Embryonic Gene Regulatory Network Research Articles

Feedback circuits are numerous in embryonic gene regulatory networks and offer a stabilizing influence on evolution of those networks

The developmental gene regulatory networks (dGRNs) of two sea urchin species, Lytechinus variegatus (Lv) and Strongylocentrotus purpuratus (Sp), have remained remarkably similar despite about 50 million years since a common ancestor. Hundreds of parallel experimental perturbations of transcription factors with similar outcomes support this conclusion. A recent scRNA-seq analysis suggested that the earliest expression of several genes within the dGRNs differs between Lv and Sp. Here, we present a careful reanalysis of the dGRNs in these two species, paying close attention to timing of first expression. We find that initial expression of genes critical for cell fate specification occurs during several compressed time periods in both species. Previously unrecognized feedback circuits are inferred from the temporally corrected dGRNs. Although many of these feedbacks differ in location within the respective GRNs, the overall number is similar between species. We identify several prominent differences in timing of first expression for key developmental regulatory genes; comparison with a third species indicates that these heterochronies likely originated in an unbiased manner with respect to embryonic cell lineage and evolutionary branch. Together, these results suggest that interactions can evolve even within highly conserved dGRNs and that feedback circuits may buffer the effects of heterochronies in the expression of key regulatory genes.

Origin of the Chordate Notochord

The phylum of Chordata is defined based on the discovery of a coelom-like dorsal notochord in ascidian and amphioxus embryos. Chordata can be classified into three subphylums, Cephalochordata, Urochordata, and Vertebrata, united by the presence of a notochord at some point during development. The origin of the notochord, the signature anatomical structure of chordates, has been under debate since the publication of Alexander Kovalevsky’s work in the mid-19th century that placed ascidians close to the vertebrates on the phylogenetic tree. During the late 20th century, the development of molecular and genetic tools in biology brought about a revival of studies on the evolutionary path of notochord development. Two main hypotheses for the origin of the notochord were proposed, the de novo theory and the axochord theory. The former states that notochord has developed de novo from the mid-dorsal archenteron of a chordate ancestor with simple morphology and no central nervous system nor notochord homolog. The putative notochord along the dorsal side of the animal is proposed to take on the signal functions later from the endoderm and ectoderm. An alternative hypothesis, the axochord theory, proposes that notochord has evolved from the mid-line muscle tissue, the so-called axochord, in annelids. Structural and molecular evidence point to the midline muscle of annelids as a distant homolog of the notochord. This hypothesis thus suggests a notochord-like structure in the urbilaterian ancestor, opposed to the consensus that notochord is a chordate-specific feature. In this review, we introduce the history of the formation of these views and summarize the current understandings of embryonic development, molecular profile, and gene regulatory networks of notochord and notochord-like structures.

Natural cryptic variation in epigenetic modulation of an embryonic gene regulatory network

Gene regulatory networks (GRNs) that direct animal embryogenesis must respond to varying environmental and physiological conditions to ensure robust construction of organ systems. While GRNs are evolutionarily modified by natural genomic variation, the roles of epigenetic processes in shaping plasticity of GRN architecture are not well understood. The endoderm GRN in Caenorhabditis elegans is initiated by the maternally supplied SKN-1/Nrf2 bZIP transcription factor; however, the requirement for SKN-1 in endoderm specification varies widely among distinct C. elegans wild isotypes, owing to rapid developmental system drift driven by accumulation of cryptic genetic variants. We report here that heritable epigenetic factors that are stimulated by transient developmental diapause also underlie cryptic variation in the requirement for SKN-1 in endoderm development. This epigenetic memory is inherited from the maternal germline, apparently through a nuclear, rather than cytoplasmic, signal, resulting in a parent-of-origin effect (POE), in which the phenotype of the progeny resembles that of the maternal founder. The occurrence and persistence of POE varies between different parental pairs, perduring for at least 10 generations in one pair. This long-perduring POE requires piwi-interacting RNA (piRNA) function and the germline nuclear RNA interference (RNAi) pathway, as well as MET-2 and SET-32, which direct histone H3K9 trimethylation and drive heritable epigenetic modification. Such nongenetic cryptic variation may provide a resource of additional phenotypic diversity through which adaptation may facilitate evolutionary changes and shape developmental regulatory systems.

