T

sPi Wing

Ventral lab pb Díd Scr Antp Ubx abd-A Abd-B

Scr,

Ubx, abd-A

Figure 3.12

Regulation of organ primordia number and position by intercellular signals and Hox proteins

The salivary gland (sg), limb (lp), wing (w), and haltere (h) primordia arise in specific segments at particular positions along the D/V axis. The formation of these primordia is marked by expression of specific genes (for example, fkh, Dll, vg). The position of each primordium and the expression of marker genes are regulated by signals along the D/V axis of each segment. Specific Hox proteins regulate the segment-specific expression of marker genes and primordium formation.

The Dll gene possesses many cis-regulatory elements that function in limb primordia in parts of the embryonic head and/or thorax. Some of these elements are regulated by other Hox genes, others appear to be redundant. The existence of several elements allows for the independent control of the timing, level, and pattern of Dll expression in different segments and at different stages of development.

The wing primordia

The formation of the wing primordia is also regulated by intrasegmental signals and repressed in specific segments by Hox proteins. Cells that form the wing primordia migrate out of the limb field and are specified later and at a more dorsal position than are the limb primordia. Unlike the limb primordia, the wing primordia form in a region characterized by high levels of Dpp signaling (Fig. 3.12). Primordia formation is selectively repressed in the first thoracic and all abdominal segments by the Scr, Ubx, and Abd-A proteins (Fig. 3.12). We will have more to say about the role of Hox genes in the evolution of insect limb and wing number in Chapter 5.

The salivary gland

The salivary gland primordia form within the ventral epidermis of parasegment 2 in the developing embryo. The positioning of this organ and its restriction to parasegment 2 follow a similar logic, as we have described for the appendage primordia. Expression of the transcription factor Forkhead (Fkh) is an early event in salivary gland development. The regulation of the fkh gene parallels the regulation of salivary gland organogenesis. Although the fkh gene and salivary gland development are activated by the Hox protein Scr in parasegment 2, they are limited to the ventral epidermis by negative regulation, from the Dpp signal dorsally, and the EGF receptor pathway more ventrally (Fig. 3.12).

Neural and muscle precursors

The patterns of embryonic neural and muscle precursors in the ectoderm and mesoderm, respectively, are also generated by combinatorial inputs of A/P and D/V signals and modified by Hox regulatory inputs. Rows of proneural clusters that will give rise to segmentally iterated patterns of neuroblasts in the early embryo are marked by the expression of Achaete-Scute Complex genes, whose regulation is a nodal point for the patterning of the central and peripheral nervous system. In the AS-C, cis-regulatory elements integrate inputs from segmentation proteins and dorsoventral-axis patterning proteins that establish proneural clusters. Later in embryogenesis, segment-specific patterns of peripheral neural precursors arise from combinatorial regulation of AS-C genes by segmentation, dorsoventral, and Hox regulatory inputs.

The AS-C contains many cz's-regulatory elements that control discrete subpatterns of AS-C gene expression. Muscle precursors, marked by the expression of the nautilus gene, are positioned by combinatorial signals within the mesoderm as well as by Hox inputs that regulate segment-specific muscle patterns.

Patterning within secondary fields: organizing signals and selector genes

The intrasegmental coordinate systems in the embryo and the Hox ground plan control the initial position, size, and number of developing Drosophila fields. As these fields grow and begin to differentiate, regulatory hierarchies within these fields establish new coordinate systems and organizers that control the formation and morphogenesis of the final structure.

Perhaps the best-studied genetic regulatory program of all secondary fields is that of the Drosophila wing. This wing develops from a portion of the larval imaginal disc that will also give rise to the body wall of the second thoracic segment. The wing disc grows from roughly 30-40 cells in the first instar larva to approximately 50,000 cells in the third instar larva before morphogenesis transforms it into the adult wing and thoracic body wall. Coordinate systems and selector genes operate within the wing field to control the position, number, and differentiation of the various pattern elements.

The regulatory logic and several of the molecular mechanisms involved in the hierarchies that control wing formation and patterning have been described in sufficient detail that some general concepts regarding the patterning of secondary fields have emerged. These concepts, which apply to other insect appendages as well as to vertebrate limbs, involve regulatory mechanisms that are similar to those operating to position and regulate the formation of pattern elements in other cellular fields. Because the number, position, size, and morphology of pattern elements within secondary fields are important aspects of morphological diversity, understanding these regulatory mechanisms is crucial to developing a picture of the evolution of gene expression and morphology within a field.

