Drosophila And Distal-less

FIGURE 4.5 Rhombomere specification in hindbrain: establishment of essential spatial regulatory states. (A) Correlation of domains of spatial expression of indicated genes in the amniote hindbrain with respect to specific rhombomeres

From this diagram alone one can see that each rhombomere expresses a unique combination of developmentally important genes. The remainder of Fig. 4.5 concerns the functional significance of the two genes shown at the top of Fig. 4.5A, i.e., krox20and kreisler. The krox20gene encodes a Zn finger transcription factor, and its expression in the hindbrain is sharply confined to r3 and r5, as illustrated in Fig. 4.5B. The downstream consequences of knockout of this gene are illustrated in Fig. 4.5C: essentially the structures to which r3 and r5 normally give rise are just missing (Schneider-Maunoury et al., 1997). Some neuronal populations are absent, others redirected, others respecified, a combination of

(r2-r6). These are the transient metameric units of the neuraxis in the hindbrain region. Genes encoding transcription factors are shown above and genes encoding signaling components below. Levels of expression are indicated by shading. Abbreviations for genes: crabpl, retinoic acid binding protein; rar's, retinoic acid receptors; sek's and ebk, Eph-related tyrosine kinase receptors; and elk's, elf2, ligands thereof, kreisler and krox20 encode transcription factors. [(A) From Lumsden and Krumlauf (1996) Science 274, I 109-1115; copyright American Association for the Advancement of Science.] (B) Patterns of expression of krox20 transcription factor in developing mouse hindbrain. (Bl) 8-somite (8s) stage; (B2) 12-somite (12s) stage. Expression is sharply confined to prospective rhombomeres (pr) (Bl) and then rhombomeres (r) 3 and 5 (B2). [(B) From Mechta-Grigoriou et al. (2000) Development ¡27, 119-128 and The Company of Biologists Ltd.] (C) Effects of krox20 gene knockout on subsequent neural organization in the domains to which the rhombomeres give rise. The diagrams show mouse embryos at 10.5 dpc. Color coding indicates neuronal pools deriving from each rhombomere. The major components in normal embryos (left; wt, wild type) are the motor neuron pool, in red, from r2 and r3; facial motor neurons, dark blue, from r4; salivary neurons and neurons that enervate portions of the ear, light blue, from r5; [Xth (green) and Xth (pink) motor nerves, from r6 and r7, respectively. In krox20 knockout embryos most of the specific neuronal pools of r3 and r5 are missing or redirected, i.e., those shown in red in wild-type r3 and light blue in wild-type r5. BA, branchial arch; ov, otic vesicle, the developing ear. [(C) From Schneider-Maunoury et al. (1997) Development 124, 1215-1226 and The Company of Biologists Ltd.] (D) Regional specification of posterior rhombomeres is controlled by expression of the kreisler (kr) gene. This gene encodes a leucine zipper transcription factor, normally expressed in r5 and r6 (Cordes and Barsh, 1994). Kr knockout embryos display altered spatial domains of several downstream regulatory genes, e.g., hoxb3 (green) where rhombomeric expression is lost altogether; and hoxb2 (red) where it is lost in r5, since r5 does not form. Hoxa3 expression (orange) is ectopic as well. In addition, expression of Eph/ ephrin receptors and ligands is affected (light and dark green and light and dark pink); levels of expression are represented by shades of color. The concentric circles are otic vesicles. Migrating neural crest patterns (purple) are also altered in the modified r2 which results from the mutation (r6 ). The diagram represents embryos at 9.5 dpc. [(D) From Manzanares et al. (1999a) Dev. Biol. 211, 220-237.]

Coxa

Trochanter

Femur

Tarsal segments

Claws

Drosophila And Distal Less

Dorsal wing surface

Ventral wing surface

FIGURE 4.6 Transcriptional regulatory domains in the regional specification of thoracic imaginal discs in Drosophila. (A) Diagram of leg imaginal disc. Distal structures are generated from central regions of disc and more proximal structures from concentric peripheral domains. The coxa and adjoining regions are genetically defined by expression of extradentide (exd) and homothorax (hth) genes (González-Crespo and Morata, 1996; Wu and Cohen, 1999), and the leg proper by expression of the distal-less (dll) regulatory gene together with other genes. [(A) From Lecuit and Cohen (1997) Nature 388, 139-145; copyright Macmillan Magazines Ltd.] (B) Composite confocal images displaying domains of expression in 3rd instar discs of hth (red), dll (green), hth + dll overlap (yellow), dachshund (doc), dark blue; dac + dll (light blue) genes. Dac is a nuclear protein required for formation of femur, tibia, and the proximal three tarsi (Mardon et al., 1994; Lecuit and Cohen, 1997). Images were obtained by immunocytology. (Bl) Horizontal optical section; (B2) three images of an optical cross section (at arrow) of disc shown in (Bl). The top image displays expression of all three factors; the center image of Hth and Dll only; the bottom image of Hth and Dac only. The convoluted form of the central (distal) region of the disc at this stage is here evident. Arrows indicate regions where expression domains overlap.

