The endoderm gene regulatory network in sea urchin embryos up to mid-blastula stage

As the result of early specification processes, sea urchin embryos eventually form various mesodermal cell lineages and a gut consisting of fore-, mid- and hindgut. The progression of specification as well as the overall spatial organization of the organism is encoded in its gene regulatory networks (GRNs). We have analyzed the GRN driving endoderm specification up to the onset of gastrulation and present in this paper the mechanisms which determine this process up to mid-blastula stage. At this stage, the embryo consists of two separate lineages of endoderm precursor cells with distinct regulatory states. One of these lineages, the veg2 cell lineage, gives rise to endoderm and mesoderm cell types. The separation of these cell fates is initiated by the spatially confined activation of the mesoderm GRN superimposed on a generally activated endoderm GRN within veg2 descendants. Here we integrate the architecture of regulatory interactions with the spatial restriction of regulatory gene expression to model the logic control of endoderm development.     

The role of the NK-homeobox gene slouch (S59) in somatic muscle patterning.

In the Drosophila embryo, a distinct class of myoblasts, designated as muscle founders, prefigures the mature pattern of somatic body wall muscles. Each founder cell appears to be instrumental in generating a single larval muscle with a defined identity. The NK homeobox gene S59 was the first of a growing number of proposed 'identity genes' that have been found to be expressed in stereotyped patterns in specific subsets of muscle founders and their progenitor cells and are thought to control their developmental fates. In the present study, we describe the effects of gain- and loss-of-function experiments with S59. We find that a null mutation in the gene encoding S59, which we have named slouch (slou), disrupts the development of all muscles that are derived from S59-expressing founder cells. The observed phenotypes upon mutation and ectopic expression of slouch include transformations of founder cell fates, thus confirming that slouch (S59) functions as an identity gene in muscle development. These fate transformations occur between sibling founder cells as well as between neighboring founders that are not lineage-related. In the latter case, we show that slouch (S59) activity is required cell-autonomously to repress the expression of ladybird (lb) homeobox genes, thereby preventing specification along the lb pathway. Together, these findings provide new insights into the regulatory interactions that establish the somatic muscle pattern.     

Cell lineage determination in the mouse

During the peri-implantation development of the mouse embryo from the blastocyst through gastrulation, Pou5f1 (OCT-4) down-regulation is closely linked to the initial step of lineage allocation to extraembryonic and embryonic somatic tissues. Subsequently, differentiation of the lineage precursors is subject to inductive tissue interactions and intercellular signalling that regulate cell proliferation and the acquisition of lineage-specific morphological and molecular characteristics. A notable variation of this process of lineage specification is the persistence of Pou5f1 activity throughout the differentiation of the primordial germ cells, which may underpin their ability to produce pluripotent progeny either as stem cells (embryonic germ cells) in vitro or as gametes in vivo. Nevertheless, intercellular signalling still plays a critical role in the specification of the primordial germ cells. The findings that primordial germ cells can be induced from any epiblast cells and that they share common progenitors with other somatic cells provide compelling evidence for the absence of a pre-determined germ line in the mouse embryo.     

Spatial mechanisms of gene regulation in metazoan embryos.

The basic characteristics of embryonic process throughout Metazoa are considered with focus on those aspects that provide insight into how cell specification occurs in the initial stages of development. There appear to be three major types of embryogenesis: Type 1, a general form characteristic of most invertebrate taxa of today, in which lineage plays an important role in the spatial organization of the early embryo, and cell specification occurs in situ, by both autonomous and conditional mechanisms; Type 2, the vertebrate form of embryogenesis, which proceeds by mechanisms that are essentially independent of cell lineage, in which diffusible morphogens and extensive early cell migration are particularly important; Type 3, the form exemplified by long germ band insects in which several different regulatory mechanisms are used to generate precise patterns of nuclear gene expression prior to cellularization. Evolutionary implications of the phylogenetic distribution of these types of embryogenesis are considered. Regionally expressed homeodomain regulators are utilized in all three types of embryo, in similar ways in later and postembryonic development, but in different ways in early embryonic development. A specific downstream molecular function for this class of regulator is proposed, based on evidence obtained in vertebrate systems. This provides a route by which to approach the comparative regulatory strategies underlying the three major types of embryogenesis.     

