Gut Regulators





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Downstream differentiation genes

FIGURE 3.7 Cis-regulatory control of E lineage specification in C. e/e-

gans. (A) Observed target sites in cis-regulatory domains of the zygotic E lineage regulatory genes end I and end3, and the related end I gene of C. briggsae. All three cis-regulatory systems have multiple sites for Sknl, Tcf/Lefl, and Gata factors, plus a set of conserved sequence motifs (A, B, C, D) that may bind additional factors (see key). [(A) From Kasmir et al., (2001), in preparation.] (B) Target sites identified immediately upstream of medl and med2 genes, which also encode early zygotic Gata transcription regulators (see text). The two genes are almost identical in sequence though located on different chromosomes. Small vertical lines represent the only base pairs that are not identical between these genes. [(B) J. M. Rothman, personal communication.] (C) c/s-Regulatory network for E lineage specification and immediately following events. For sources and evidence in addition to the following, see text. Maternal factors are shown in colored boxes. For the Tcf/Lefl factor, green is used for repression and tan for activation functions. Red lines represent zygotic interactions. Where the red lines are solid there is evidence for direct interactions; dashed red lines are inferred interactions. c/s-Regulatory elements of the med genes bind Skn I protein in vitro and are also occupied by Skn I factor in vivo, according to an in situ assay. Furthermore, medl expression constructs are dependent on presence of the Sknl factor for activity, and ectopic expression of Sknl causes a parallel ectopic expression of such constructs (Maduro et al., 2001; M.F. Madura and J.M. Rothman, personal communication). Presence of Gata sites (cf. B) suggest an autostimulatory feedback. As described in text the Tcf/Lefl factor represses endl and end3 (in MS and MS progeny), unless it is altered by the signal from P2, which converts it into an activator (in E lineage) (Kasmir et al., 2001). Binding of maternal Sknl protein to endl and end3 target sites (cf. A) is observed in vitro, and binding of Medl protein at Gata target sites is found in vivo as well (J. M. Rothman, personal communication). The activation function of the Tcf/Lefl factor which is the product of the pop I gene is required for expression. The normal level of output depends on the synergistic behavior of bound Sknl and Med proteins (Kasmir et al., 2001; Maduro et al., 2001.). The endl and end3 genes are expressed only transiently, during the initial few cleavages of the E cell. Therefore they are unlikely to be equipped with a self-sufficient autoregulatory device, depending rather on simultaneous inputs of the factors shown in (A), which are also present transiently. Autoregulation of the elt2 gene was reported by Fukushige et al. (1998), and binding of the factor to its own cis-regulatory element has been visualized in vivo (Fukushige et al., 1999). Genetic and molecular evidence require that elt2 is downstream of the endl/end3 genes: while ectopic expression of elt2 causes ectopic gut-specific gene expression, including of the gesl gene (Fukushige et al., 1998); elimination of endl and end3 expression by use of RNAi prevents all endoderm differentiation (Zhu et al., 1998; Kasmir et al., 2001). elt7 may function like elt2. The elt2 gene product evidently activates gesI, according both to in vitro and genetic evidence, by interactions at a crucial pair of Gata target sites in the c/s-regulatory element of the gesl gene (Fukushige et al., 1996, 1998, 1999; Egan et al., 1995). Initially the gesl gene might respond to regulation by endl/end3 gene products,


popl gene is present (Maduro et al., 2000; Kasmir et al., 2000; M.F. Maduro and J.M. Rothman, personal communication). When it is absent, as in the MS cell, the repressor form is dominant.

The end genes may directly initiate expression of early gut-specific differentiation genes such as gesl, but they are not the major activators that drive differentiation of E-lineage progeny. Their role is specification. Immediately downstream of the end genes is another Gata-type regulatory gene called elt2 (there may again be a pair of such genes; elt7 is a likely candidate). The elt2 gene has a strongly autostimulatory regulatory system which binds its own product in vivo (Fukushige et al., 1998, 1999). Its role in the network is clearly to produce an amplified, gut cell-specific regulatory output. It is activated once the E blastomeres have divided to produce the first two E lineage daughters. cis-Regulatory analysis of the gesl gene shows that its expression depends on two Gata factor target sites (Aamodt et al., 1991; Egan et al., 1995). The gesl gene is activated by an elt2 gene product which binds at these sites, or at least can bind there, since ectopic elt2 expression results directly in ectopic gesl expression (Fukushige et al., 1998). As Fig. 3.7C further indicates, the full program of intestine cell differentiation depends on a suite of other regulatory genes that operate further downstream. These must control expression of additional gene batteries that encode all the functions required for morphogenetic organization of the intestine (cf. Leung et al., 1999), as well as for digestive operations. Some of these regulatory genes are gut specific (end2, nhr80, elt4), others not; and expression in the gut of at least one, the forkhead/hnf3$ gene, pha4 (Mango et al., 1994), appears to be controlled by the factor encoded by the elt2 gene (Kalb et al., 1998).