Coxa

Trochanter

Femur

Tarsal segments

Claws

Larva

Pteropieura

Ventral wing surface

Mesopfeura

Dorsal wing surface

Wing = margin cu

Dorsal wing surface

Mesopleura

K Ptero- Wv f Postnotum p|euraT>w

Post- ^ Scutellum pieropleura

Prescutum-

Ventral wing surface

Scutum

Postnotum autonomous and indirect or nonautonomous developmental failures. In Fig. 4.5D the effects on rhombomeric gene expression patterns of a kreisler {kr) gene knockout are shown (Manzanares et al., 1999a). The kr gene is normally expressed in r5 and r6, and the main effects of the knockout are in these domains, but again there are nonautonomous effects as well. Although not illustrated here, the subsequent consequence is again the development of defective neuronal architecture. In case there were any doubt, these examples show the essential role of the transient early transcriptional states by which specific downstream regulatory functions are brought into play in individual rhombomeres.

Appendage Parts and Transcriptional Patterns in Drosophila Imaginal Discs

Drosophila affords many excellent examples of the institution of spatial transcriptional domains at the beginning of a developmental process, the function of which is to specify subpopulations of cells that will give rise to particular components of a body part. But none so transparently reveal the nature of the mechanism as does transcriptional pattern formation in imaginal discs. This is partly because the cell populations of these anlagen are confined from the beginning; the imaginal disc cells are set aside from the rest of the epidermis as in Fig. 4.IE, and in terms of transcriptional state and morphology remain distinct. A major simplifying factor is the two-dimensional nature of their fate maps. Figure 4.6 illustrates such fate maps, and their relations to the three-dimensional structures to which the discs give rise, for leg (Fig. 4.6A) and wing (Fig. 4.6C). The centers of the discs evaginate

[(B) From Wu and Cohen (1999) Development 126, 109-117 and The Company of Biologists Ltd.] (C) Diagrammatic display of 3rd instar wing disc (anterior left), and of the dorsal and ventral surfaces of the adult wing. [(C) From Williams and Carroll (1993) BioEssays 15, 567-577, copyright Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.] (D) Transcriptional regulatory states that mark the dorsal, ventral, and anterior domains of the wing disc, oriented as in (C), and displayed immunocytologically. The vestigial (vg) gene, which encodes a transcriptional cofactor (Haider et al., 1998) is expressed throughout the wing blade (green), under control of the boundary and quadrant enhancers discussed in Chapter 2 (cf. Fig. 2.7); and the apterous (ap) gene, which encodes a Lim domain transcription factor, is expressed in the future dorsal domain of the wing blade and in more proximal regions (red). Thus the dorsal side of the future wing blade is denoted by ap + vg expression and the ventral side by expression of vg without ap. All cells of the anterior portion of the disc express the cubitus interruptus (ci) gene (blue), which is activated downstream of Hedgehog signaling on the anterior side of the A/P boundary (Jiang and Struhl, 1996). The A/P boundary itself is marked by a stripe of Dpp expression is not shown here. [(D) From Williams et al. (1994) Nature 368, 299-305; copyright Macmillan Magazines Ltd.]

to form the distal structures of the appendages and the peripheral regions give rise to the structures that join the appendage to the body wall and the adjacent portions thereof. By comparison to the fate maps, the images of the transcriptional domains set up in these discs say it all: for leg discs see Fig. 4.6B (Lecuit and Cohen, 1997; Wu and Cohen, 1999); for wing discs, Fig. 4.6D (Williams et al., 1994). The transcriptional domains mark out the regions of the discs from which the subparts of the emergent appendages will form, according to the fate map. As many mutational studies show {ibid, for references), expression of the genes is indeed essential for the development of the respective subparts, though many subsequent patterning steps intervene before institution of the final, detailed morphogenetic processes (see below).

