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Knockout phenotype Phase III expression

Phase III •Hox a-13d13

O Hox a-13, d-13 O Hox a-13, d-13, d-12, d-11, d-10

Hoxd-9 Hoxd-10

Phase III •Hox a-13d13

O Hox a-13, d-13 O Hox a-13, d-13, d-12, d-11, d-10

Hoxd-9 Hoxd-10

Figure 3.22

Hox gene expression and function in vertebrate limb development

(a—c) Three temporal phases of Hox gene expression in the limb bud correlate with the elaboration of three distinct elements of the limb. (a) Phase I involves a more limited set of Hox genes expressed across the entire bud in the region that will give rise to the upper arm or thigh (stylopod). Mutations in these genes primarily affect this structure. (b) Phase II expression is initiated in response to Sonic hedgehog signaling and is polarized with respect to the A/P axis of the limb bud. Disruption of multiple Hox genes that have pronounced phase II patterns affects formation of the bones corresponding to the lower arm or calf (zeugopod). (c) Phase III expression occurs in the hand and foot (autopod). Disruption of certain Hox genes primarily affects autopod formation and patterning. (d) The c/s-regulatory elements of the Hoxd9-13 genes. Both global and local elements regulate Hox expression in the limb bud. Multiple Hox genes are influenced by remote elements active in the zeugopod and autopod. Local elements, such as RXI (red), affect expression of individual genes in restricted domains.

Source: Shubin N, Tabin C, Carroll S. Nature 1997; 388: 639-648; Hérault Y, Beckers J, Kondo T, et al. Development 1998; 125: 1669-1677.

b c genes in the limb bud, respectively. For example, phase II expression of the Hoxd12 gene is regulated by both a nearby element and a remote global element, whereas phase III expression is controlled by a remote global element (Fig. 3.22d). Thus the complex patterns of Hox gene expression are built up from many potentially independent inputs, which creates the potential for morphological diversification through changes in the relative timing and spatial regulation of individual elements.

Regulatory networks controlling the differentiation of major limb pattern elements

The skeletal defects caused by Hox mutations indicate that one function of Hox proteins may be to regulate the position and timing of cartilage development and subsequent growth and differentiation in the limb field. Evidence suggests that multiple signaling molecules (Bmps, Wnts, Fgfs, another member of the Hedgehog signal family, Indian hedgehog-Ihh), Sox transcription factors, and Hox genes regulate the progression of bone differentiation and thus major features of limb pattern formation. Certain Hox proteins appear to act early in the differentiation pathway to prevent its progression, either by promoting the proliferation of bone forming cells or by preventing their differentiation.

The positions of the joints between limb skeletal elements are also spatially regulated. One regulator of this process, the GDF5 signaling protein (a member of the large TGF-P super-family), is specifically expressed in regions where bone development does not occur. GDF5 expression presages the location of joints throughout the limb field (as well as elsewhere in the body). Clearly, the regulation of the spatial expression of these various cartilage and bone promoting and inhibiting factors is key to the skeletal pattern formed.

The regulation of forelimb and hindlimb identity by selector genes

The serially homologous tetrapod forelimb and hindlimb are believed to have evolved from the paired pectoral and pelvic fins of fish, respectively. Although the developing forelimbs and hindlimbs both utilize organizing signals that are necessary for appendage outgrowth, no single transcription factor required for limb outgrowth has been identified as yet. The Hox genes, for example, do not play the role of selector genes for limb field formation or identity. No Hox mutations result in homeotic transformations between limb types.

A few other transcription factors have, however, been identified as selectors of forelimb and hindlimb identity in vertebrate limbs. The paired homeodomain-containing protein Pitx-1 and the T-box-containing protein Tbx4 are expressed specifically in the hindlimb mesenchyme, and the T-box gene Tbx5 is expressed specifically in the forelimb mesenchyme (Fig. 3.23). In addition to the early requirement of these genes for limb outgrowth, the differentiation between forelimb and hindlimb identity is regulated, at least in part, by these selector genes. Although each is required for proper identity, none of the three genes is sufficient by itself to confer limb-specific identity. Hence, additional regulators of limb identity may act upstream of these genes, or perhaps limb identity results from a number of field-specific inputs.

These and other potential field-specific regulators presumably function to control the differential expression of genes between the two fields (Fig. 3.23). Several Hox genes, including HoxclO and Hoxcll, are differentially expressed between limb types and are potential

Figure 3.23

Selector genes controlling vertebrate limb identity

The differential development of the vertebrate forelimb and hindlimb is under the control of selector genes that are differentially expressed in the limb mesenchyme. The Tbx5 gene is expressed in the forelimb; the Pitx-1 and Tbx-4 genes are expressed in the developing hindlimb. In the chick, products of these genes regulate wing versus leg identity.