Evolution and Developmental System Drift in the Endoderm Gene Regulatory Network of Caenorhabditis and Other Nematodes.

Developmental gene regulatory networks (GRNs) underpin metazoan embryogenesis and have undergone substantial modification to generate the tremendous variety of animal forms present on Earth today. The nematode Caenorhabditis elegans has been a central model for advancing many important discoveries in fundamental mechanistic biology and, more recently, has provided a strong base from which to explore the evolutionary diversification of GRN architecture and developmental processes in other species. In this short review, we will focus on evolutionary diversification of the GRN for the most ancient of the embryonic germ layers, the endoderm. Early embryogenesis diverges considerably across the phylum Nematoda. Notably, while some species deploy regulative development, more derived species, such as C. elegans, exhibit largely mosaic modes of embryogenesis. Despite the relatively similar morphology of the nematode gut across species, widespread variation has been observed in the signaling inputs that initiate the endoderm GRN, an exemplar of developmental system drift (DSD). We will explore how genetic variation in the endoderm GRN helps to drive DSD at both inter- and intraspecies levels, thereby resulting in a robust developmental system. Comparative studies using divergent nematodes promise to unveil the genetic mechanisms controlling developmental plasticity and provide a paradigm for the principles governing evolutionary modification of an embryonic GRN.

Extensive intraspecies cryptic variation in an ancient embryonic gene regulatory network.

Innovations in metazoan development arise from evolutionary modification of gene regulatory networks (GRNs). We report widespread cryptic variation in the requirement for two key regulatory inputs, SKN-1/Nrf2 and MOM-2/Wnt, into the C. elegans endoderm GRN. While some natural isolates show a nearly absolute requirement for these two regulators, in others, most embryos differentiate endoderm in their absence. GWAS and analysis of recombinant inbred lines reveal multiple genetic regions underlying this broad phenotypic variation. We observe a reciprocal trend, in which genomic variants, or knockdown of endoderm regulatory genes, that result in a high SKN-1 requirement often show low MOM-2/Wnt requirement and vice-versa, suggesting that cryptic variation in the endoderm GRN may be tuned by opposing requirements for these two key regulatory inputs. These findings reveal that while the downstream components in the endoderm GRN are common across metazoan phylogeny, initiating regulatory inputs are remarkably plastic even within a single species.

A bipolar role of the transcription factor ERG for cnidarian germ layer formation and apical domain patterning

Germ layer formation and axial patterning are biological processes that are tightly linked during embryonic development of most metazoans. In addition to canonical WNT, it has been proposed that ERK-MAPK signaling is involved in specifying oral as well as aboral territories in cnidarians. However, the effector and the molecular mechanism underlying latter phenomenon is unknown. By screening for potential effectors of ERK-MAPK signaling in both domains, we identified a member of the ETS family of transcription factors, Nverg that is bi-polarily expressed prior to gastrulation. We further describe the crucial role of NvERG for gastrulation, endomesoderm as well as apical domain formation. The molecular characterization of the obtained NvERG knock-down phenotype using previously described as well as novel potential downstream targets, provides evidence that a single transcription factor, NvERG, simultaneously controls expression of two different sets of downstream targets, leading to two different embryonic gene regulatory networks (GRNs) in opposite poles of the developing embryo. We also highlight the molecular interaction of cWNT and MEK/ERK/ERG signaling that provides novel insight into the embryonic axial organization of Nematostella, and show a cWNT repressive role of MEK/ERK/ERG signaling in segregating the endomesoderm in two sub-domains, while a common input of both pathways is required for proper apical domain formation. Taking together, we build the first blueprint for a global cnidarian embryonic GRN that is the foundation for additional gene specific studies addressing the evolution of embryonic and larval development.

