Modeling DNA sequence-based cis-regulatory gene networks

Gene network analysis requires computationally based models which represent the functional architecture of regulatory interactions, and which provide directly testable predictions. The type of model that is useful is constrained by the particular features of developmentally active cis-regulatory systems. These systems function by processing diverse regulatory inputs, generating novel regulatory outputs. A computational model which explicitly accommodates this basic concept was developed earlier for the cis-regulatory system of the endo16 gene of the sea urchin. This model represents the genetically mandated logic functions that the system executes, but also shows how time-varying kinetic inputs are processed in different circumstances into particular kinetic outputs. The same basic design features can be utilized to construct models that connect the large number of cis-regulatory elements constituting developmental gene networks. The ultimate aim of the network models discussed here is to represent the regulatory relationships among the genomic control systems of the genes in the network, and to state their functional meaning. The target site sequences of the cis-regulatory elements of these genes constitute the physical basis of the network architecture. Useful models for developmental regulatory networks must represent the genetic logic by which the system operates, but must also be capable of explaining the real time dynamics of cis-regulatory response as kinetic input and output data become available. Most importantly, however, such models must display in a direct and transparent manner fundamental network design features such as intra- and intercellular feedback circuitry; the sources of parallel inputs into each cis-regulatory element; gene battery organization; and use of repressive spatial inputs in specification and boundary formation. Successful network models lead to direct tests of key architectural features by targeted cis-regulatory analysis.     

R11: a cis-regulatory node of the sea urchin embryo gene network that controls early expression of SpDelta in micromeres

A gene regulatory network (GRN) controls the process by which the endomesoderm of the sea urchin embryo is specified. In this GRN, the program of gene expression unique to the skeletogenic micromere lineage is set in train by activation of the pmar1 gene. Through a double repression system, this gene is responsible for localization of expression of downstream regulatory and signaling genes to cells of this lineage. One of these genes, delta, encodes a Notch ligand, and its expression in the right place and time is crucial to the specification of the endomesoderm. Here we report a cis-regulatory element R11 that is responsible for localizing the expression of delta by means of its response to the pmar1 repression system. R11 was identified as an evolutionarily conserved genomic sequence located about 13 kb downstream of the last exon of the delta gene. We demonstrate here that this cis-regulatory element is able to drive the expression of a reporter gene in the same cells and at the same time that the endogenous delta gene is expressed, and that temporally, spatially, and quantitatively it responds to the pmar1 repression system just as predicted for the delta gene in the endomesoderm GRN. This work illustrates the application of cis-regulatory analysis to the validation of predictions of the GRN model. In addition, we introduce new methodological tools for quantitative measurement of the output of expression constructs that promise to be of general value for cis-regulatory analysis in sea urchin embryos.     

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.     

Developmental gene network analysis

The developmental process is controlled by the information processing functions executed by the cis-elements that regulate the expression of the participating genes. A model of the network of cis-regulatory interactions that underlies the specification of the endomesoderm of the sea urchin embryo is analyzed here. Although not all the relevant interactions have yet been uncovered, the model shows how the information processing functions executed by the cis-regulatory elements involved can control essential functions of the specification process, such as transforming the localization of maternal factors into a domain-specific program of gene expression; refining the specification pattern; and stabilizing states of specification. The analysis suggests that the progressivity of the developmental process is also controlled by the cis-regulatory interactions unraveled by the network model. Given that evolution occurs by changing the program for development of the body plan, we illustrate the potential of developmental gene network analysis in understanding the process by which morphological features are maintained and diversify. Comparison of the network of cis-regulatory interactions with a portion of that underlying the specification of the endomesoderm of the starfish illustrates how the similarities and differences provide insights into how the programs for development work and how they evolve.     

Multiple signaling pathways and a selector protein sequentially regulate Drosophila wing development

Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields (Weatherbee, S. D. and Carroll, S. B. (1999) Cell 97, 283-286) and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, we studied the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs. Here, we show that a 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, we demonstrate that Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, can sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, we show that later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates.     

Perturbation analysis analyzed—mathematical modeling of intact and perturbed gene regulatory circuits for animal development

Gene regulatory networks for animal development are the underlying mechanisms controlling cell fate specification and differentiation. The architecture of gene regulatory circuits determines their information processing properties and their developmental function. It is a major task to derive realistic network models from exceedingly advanced high throughput experimental data. Here we use mathematical modeling to study the dynamics of gene regulatory circuits to advance the ability to infer regulatory connections and logic function from experimental data. This study is guided by experimental methodologies that are commonly used to study gene regulatory networks that control cell fate specification. We study the effect of a perturbation of an input on the level of its downstream genes and compare between the cis-regulatory execution of OR and AND logics. Circuits that initiate gene activation and circuits that lock on the expression of genes are analyzed. The model improves our ability to analyze experimental data and construct from it the network topology. The model also illuminates information processing properties of gene regulatory circuits for animal development.     

Developmental effector gene regulation: Multiplexed strategies for functional analysis

The staggering complexity of the genome controls for developmental processes is revealed through massively parallel cis-regulatory analysis using new methods of perturbation and readout. The choice of combinations of these new methods is tailored to the system, question and resources at hand. Our focus is on issues that include the necessity or sufficiency of given cis-regulatory modules, cis-regulatory function in the normal spatial genomic context, and easily accessible high throughput and multiplexed analysis methods. In the sea urchin embryonic model, recombineered BACs offer new opportunities for consecutive modes of cis-regulatory analyses that answer these requirements, as we here demonstrate on a diverse suite of previously unstudied sea urchin effector genes expressed in skeletogenic cells. Positively active cis-regulatory modules were located in single Nanostring experiments per BAC containing the gene of interest, by application of our previously reported “barcode” tag vectors of which>?100 can be analyzed at one time. Computational analysis of DNA sequences that drive expression, based on the known skeletogenic regulatory state, then permitted effective identification of functional target site clusters. Deletion of these sub-regions from the parent BACs revealed module necessity, as simultaneous tests of the same regions in short constructs revealed sufficiency. Predicted functional inputs were then confirmed by site mutations, all generated and tested in multiplex formats. There emerged the simple conclusion that each effector gene utilizes a small subset of inputs from the skeletogenic GRN. These inputs may function to only adjust expression levels or in some cases necessary for expression. Since we know the GRN architecture upstream of the effector genes, we could then conceptually isolate and compare the wiring of the effector gene driver sub-circuits and identify the inputs whose removal abolish expression.     

Gene regulatory network controlling embryonic specification in the sea urchin

The current state of the gene regulatory network for endomesoderm specification in sea urchin embryos is reviewed. The network was experimentally defined, and is presented as a predictive map of cis-regulatory inputs and functional regulatory gene interconnections (updated versions of the network and most of the underlying data are at ). The network illuminates the 'whys' of many aspects of zygotic control in early sea urchin development, both spatial and temporal. The network includes almost 50 genes, and these are organized in subcircuits, each of which executes a particular regulatory function.     

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.