The development of secondary fields from primordia established in the embryo involves three major processes:

1. The generation of new coordinate systems during the growth of the field

2. The placement and specification of pattern elements within the field

3. The differentiation of field identity from other, serially homologous fields

The genetic regulatory mechanisms governing these processes involve the integration of combinatorial inputs by cis-regulatory elements, similar to those described earlier for the generation of body axes and the initial specification of the fields. One set of regulators includes short- and long-range signaling proteins (morphogens) that determine the area within a field in which a given gene is activated or repressed. The sources of these signals are usually oriented with respect to one patterning axis of the field (anteroposterior, dorsoventral, or prox-imodistal). A second regulator controls the field- or cell-type-specific response to these signals, such as a selector gene. The identity of serially homologous fields is determined by another layer of regulation superimposed on the signaling inputs and field-specific selector genes by Hox genes or analogous regulators that are expressed in only one serial homolog. The integration of signaling, field-specific selector, and Hox inputs through cis-regulatory elements of target genes controls gene expression patterns in a particular field.

In the Drosophila wing and haltere (the wing's serial homolog), the identity and function of the major signaling (Dpp, Hh, Wg, N), wing-specific selector (Vg, Sd), and Hox (Ubx) inputs in the wing patterning hierarchies have been studied extensively. Three coordinate systems operate in the wing field:

1. The first coordinate system is involved with patterning along the A/P axis.

2. The second coordinate system acts along the D/V axis.

3. The third coordinate system, the proximodistal (P/D) axis, affects the integration of these inputs and the specification of the entire wing field.

Next, we examine the genetic regulatory hierarchies that govern the formation and operating of these coordinate systems and see how they control gene expression and wing pattern.

The anteroposterior coordinate system

The A/P coordinate system encompasses the sequential organizing activities of the Engrailed, Hedgehog, and Dpp proteins. Patterning along the A/P axis of the wing is controlled by a hierarchy involving compartment selector genes and multiple signaling pathways.

Posterior cells are segregated from anterior cells by a lineage restriction imposed by the engrailed gene, which is expressed in all posterior cells but not in anterior cells. This lineage restriction creates a smooth boundary between the anterior and posterior compartments, across which important inductive events take place. Due to the regulatory activity of engrailed, cells in the posterior (but not the anterior) compartment express the short-range signaling molecule Hedgehog (Fig. 3.13a). Cells in the anterior compartment express the Patched protein (see Table 2.2), a receptor for the Hh signal. Transduction of Hh signaling induces a stripe of cells on the anterior side of the A/P boundary to express the Dpp signaling protein (Fig. 3.13a,b). Dpp, in turn, acts as a morphogen and diffuses across both the anterior and posterior compartments from its source along the A/P compartment boundary, thereby regulating gene expression in a concentration-dependent manner.

The dorsoventral coordinate system

The D/V coordinate system includes the sequential organizing activities of Apterous and the Notch and Wingless pathways.

The organization of the D/V axis is regulated in a fashion somewhat analogous to the regulation of the A/P axis organization. Dorsal cells are segregated from ventral cells by a lineage restriction imposed by the apterous compartment selector gene (see Table 2.1), which is expressed in all dorsal cells (Fig.3.13a). The Apterous protein regulates the expression of Serrate and Fringe (Fig. 3.13b & see Table 2.2), two proteins that interact with the ubiquitously expressed Notch receptor protein. Along both sides of the D/V boundary, Notch-mediated signaling between cells induces expression of the Wg signaling protein in a stripe that straddles the D/V boundary. Wg acts as a morphogen, diffusing from its source to regulate gene expression in both the dorsal and ventral compartments.

Signal integration by the vestigial field-specific selector gene

One important downstream target of the Dpp, N, and Wg organizing signals emanating from the compartment boundaries is the vg field-specific selector gene. The vg gene is expressed in all of the cells in the wing disc that will form the wing. The regulation of the overall pattern of its expression is the sum of inputs from two separate cis-regulatory elements that respond to signals from each coordinate system (Fig. 3.13c). The "boundary" cis-element is directly activated along the D/V boundary through the Notch pathway. The "quadrant" cis-element is expressed in the complementary pattern in the rest of the wing field through activation by the Dpp pathway and other regulators (Fig. 3.13c). Expression of this field-specific selector gene depends directly on the signaling sources of the wing field and indirectly on the compart-mental selectors that establish the expression of these signals.

