SUMMARY Alteration of the functional organization of the gene regulatory networks (GRNs) that control development of the body plan causes evolutionary change in animal morphology. A major mechanism of evolutionary change in GRN structure is alteration of cis-regulatory modules that determine regulatory gene expression. Both evolutionary conservation and evolutionary innovation must be considered in terms of GRN structure. Here we consider the causes and consequences of GRN evolution, both from an a priori point of view, and in light of extensive recent research on developmental regulatory alterations occurring at different levels of GRN hierarchy. Some GRN subcircuits are of great antiquity while other aspects are highly flexible and thus in any given genome more recent. Both evolutionary conservation and evolutionary innovation occur at the level of whole GRN subcircuits. This mosaic view of the evolution of GRN structure explains major aspects of evolutionary process, such as hierarchical phylogeny and discontinuities of paleontological change and stasis.

Specification of endoderm is the prerequisite for gut formation in the embryogenesis of bilaterian organisms. Modern lineage labelling studies 1–3 have shown that in the sea urchin embryo model system, descendants of the veg1 and veg2 cell lineages produce the endoderm, and that the veg2 lineage also gives rise to mesodermal cell types. It is known that Wnt/β-catenin signalling is required for endoderm specification4–6 and Delta/Notch signalling is required for mesoderm specification7–9. Some direct cis-regulatory targets of these signals have been found10,11 and various phenomenological patterns of gene expression have been observed in the pre-gastrular endomesoderm. However, no comprehensive, causal explanation of endoderm specification has been conceived for sea urchins, nor for any other deuterostome. Here we propose a model, on the basis of the underlying genomic control system, that provides such an explanation, built at several levels of biological organization. The hardwired core of the control system consists of the cis-regulatory apparatus of endodermal regulatory genes, which determine the relationship between the inputs to which these genes are exposed and their outputs. The architecture of the network circuitry controlling the dynamic process of endoderm specification then explains, at the system level, a sequence of developmental logic operations, which generate the biological process. The control system initiates noninteracting endodermal and mesodermal gene regulatory networks in veg2-derived cells and extinguishes the endodermal gene regulatory network in mesodermal precursors. It also generates a cross-regulatory network that specifies future anterior endoderm in veg2 descendants and institutes a distinct network specifying posterior endoderm in veg1-derived cells. The network model provides an explanatory framework that relates endoderm specification to the genomic regulatory code.

Adenovirus (AdV)-mediated gene expression of immune stimulators represents a valuable in vivo approach for gene therapy of human cancer. The expression level of the therapeutic gene is of crucial importance for the efficacy of this type of treatment. Entry of AdV is dependent on the primary adenovirus receptor CAR and the secondary AdV receptor identified earlier to be a member of the integrin family of surface molecules. We have analyzed 14 different human melanoma cell cultures from different stages together with one melanoma cell line for their AdV-mediated transduction and expression efficiency. Recombinant viruses at various concentrations were used for expression of the B7-1 costimulatory molecule under the control of different promoters and the expression levels of B7-1 were analyzed by flow cytometry. AdV-mediated IL-12 expression was measured using a commercial ELISA. Levels of transgene expression were compared with the expression levels of HCAR, the alpha(v)beta3 and alpha(v)beta5 integrins, and HLA class I. In 4 of 14 cell cultures tested, the presence of the primary virus receptor CAR was associated with the high transduction efficiency phenotype when using the B7-1- and IL-12-expressing viruses at a relatively low multiplicity of infection (MOI) of 50. Immunohistochemistry on cryosections from the original biopsies yielded a strong signal specific for CAR. In contrast, cell cultures expressing low or undetectable levels of CAR needed a 20- to 40-fold higher viral input to show comparable expression level of B7-1 or IL-12. Expression levels of the transgenes hardly varied when using different promoters and no association was observed with the presence or absence of HLA class I molecules or with the expression levels of integrins.

Gene regulatory networks (GRNs) control the dynamic spatial patterns of regulatory gene expression in development. Thus, in principle, GRN models may provide system-level, causal explanations of developmental process. To test this assertion, we have transformed a relatively well-established GRN model into a predictive, dynamic Boolean computational model. This Boolean model computes spatial and temporal gene expression according to the regulatory logic and gene interactions specified in a GRN model for embryonic development in the sea urchin. Additional information input into the model included the progressive embryonic geometry and gene expression kinetics. The resulting model predicted gene expression patterns for a large number of individual regulatory genes each hour up to gastrulation (30 h) in four different spatial domains of the embryo. Direct comparison with experimental observations showed that the model predictively computed these patterns with remarkable spatial and temporal accuracy. In addition, we used this model to carry out in silico perturbations of regulatory functions and of embryonic spatial organization. The model computationally reproduced the altered developmental functions observed experimentally. Two major conclusions are that the starting GRN model contains sufficiently complete regulatory information to permit explanation of a complex developmental process of gene expression solely in terms of genomic regulatory code, and that the Boolean model provides a tool with which to test in silico regulatory circuitry and developmental perturbations.gene regulatory logic | transcriptional control system | sea urchin embryogenesis | Boolean gene expression G ene regulatory network (GRN) models formalize the manner in which specification of cellular domains during development is controlled by spatial and temporal gene expression (1). Each cell fate depends on expression of a specific set of regulatory genes, that is, genes encoding transcription factors and signaling molecules. In each domain of the developing organism and at each point in time, the genetic activities and therefore the fates of the cells are determined directly by the regulatory gene products present in the nuclei. The regulatory states constituted by these regulatory gene products are themselves the output of transcriptional control systems encoded in the genome. GRNs thus capture the transcriptional control functions that specify the spatial regulatory states of the embryo. GRNs consist of regulatory genes and the transcriptional interactions that determine their specific patterns of expression. Models derived from experimental studies of developmental GRNs conceptually relate genomic regulatory sequence information to developmental process. Every node in such network models represents a regulatory gene, which is controlled by interactions encoded in genomic cis-regulatory binding sites.GRN models represent intellectual syntheses of experimental gene expression and cis-and trans-perturbation data, as well as information regard...

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.