The cis-regulatory network shown in Fig. 3-7C is simple, direct, and one might say, very nicely designed. It uses a pair of very similar cis-regulatory systems, those of the endl and end3 genes, to transduce the signal that distinguishes E from MS and output a zygotic specification function. Another system, that of the med genes, transduces the localization pattern of the maternal Sknl factor into a zygotic output that defines the EMS cell as endomesodermal. The two are combined in the synergistic endl/end3 cis-regulatory elements. And as soon as E is specified, the endl/end3 products turn on elt2, a gene encoding an endoderm-specific regulator which is sharply stimulated by its own product. By the time i.e., before institution of the e/t2 autoamplification circuit. Later differentiation of intestine cells involves other transcriptional regulators, some endoderm-specific, some not (for Nhr28, Miyabayashi et al., 1999; for Pha4, Kalb et al., 1998; for zSknl, i.e., zygotically expressed Sknl, which is also required for normal gut development, Bowerman et al., 1992; for others listed, J.M. Rothman, unpublished data). The dashed arrows from the e/t2 gene to the box containing the names of downstream transcriptional regulatory genes expressed in gut, indicates that for some genes such as pha4 there is evidence for direct control by e/t2 (Kalb et al., 1998), while for others it is not known whether control is direct or indirect.

there are only two E-lineage cells, a self-reinforcing gut-specific regulatory state has been generated. A downstream differentiation gene, gesl (and no doubt others), begins to be expressed when there are only four E-lineage cells, followed by all the other specific gene batteries required for differentiation of the intestine. The network shown in Fig. 3-7 is again a direct cell-type specification system that within a few divisions produces differentiated cell types from the progeny of unspecified early cleavage blastomeres.


In this chapter we have traversed regulatory mechanisms underlying the simplest and most direct processes of bilaterian embryogenesis, from the cytoplasmic interactions that mediate the initial specification events to the internal programs wired into the genome. Type 1 embryos display common phenomenological features, such as the early activation of embryonic genomes, and territorial specification during cleavage leading at once to differentiation. These features are general because they utilize a common regulatory "deep structure."

The most important feature of the architecture of the genomic regulatory networks that initiate Type 1 embryonic processes is that they are remarkably shallow. They do not have a deeply hierarchical or multilayered structure. The regulatory transactions that these networks mediate occur during cleavage. Essentially they depend on the information processing capabilities of key czs-regulatory elements, the job of which is to transduce the spatial inputs available in the early embryo into blastomere (and hence lineage- and territory-specific) transcriptional regulatory states. Thereby they execute what we call blastomere specification. Spatial inputs for specification derive from maternal cytoarchitectural location or from the regional activation of maternal components, sometimes caused by early interblastomere signaling. As specification states are established they are often supported by autostimulatory loops. The output of the specification process soon results in direct activation of differentiation gene batteries. The relation between specification state and the cellular organization of the Type 1 embryo is simplified by use of lineage relations: specification occurs in founder cells, and in later cleavage, these are divided up into polyclonal territories of differentiated progeny, all without net growth. From these territories the structures of the embryo arise. The complexity of the process of embryogenesis in Type 1 systems lies in the organization and operation of the cytoplasmic mechanisms that regionalize the activities of maternal regulatory molecules in the very early embryo; and in the internal organization of the key genomic as-regulatory elements which respond to these regulators (cf. Chapter 2). But at least in relative terms the complexity is not in the number of layers in the network of intergenic regulatory connections, particularly in comparison to what we are to encounter in the next chapter.

Our task now is to approach the development of adult body parts. Here the answer to the same questions are quite different. Egg cytoarchitecture and sub-lineages of blastomeres that occupy fixed spatial positions are no longer relevant. The developmental process is itself far more complex, and it depends on far more elaborate forms of intergenic regulatory architecture.

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