRN, unlike its deployment in the micromere-PMC lineage, is independent of the transcriptional repressor Pmar1. In this work, we identify several additional differences in the upstream regulation of the GRN during normal and regulative development. We provide evidence that, despite these changes in the upstream regulation of the network, downstream regulatory genes and key morphoregulatory genes are deployed in transfating NSM cells in a fashion that recapitulates the normal deployment of the GRN, and which can account for the striking changes in migratory behavior that accompany NSM transfating. Finally, we report that mitotic cell division is not required for genomic reprogramming in this system, either within a germ layer (NSM transfating) or across a germ layer boundary (endoderm transfating).

The number of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) cases is increasing in India. This study looks upon the geographic distribution of the virus clades and variants circulating in different parts of India between January and August 2020. The NPS/OPS from representative positive cases from different states and union territories in India were collected every month through the VRDLs in the country and analyzed using next-generation sequencing. Epidemiological analysis of the 689 SARS-CoV-2 clinical samples revealed GH and GR to be the predominant clades circulating in different states in India. The northern part of India largely reported the ‘GH’ clade, whereas the southern part reported the ‘GR’, with a few exceptions. These sequences also revealed the presence of single independent mutations—E484Q and N440K—from Maharashtra (first observed in March 2020) and Southern Indian States (first observed in May 2020), respectively. Furthermore, this study indicates that the SARS-CoV-2 variant (VOC, VUI, variant of high consequence and double mutant) was not observed during the early phase of virus transmission (January–August). This increased number of variations observed within a short timeframe across the globe suggests virus evolution, which can be a step towards enhanced host adaptation.

The analysis of temporal and spatial patterns of gene expression is critically important for many kinds of developmental studies, including the construction of gene regulatory networks. Recently, multiplex, fluorescent, whole mount in situ hybridization (multiplex F-WMISH), applied in combination with confocal microscopy, has emerged as the method of choice for high-resolution, three-dimensional (3D) mapping of gene expression patterns in developing tissues. We have developed an image analysis tool, GeneExpressMap (GEM), that facilitates the rapid, 3D analysis of multiplex F-WMISH data at single-cell resolution. GEM assigns F-WMISH signal to individual cells based upon the proximity of cytoplasmic hybridization signal to cell nuclei. Here, we describe the features of GEM and, as a test of its utility, we use GEM to analyze patterns of regulatory gene expression in the non-skeletogenic mesoderm of the early sea urchin embryo. GEM greatly extends the power of multiplex F-WMISH for analyzing patterns of gene expression and is a valuable tool for gene network analysis and many other kinds of developmental studies.