Direct and indirect control of oral ectoderm regulatory gene expression by Nodal signaling in the sea urchin embryo

The Nodal signaling pathway is known from earlier work to be an essential mediator of oral ectoderm specification in the sea urchin embryo, and indirectly, of aboral ectoderm specification as well. Following expression of the Nodal ligand in the future oral ectoderm during cleavage, a sequence of regulatory gene activations occur within this territory which depend directly or indirectly on nodal gene expression. Here we describe additional regulatory genes that contribute to the oral ectoderm regulatory state during specification in Strongylocentrotus purpuratus, and show how their spatial expression changes dynamically during development. By means of system wide perturbation analyses we have significantly improved current knowledge of the epistatic relations among the regulatory genes of the oral ectoderm. From these studies there emerge diverse circuitries relating downstream regulatory genes directly and indirectly to Nodal signaling. A key intermediary regulator, the role of which had not previously been discerned, is the not gene. In addition to activating several genes earlier described as targets of Nodal signaling, the not gene product acts to repress other oral ectoderm genes, contributing crucially to the bilateral spatial organization of the embryonic oral ectoderm.     

How embryos work: a comparative view of diverse modes of cell fate specification

Embryonic processes in the nematode C. elegans, the gastropod mollusc Ilyanassa, the dipteran Drosophila, the echinoid Strongylocentrotus purpuratus, the ascidian Ciona, the anuran Xenopus, the teleost Brachydanio and mouse are compared with respect to a series of parameters such as invariant or variable cleavage, the means by which the embryonic axes are set up, egg anisotropies and reliance on conditional or on autonomous specification processes. A molecular interpretation of these modes of specification of cell fate in the embryo is proposed, in terms of spatial modifications of gene regulatory factors. On this basis, classically defined phenomena such as regulative development and cytoplasmic localization can be interpreted at a mechanistic level, and the enormous differences between different forms of embryogenesis in the Animal Kingdom can be considered within a common mechanistic framework. Differential spatial expression of histospecific genes is considered in terms of the structure of the gene regulatory network that will be required in embryos that utilize cell-cell interaction, autonomous vs conditional specification and maternal spatial information to differing extents. It is concluded that the regulatory architectures according to which the programs of gene expression are organized are special to each form of development, and that common regulatory principles are to be found only at lower levels, such as those at which the control regions of histospecific structural genes operate.     

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells ^1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.5^4,5. Myogenin is a regulatory factor required for skeletal muscle differentiation ^6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development ^6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.     

Complexity of developmental control: analysis of embryonic cell lineage specification in Caenorhabditis elegans using pes-1 as an early marker.

In the early Caenorhabditis elegans embryo five somatic founder cells are born during the first cleavages. The first of these founder cells, named AB, gives rise to 389 of the 558 nuclei present in the hatching larva. Very few genes directly involved in the specification of the AB lineage have been identified so far. Here we describe a screen of a large collection of maternal-effect embryonic lethal mutations for their effect on the early expression of a pes-1::lacZ fusion gene. This fusion gene is expressed in a characteristic pattern in 14 of the 32 AB descendants present shortly after the initiation of gastrulation. Of the 37 mutations in 36 genes suspected to be required specifically during development, 12 alter the expression of the pes-1::lacZ marker construct. The gene expression pattern alterations are of four types: reduction of expression, variable expression, ectopic expression in addition to the normal pattern, and reduction of the normal pattern together with ectopic expression. We estimate that approximately 100 maternal functions are required to establish the pes-1 expression pattern in the early embryo.     

Capturing pluripotency

In this Essay, we argue that pluripotent epiblast founder cells in the embryo and embryonic stem (ES) cells in culture represent the ground state for a mammalian cell, signified by freedom from developmental specification or epigenetic restriction and capacity for autonomous self-replication. We speculate that cell-to-cell variation may be integral to the ES cell condition, safe-guarding self-renewal while continually presenting opportunities for lineage specification.