An Integrative Developmental Genomics and Systems Biology Approach to Identify an In Vivo Sox Trio-Mediated Gene Regulatory Network in Murine Embryos.

Embryogenesis is an intricate process involving multiple genes and pathways. Some of the key transcription factors controlling specific cell types are the Sox trio, namely, Sox5, Sox6, and Sox9, which play crucial roles in organogenesis working in a concerted manner. Much however still needs to be learned about their combinatorial roles during this process. A developmental genomics and systems biology approach offers to complement the reductionist methodology of current developmental biology and provide a more comprehensive and integrated view of the interrelationships of complex regulatory networks that occur during organogenesis. By combining cell type-specific transcriptome analysis and in vivo ChIP-Seq of the Sox trio using mouse embryos, we provide evidence for the direct control of Sox5 and Sox6 by the transcriptional trio in the murine model and by Morpholino knockdown in zebrafish and demonstrate the novel role of Tgfb2, Fbxl18, and Tle3 in formation of Sox5, Sox6, and Sox9 dependent tissues. Concurrently, a complete embryonic gene regulatory network has been generated, identifying a wide repertoire of genes involved and controlled by the Sox trio in the intricate process of normal embryogenesis.

Reversion to an Embryonic Alternative Splicing Program Enhances Leukemia Stem Cell Self-Renewal

BackgroundFormative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. However, its role in malignant reprogramming of progenitors into self-renewing leukemia stem cells (LSCs) had not been established.MethodsWhole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified progenitors from normal, chronic phase and blast crisis chronic myeloid leukemia samples and analyzed using Cuff-links, GSEA and IPA software. Splice isoform specific qRT-PCR, confocal microscopy, lentiviral overexpression and shRNA knockdown experiments were performed according to published methods (Jamieson NEJM 2004; Geron et al Cancer Cell 2008; Goff et al Cell Stem Cell 2013).ResultsWe performed LSC RNA-seq, lentiviral overexpression and knockdown and discovered that decreased expression of MBNL3, a repressor of an embryonic alternative splicing program and reprogramming, activated a pluripotency network and increased expression of a pro-survival isoform of CD44v3, which is more commonly expressed in human embryonic stem cells. This resulted in malignant reprogramming of progenitors in blast crisis CML endowing them with unbridled survival and self-renewal capacity. This is the first description of MBNL3 downregulation as a mechanism of reversion to an embryonic alternative splicing program, which elicits malignant progenitor reprogramming of progenitors into self-renewing leukemia stem cells. While isoform specific lentiviral CD44v3 overexpression enhanced chronic phase CML progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. In keeping with activation of a stem cell reprogramming network, CD44v3 upregulation was associated with increased expression of pluripotency transcription factors, including OCT4, SOX2 and b-catenin in addition to the pro-survival long isoforms of MCL1 and BCLX resulting in increased self-renewal and apoptosis resistance.ConclusionIn summary, MBNL3 downregulation activates an embryonic alternative splicing program, typified by CD44v3 overexpression, and represents a novel mechanism governing LSC generation in malignant microenvironments. Reversal of malignant reprogramming by epigenetic modulation of embryonic alternative splicing or via monoclonal antibody targeting of CD44v3 splice isoform may represent a pivotal opportunity for selective BC LSC eradication. DisclosuresJamieson:Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.

Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8-10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation-related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.

Eric Harris Davidson: A systems biologist who studied how genomes function.