Combinatorial regulation of wing patterning by signaling proteins and the Vg/Sd selector proteins

Within the wing field, a large number of genes function to pattern various features of the wing. For example, the AS-C genes are activated along the anterior extent of the D/V boundary to generate the sensory organs of the leading edge of the adult wing. The blistered or Drosophila Serum Response Factor (D-SRF) gene is expressed in all of the cells that lie between the developing wing veins. Many genes are involved in the positioning and differentiation of the

Figure 3.13

The genetic regulatory hierarchy in the Drosophila wing

Figure 3.13

The genetic regulatory hierarchy in the Drosophila wing

(a) Two systems organize the pattern along the dorsoventral (left) and anteroposterior (right) axes of the wing. (b) Compartmental selector genes establish signaling sources along each compartment boundary. Transduction of these signals leads to the activation of downstream target genes. (c) One important target that is directly regulated by these signals is the vestigial wing selector gene. The vg gene is expressed in all wing cells through the sum of the activities of two cis-regulatory elements—one active along the compartment boundaries, and one active in the remaining four quadrants of the wing field.

veins that provide the structural support for and the fluid transport system of the wing. The expression of each gene required for the development of wing pattern elements depends on combinatorial inputs from signaling pathways and the wing selector proteins Vg and Scalloped (the DNA binding of partner of Vg). Specifically, the signaling proteins regulate where within the wing field a given gene is activated or repressed, and the activities of Vg/Sd make the response to these signals specific to the wing field.

The response to organizing signals

Various ligands for the major signaling pathways (Serrate, Dpp, Wnt, Hedgehog, and so on) are deployed in dynamic patterns in the wing field. Some signals appear to have a greater range of influence than others do. The Hedgehog protein, for example, induces Dpp expression over a few cell diameters, whereas the Dpp signal produced within these cells influences cells as many as 20 or more cell diameters away.

At the level of gene regulation, the response to signaling inputs may be graded or threshold. In addition, some genes are activated at high concentrations of signal, whereas others are activated at a wider range of concentrations. The differential response of genes can create nested patterns of gene expression along a given axis of the wing field. For example, the spalt gene is activated at high Dpp levels, the omb gene at lower Dpp levels, and the vg quadrant element at still lower levels; consequently, the three genes are expressed in nested, partially overlapping domains centered on the A/P axis (Fig. 3.14). The different responses of target genes to levels of the Dpp signal fit the classic description of a morphogen.

Figure 3.14

Activation of target genes by the Dpp morphogen gradient

The Dpp protein is expressed along the anterior/posterior compartment boundary of the wing, and spreads from this source to establish a concentration gradient. The spalt (purple), optomotor-blind(omb; yellow), and vg (red) target genes are activated in nested patterns centered along this boundary. The differential response of target genes to a common signal is critical to pattern formation in cellular fields. Source: Courtesy of Jaeseob Kim.

jpfP DPP Source

Figure 3.14

Activation of target genes by the Dpp morphogen gradient

The Dpp protein is expressed along the anterior/posterior compartment boundary of the wing, and spreads from this source to establish a concentration gradient. The spalt (purple), optomotor-blind(omb; yellow), and vg (red) target genes are activated in nested patterns centered along this boundary. The differential response of target genes to a common signal is critical to pattern formation in cellular fields. Source: Courtesy of Jaeseob Kim.

The action of the Vg/Sd selector genes

Because the various signaling pathways deployed in the wing field are active elsewhere in the body, regulatory mechanisms must exist to impart specificity to their activities in any given tissue. This role is fulfilled by the field-specific selector genes. In the wing, the Vg and Sd proteins are required to permit appendage formation and to effect a wing-specific response of target genes to particular signals. The molecular basis for the Vg/Sd selector function is as follows: Sd acts as a sequence-specific DNA binding protein that binds to cis-regulatory elements that control patterns of target gene expression in the wing. Sd forms a complex with Vg; in this complex, the Vg protein acts as a transcriptional activator. Importantly, however, the binding of Vg/Sd is not sufficient to activate target genes. A second cue is required from one or more signaling pathways or another regulatory input. This combinatorial regulation by the selector and signaling input restricts gene activity to particular cells within the wing field (Fig. 3.15).