In summary, in the modern Bilateria body parts are composed of subparts, and following the initial transcriptional specification of the progenitor field for the morphologically distinct body part, the subparts are each defined by institution of a particular regulatory state. This requires expression of a set of genes encoding transcription factors, and then the operation of an ensuing regulatory network, within the regions from which the subparts will form. The mechanisms by which the regional expression domains of such genes are set up; what these genes do; and what happens downstream, we take up in the following.

glimpses of how it works

For our purposes "how it works" means getting as close as possible to the way pattern formation is encoded in the genome. For that is of course where the whole process is programmed. As remarked above, thereby we can approach a direct DNA-level solution to the problem of understanding what has happened in evolution to generate diverse animal morphologies, i.e., the clade-specific parts and pieces of body plans. Someday, perhaps not too far off, we will be able to trace the genomic regulatory network for formation of a typical animal body part all the way from its inception, through the imposition of all the spatial transcriptional domains which specify the future subregions of the body part, to its terminal differentiation phase. This will require knowledge of the large number of functionally linked czs-regulatory elements which mediate expression of the key genes in the network. We will need to know the relevant inputs and outputs of these cis-regulatory systems, most importantly the signal pathway termini and the connections among the many genes encoding transcription factors. But nowhere does current knowledge yet encompass a large fraction of any pattern formation regulatory network for an adult body part. We do have some reasonably well illuminated pieces and bits of such networks, for a number of different developmental systems. Five examples follow. In discussing these only the bare bones of the developmental processes themselves are given: rather the intent is to focus on regulatory properties that these systems illustrate, and that can be regarded as intrinsic, even canonical aspects of genomic pattern formation architecture.

These general regulatory properties are as follows: (1) modularity in cz's-regu-latory organization underlying the specific subparts of the future structure; (2) use of integrative czs-regulatory information processing for establishing spatial transcriptional domains; (3) depth in the regulatory network, i.e., many layers of interaction between initial specification of the progenitor field and the ultimate activation of differentiation genes; and (4) the presence of inputs from intercellular signaling pathways at each level of spatial transcriptional specification. The reader will have to wade along, as these features cannot be taken up in a completely organized fashion. Each of the five systems that we shall touch on can be used to illustrate some, but none illustrate all of these aspects of pattern formation mechanism.

Transcriptional Domains and the Pattern Program for the

Drosophila Wing Disc: Modularity and cis -Regulatory Inputs

The main pattern elements on the adult Drosophila wing are its veins and a large array of exactly positioned peripheral nervous system elements. These are stereotypic, species-specific features (for comparative consideration of veins see Biehs et al., 1998; and of the relevant sensory elements of the peripheral nervous system, Wiilbeck and Simpson, 2000). Each of the peripheral nervous system elements begins as a localized patch of cells, or a "proneural cluster," which expresses genes of the achaete-scute complex {AS-C). These genes encode a special class of bHLH transcriptional regulators. As illustrated in Fig. 4.7A2 more than two dozen proneural clusters can be detected in the 3rd instar disc as patches of AS-C expressing cells. The locations of the sensory organs to which each gives rise are shown in Fig. 4.7A3. In these figures the color coding reflects the modular organization of the AS-C czs-regulatory system (Gomez-Skarmeta et al., 1995), as indicated in Fig. 4.7A1. This fascinating relation means that the individual proneural domains are set up by the individual enhancers of the AS-C genes, each of which processes the locally incident spatial information available to it in the form of transcription factors expressed or activated there. There is no single prior proneural cluster pattern: the modular AS-C czs-regulatory system generates it. In this light one can see why such pattern formation systems are modular, since each location requires that a unique czs-regulatory information processing job be performed (cf. Chapter 2). Ultimately a single sensory organ precursor (SOP) cell is specified within each proneural cluster, as touched on later in this chapter. AS-C regulators play a role in the process by which the SOPs are chosen from amongst the other cells of the proneural clusters; but once specified all of the SOPs present the same information to the AS-C control system, and now a single AS-C module runs those genes in all of the SOPs (black box in Fig. 4.7A1). SOP specification occurs asynchronously, so at the stage illustrated in Fig. 4.7B an enhancer trap in the SOP module produces its Lacz product in only a subset of the proneural clusters (greenish color). The proneural clusters are identified by endogenous AS-C expression (purple) under control of the remainder of the modules shown in Fig. 4.7A1, i.e., the pattern formation modules.

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Training And Devolopment

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