The phrase “force of nature” has popped up repeatedly in social media responses to the announcement of Eric Davidson’s death on September 1, 2015. This sobriquet reflects his huge influence on the fields of developmental biology and evolution. His career began about the time Watson and Crick published the structure of DNA, and his contributions continued into the genomic era in which we are now immersed. But his stature wasn’t due to this timing. As Pasteur once said: “Dans les champs de l’observation le hasard ne favorise que les esprits prepares.” (“In the fields of observation chance favors only the prepared mind”). Perhaps Eric’s strong focus on the function of the genome and his great intellect also contributed to this influence. His unwavering focus on how the genome does its job must also play a role. Furthermore, he clearly took genuine delight in confronting complex biological problems, and he erected broad views of the process of nature. This point is documented in his dedication to the third edition of Gene Activity in Early Development, where he credits his father with teaching him about organizing complex perceptions. Eric Davidson was born on April 13, 1937 in New York City, New York. He is the son of Morris and Anne Davidson. His father was a renowned abstract painter of the mid-20th century who maintained a school of painting near Provincetown on Cape Cod, Massachussetts. Eric attended the University of Pennsylvania as an undergraduate, and earned a Ph.D. from Rockefeller University (then Institute). He continued at Rockefeller as a post-doc, and then as assistant professor until 1971. He then moved to the California Institute of Technology, where he remained for the remainder of his career.

Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network.

In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin-1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems-level analyses, we compiled a MKRN1-centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA-binding proteins, and ensuing RIP-chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule-resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems-level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN.

Constraint and divergence of global gene expression in the mammalian embryo.

The effects of genetic variation on gene regulation in the developing mammalian embryo remain largely unexplored. To globally quantify these effects, we crossed two divergent mouse strains and asked how genotype of the mother or of the embryo drives gene expression phenotype genomewide. Embryonic expression of 331 genes depends on the genotype of the mother. Embryonic genotype controls allele-specific expression of 1594 genes and a highly overlapping set of cis-expression quantitative trait loci (eQTL). A marked paucity of trans-eQTL suggests that the widespread expression differences do not propagate through the embryonic gene regulatory network. The cis-eQTL genes exhibit lower-than-average evolutionary conservation and are depleted for developmental regulators, consistent with purifying selection acting on expression phenotype of pattern formation genes. The widespread effect of maternal and embryonic genotype in conjunction with the purifying selection we uncovered suggests that embryogenesis is an important and understudied reservoir of phenotypic variation.

Cis-regulatory control of the nuclear receptor Coup-TF gene in the sea urchin Paracentrotus lividus embryo.

Coup-TF, an orphan member of the nuclear receptor super family, has a fundamental role in the development of metazoan embryos. The study of the gene's regulatory circuit in the sea urchin embryo will facilitate the placement of this transcription factor in the well-studied embryonic Gene Regulatory Network (GRN). The Paracentrotus lividus Coup-TF gene (PlCoup-TF) is expressed throughout embryonic development preferentially in the oral ectoderm of the gastrula and the ciliary band of the pluteus stage. Two overlapping λ genomic clones, containing three exons and upstream sequences of PlCoup-TF, were isolated from a genomic library. The transcription initiation site was determined and 5′ deletions and individual segments of a 1930 bp upstream region were placed ahead of a GFP reporter cassette and injected into fertilized P.lividus eggs. Module a (−532 to −232), was necessary and sufficient to confer ciliary band expression to the reporter. Comparison of P.lividus and Strongylocentrotus purpuratus upstream Coup-TF sequences, revealed considerable conservation, but none within module a. 5′ and internal deletions into module a, defined a smaller region that confers ciliary band specific expression. Putative regulatory cis-acting elements (RE1, RE2 and RE3) within module a, were specifically bound by proteins in sea urchin embryonic nuclear extracts. Site-specific mutagenesis of these elements resulted in loss of reporter activity (RE1) or ectopic expression (RE2, RE3). It is proposed that sea urchin transcription factors, which bind these three regulatory sites, are necessary for spatial and quantitative regulation of the PlCoup-TF gene at pluteus stage sea urchin embryos. These findings lead to the future identification of these factors and to the hierarchical positioning of PlCoup-TF within the embryonic GRN.

Sea urchin neural development and the metazoan paradigm of neurogenesis.