Figure 3.23

Selector genes controlling vertebrate limb identity

The differential development of the vertebrate forelimb and hindlimb is under the control of selector genes that are differentially expressed in the limb mesenchyme. The Tbx5 gene is expressed in the forelimb; the Pitx-1 and Tbx-4 genes are expressed in the developing hindlimb. In the chick, products of these genes regulate wing versus leg identity.

targets of the limb-specific selectors. Given the important morphological and functional diversity of vertebrate limb morphologies, it will be important to characterize the levels in the limb regulatory hierarchy at which changes have arisen between taxa.

REVIEW: THE GENERAL LOGIC AND MECHANISMS CONTROLLING GENE EXPRESSION IN CELLULAR FIELDS

This chapter has presented an overview of the genetic regulatory logic and mechanisms that operate to control development in a few model species. It concentrated on the genes of the general animal toolkit for development as well as the mechanisms that control large-scale patterning of the main body axes and secondary fields. Although we explored only a modest number of regulatory hierarchies in representatives of just two phyla, the similarities in the regulatory logic and mechanisms allowed us to identify some general themes concerning the regulation and function of toolkit genes and the architecture of the regulatory hierarchies that progressively specify pattern formation in cellular fields. The identification of these general themes is critical for understanding trends in animal evolution.

Because the regulatory mechanisms of development are themselves the product of evolution, their architecture is a reflection of the evolutionary processes that assembled them. Armed with our knowledge of regulatory architectures in one species, we can begin to make comparisons with other taxa with different morphologies to identify at what level and through what genetic mechanisms developmental and morphological diversity arises.

From this survey of regulatory hierarchies and mechanisms, we underscore three general themes with regard to the architecture of regulatory hierarchies, the molecular mechanisms controlling gene expression, and the functions of toolkit genes in controlling gene expression and patterning in cellular fields:

1. The development of the growing embryo and its body parts occurs in a spatially and temporally ordered progression through the sequential generation of coordinate systems. The position of any adult pattern element is determined through a series of hierarchies that subdivide the embryo and its morphogenetic fields into progressively finer elements. From the coordinate systems that subdivide the major embryonic axes, secondary fields are specified that contain their own autonomous coordinate systems. Domains within these fields can be further subdivided and specified through the establishment of local domains of gene expression. Ultimately, the temporal and spatial segregation of these processes allows for the modular organization of bilaterians and the individualization of body parts and pattern elements, such that morphology evolves independently from the rest of a field or the body plan.

2. The modularity of cis-regulatory elements acting on individual genes allows for the independent spatial and temporal control of discrete features of gene expression and function. Many gene expression patterns are actually the sum of the functions of many independent cis-regulatory elements. The modularity of cis-regulation is crucial to the control of the specificity of gene interactions and function during development. The independent spatial and/or temporal regulation of gene expression permits individual genes to have different but specific functions in different contexts. Thus, while operating through identical signal transduction pathways, a signaling protein can be expressed in entirely unrelated populations of cells in two different tissues (by virtue of its own ciselements) and can regulate the expression of completely different target genes (through different cis-regulatory elements of its target genes).

In this light, it is not adequate or accurate to describe a given toolkit gene function solely in terms of the protein it encodes, because the function of that protein almost always depends on the context in which it is expressed. Instead, toolkit genes should be viewed as consisting of both a functional protein and a suite of regulatory elements that control its deployment. Each element represents a separate genetic function.

The modular organization of gene-specific control elements has two profound evolutionary implications. First, this modularity is the product of evolution and therefore directly reflects the mechanisms that allow new and independent patterns of gene expression to evolve. Second, the modular control of extant genes allows for changes in one aspect of gene expression and function without affecting any other functions. In other words, modularity facilitates the dissociation of gene functions as well as the evolution of new interactions and potential morphologies.

3. Spatial patterns are the product of combinatorial regulation In the examples of regulatory hierarchies and individual genes expression patterns analyzed in this chapter, we saw that new patterns were created by the combined inputs of preceding patterns. The integration of D/V and A/P inputs to position primordia, the positioning of a pair-rule stripe, the positioning of organizers, and the field-specific pattern of a regulatory gene, for example, are all derived from the integration of multiple inputs (some positive, some negative) by cis-regulatory elements.

Combinatorial control is key to both the specificity and the diversity of gene expression. In terms of specificity, it provides a means to localize gene expression to discrete cell populations using inputs (such as signaling pathways) that are not cell-type-specific or tissue-specific. In terms of diversity, combinatorial mechanisms provide a means to generate a virtually limitless variety of spatial patterns through the overlapping inputs of positive and negative regulators and autoregulatory feedback mechanisms. As the number of discrete domains of gene activity increases in a field, the potential combinations of regulators and patterning outputs increase exponentially.

The evolutionary significance of combinatorial regulation is obvious. New gene expression patterns can evolve as new combinations of regulatory inputs are integrated. In turn, the new gene expression patterns create the potential for further change, and the cycle continues.

From these general themes, we can start to anticipate how regulatory hierarchies and gene expression patterns might be cobbled together in the course of evolution and how they might change. To begin to understand what has occurred, we must take a broad inventory of the genetic toolkit across the animal kingdom and examine the regulatory mechanisms for building animals in a phylogenetic and comparative context to see how morphological diversity evolves.

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