Many genes that are expressed in discrete patterns in the wing field are also expressed in entirely different patterns in other fields. The wing-specific aspects of these genes' expression are controlled by independent wing-specific cis-regulatory elements that respond to and contain binding sites for Vg/Sd and at least one other input. Other cz's-regulatory elements

Figure 3.15

Integration of signaling and selector protein inputs by c/s-regulatory elements

Figure 3.15

Integration of signaling and selector protein inputs by c/s-regulatory elements

Target genes are activated in the wing field by a combination of inputs from one or more signal transducers and the Vg/Sd selector proteins. Localized activation of the Hedgehog (left), Notch (middle), or Dpp (right) signaling pathways induces target gene expression (red) where the signaling input and selector input (Vg/Sd; blue stippling) overlap. Each target gene possesses wing-specific c/s-regulatory elements that integrate inputs from the appropriate signal transducer (Ci, Mad/Medea, Su(H); see Table 2.2) and Vg/Sd.

control gene expression elsewhere in the body. The modularity of the various cz's-regulatory elements allows for the independent regulation of genes in the wing field and in other regions of the body.

The modification of regulatory hierarchies in secondary fields by Hox genes

The regulatory events in the growing wing field described previously transpire in the absence of Hox gene activity. Similarly, the patterning of the antennal field does not involve any Hox gene. On the other hand, the development of the haltere (the serial homolog of the wing) and the serial homologs of the antenna (which include all limb-type appendages) does involve Hox genes. The Hox proteins act to differentiate these structures by modifying developmental programs between serially homologous fields.

Before we discuss how Hox genes modify these complex morphologies and regulatory hierarchies, it is useful to briefly summarize our understanding of Hox protein functions. All Hox genes encode homeodomain proteins with similar DNA-binding specificities. One way that Hox proteins exert specific regulatory control is by interacting with protein cofactors. The Extradenticle (Exd) protein, another homeodomain-containing protein, is a key cofactor that interacts with several Hox proteins. Certain Hox-regulated cz's-regulatory elements contain both Hox and adjacent Exd protein binding sites; occupancy of both sites and interaction between Hox and Exd molecules are required for gene regulation.

Hox proteins can act as both transcriptional activators and repressors, with their precise roles depending on a number of as yet unknown variables. In addition to cofactor interactions, post-translational modifications (such as phosphorylation) and the context of Hox binding sites can influence Hox activity. The core recognition sequence for Hox proteins is only about 6 bp long, so the genome contains many low- and high-affinity sites for these proteins.

The striking feature of Hox genes is that their expression in a new position can be sufficient to transform one structure into another. For example, expression of Antp in the antenna, where it is normally absent, causes the development of a leg. Similarly, expression of Ubx in the wing transforms that structure into a haltere. How can changes in a single gene so radically alter development? Do Hox proteins act globally to modify the expression of genes at the top of regulatory hierarchies? Or do they act throughout hierarchies, modifying some regulatory interactions but not others?

The picture that has emerged is one depicting Hox genes as "micromanagers" that act at many levels of regulatory hierarchies on selected components. The development of the haltere, for example, depends on the same major regulators (apterous, engrailed, hedgehog, vestigial, and so on) as its serial homolog, the wing. Yet, the morphology of the haltere is dramatically different in terms of its smaller size, balloon shape, lack of veins or large bristles, and details of cell architecture. The Ubx protein modifies the wing-patterning hierarchy to shape the development of the haltere by acting on a selected subset of genes that influence features of wing pattern formation (Fig. 3.16). For example, Ubx suppresses the production of the Wg protein in the posterior compartment of the haltere disc. This protein also represses AS-C genes, resulting in the suppression of bristle formation at the edge of the haltere. Along the A/P axis of the haltere, certain Dpp-regulated genes are specifically repressed by Ubx, while others are expressed in patterns similar to those in the wing.

Figure 3.16

The Ultrabithorax-regulated hierarchy in the Drosophila haltere

The haltere is a serial homolog of the wing. The Ubx protein regulates the morphological differentiation of the haltere by selectively modifying the wing regulatory hierarchy at many levels. (top) Genes or regulatory elements that are Ubx-regulated in the haltere are shown in red. (bottom) The expression patterns of genes in the wing and haltere imaginal discs. (top row, left to right) engrailed, apterous, and dpp expression are similar in the two fields. (bottom row, left to right) spalt, vgquadrant enhancer, and achaete expression differ between the wing and haltere discs due to the actions of Ubx. Source: Modified from Weatherbee SD, Halder G, Hudson A, et al. Genes Dev 1998;12:1474-1482.

How is the selective regulation of genes controlled by Ubx in the haltere? Once again, the key is cis-regulatory elements. Recall that wing-specific patterns of gene expression are controlled by discrete elements. In genes that are directly controlled by Ubx in the haltere, wing-specific cis-regulatory elements also contain functional sites for the Ubx protein, which generally acts to repress wing-patterning genes in the developing haltere field. The modularity of cis-regulatory elements enables the selective and differential regulation of gene expression by Hox proteins between serially homologous fields.

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