Summary:Urchin embryos continue to prove useful as a means of studying embryonic signaling and gene regulatory networks, which together control early development. Recent progress in understanding the molecular mechanisms underlying the patterning of ectoderm has renewed interest in urchin neurogenesis. We have employed an emerging model of neurogenesis that appears to be broadly shared by metazoans as a framework for this review. We use the model to provide context and summarize what is known about neurogenesis in urchin embryos. We review morphological features of the differentiation phase of neurogenesis and summarize current understanding of neural specification and regulation of proneural networks. Delta-Notch signaling is a common feature of metazoan neurogenesis that produces committed progenitors and it appears to be a critical phase of neurogenesis in urchin embryos. Descriptions of the differentiation phase of neurogenesis indicate a stereotypic sequence of neural differentiation and patterns of axonal growth. Features of neural differentiation are consistent with localized signals guiding growth cones with trophic, adhesive, and tropic cues. Urchins are a facile, postgenomic model with the potential of revealing many shared and derived features of deuterostome neurogenesis.

Select microRNAs are essential for early development in the sea urchin

microRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene regulation and have emerged as essential regulators of many developmental events. The transcriptional network during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus, is well described and can serve as an excellent model to test functional contributions of miRNAs in embryogenesis. We examined the loss of function phenotypes of major components of the miRNA biogenesis pathway. Inhibition of de novo synthesis of Drosha and Dicer in the embryo led to consistent developmental defects, a failure to gastrulate, and embryonic lethality, including changes in the steady state levels of transcription factors and signaling molecules involved in germ layer specification. We annotated and profiled small RNA expression from the ovary and several early embryonic stages by deep sequencing followed by computational analysis. miRNAs as well as a large population of putative piRNAs (piwi-interacting RNAs) had dynamic accumulation profiles through early development. Defects in morphogenesis caused by loss of Drosha could be rescued with four miRNAs. Taken together our results indicate that post-transcriptional gene regulation directed by miRNAs is functionally important for early embryogenesis and is an integral part of the early embryonic gene regulatory network in S. purpuratus.

Integrating post‐transcriptional regulation into the embryonic stem cell gene regulatory network

Stem cell behavior is orchestrated as a multilayered, concert of gene regulatory mechanisms collectively referred to as the gene regulatory network (GRN). Via cooperative mechanisms, transcriptional, epigenetic, and post-transcriptional regulators activate and repress gene expression to finely regulate stem cell self-renewal and commitment. Due to their tractability, embryonic stem cells (ESCs) serve as the model stem cell to dissect the complexities of the GRN, and discern its relation to stem cell fate. By way of high-throughput genomic analysis, targets of individual gene regulators have been established in ESCs. The compilation of these discrete networks has revealed convergent, multi-dimensional gene regulatory mechanisms involving transcription factors, epigenetic modifiers, non-coding RNA (ncRNA), and RNA-binding proteins. Here we highlight the seminal genomic studies that have shaped our understanding of the ESC GRN and describe alternate post-transcriptional gene regulatory mechanisms that require in depth analyses to draft networks that fully model ESC behavior.

Evolution of the mammalian embryonic pluripotency gene regulatory network

Embryonic pluripotency in the mouse is established and maintained by a gene-regulatory network under the control of a core set of transcription factors that include octamer-binding protein 4 (Oct4; official name POU domain, class 5, transcription factor 1, Pou5f1), sex-determining region Y (SRY)-box containing gene 2 (Sox2), and homeobox protein Nanog. Although this network is largely conserved in eutherian mammals, very little information is available regarding its evolutionary conservation in other vertebrates. We have compared the embryonic pluripotency networks in mouse and chick by means of expression analysis in the pregastrulation chicken embryo, genomic comparisons, and functional assays of pluripotency-related regulatory elements in ES cells and blastocysts. We find that multiple components of the network are either novel to mammals or have acquired novel expression domains in early developmental stages of the mouse. We also find that the downstream action of the mouse core pluripotency factors is mediated largely by genomic sequence elements nonconserved with chick. In the case of Sox2 and Fgf4, we find that elements driving expression in embryonic pluripotent cells have evolved by a small number of nucleotide changes that create novel binding sites for core factors. Our results show that the network in charge of embryonic pluripotency is an evolutionary novelty of mammals that is related to the comparatively extended period during which mammalian embryonic cells need to be maintained in an undetermined state before engaging in early differentiation events.

P71. Stem cells and gene expression during hemichordate head regeneration

Hemichordate acorn worms grow a new head on the anterior wound surface when the body is transected. In Ptychodera flava, a blastema is evident by 3 days and a new full-sized, functional head differentiates on the cut stump by 12 days. The hemichordate nervous system provides the most ancestral extant example of the dorsal hollow brain, formed by neurulation, characteristic of chordates and humans. Only in hemichordates does such a nervous system regenerate significantly. Animals throughout the phylogenetic scale share similar developmental gene regulatory networks, and our work, along with that of others, supports the conclusion that the same gene regulatory networks occur in hemichordate and chordate neural differentiation. Examination of the regeneration process by whole mount in situ hybridization (WISH) of sequences from Yamanaka pluripotency factors or “stem cell” genes, as defined by genes inducing pluripotency in mammalian somatic cells, and developmental organizer genes reveals a cascade of gene expression associated with the steps of regeneration. A subset of the stem cell genes produce the first WISH signals before a blastema is formed. The second set of stem cell genes begins to generate signal as the blastema is established and this signal persists in the blastema as organizer and early development gene signals appear as differentiation proceeds. These results suggest that activation of cells with regenerative capability involves the early expression of stem cell genes in the cells that will form the blastema. Signal from some stem cell genes continues as the expression of typical embryonic gene regulatory networks are activated for the differentiation of the tissues. In planarians, regeneration is based on mobilization of stem cells residing in the tissue of the animal to form a regeneration blastema. During regeneration of an amphibian limb, a blastema is also formed, apparently by dedifferentiation of tissue cells in the cut limb to stem cells which accumulate to establish the blastema. The nature of the cells and the tissue origin of the cells that participate in hemichordate blastema formation and head regeneration are being investigated. Supported by the PBRC Biomedical Fund UHF#12-209-04.

Repression of mesodermal fate byfoxa, a key endoderm regulator of the sea urchin embryo

The foxa gene is an integral component of the endoderm specification subcircuit of the endomesoderm gene regulatory network in the Strongylocentrotus purpuratus embryo. Its transcripts become confined to veg2, then veg1 endodermal territories, and, following gastrulation, throughout the gut. It is also expressed in the stomodeal ectoderm. gatae and otx genes provide input into the pregastrular regulatory system of foxa, and Foxa represses its own transcription, resulting in an oscillatory temporal expression profile. Here, we report three separate essential functions of the foxa gene: it represses mesodermal fate in the veg2 endomesoderm; it is required in postgastrular development for the expression of gut-specific genes; and it is necessary for stomodaeum formation. If its expression is reduced by a morpholino, more endomesoderm cells become pigment and other mesenchymal cell types, less gut is specified, and the larva has no mouth. Experiments in which blastomere transplantation is combined with foxa MASO treatment demonstrate that, in the normal endoderm, a crucial role of Foxa is to repress gcm expression in response to a Notch signal, and hence to repress mesodermal fate. Chimeric recombination experiments in which veg2, veg1 or ectoderm cells contained foxa MASO show which region of foxa expression controls each of the three functions. These experiments show that the foxa gene is a component of three distinct embryonic gene regulatory networks.

Same Difference: Sea Urchins or Starfish

DEVELOPMENT Evolutionary forces are known to work on the gene at many levels, from sequence to organization. In the sea urchin, there have been extensive efforts to map the network responsible for endoderm formation. Despite their differences—including the ability of sea urchin larvae to form a skeleton during embryogenesis—endoderm formation shows similarities between sea urchins and starfish. Hinman et al. studied the conservation and changes in embryonic gene regulatory networks during the course of metazoan evolution. They compared the expression patterns of genes involved in endoderm formation in embryo development and the effects of perturbing expression profiles. A crucial feedback loop involving three transcription factors was found to be almost identical in both of these evolutionarily distant species. — BJ Proc. Natl. Acad. Sci. U.S.A. 100, 13356 (2003).