FIGURE 4.12 Specification of sensory organ precursors and individual cell types in the Drosophila wing imaginal disc. (A) c/s-Regulatory elements controlling expression of some Enhancer of split (E(spl)) Complex genes; solid symbols
indicate interactions established by direct DNA-protein binding studies; open symbols, probable sites based on their similar sequence (see color key for identity). Each gene has sites for AS-C proneural activators (green triangles); for bHLH repressors (red circles); and for the Suppressor of Hairless (Su(H)) factor (blue square). Together with the intracellular domain of Notch (N), which is released on ligand binding, the Su(H) protein acts as a transcriptional activator (Struhl and Adachi, 1998; Artavanis-Tsakonis et al., 1999; Lecourtois and Schweisguth, 1998). Other genes of the E(spl) Complex share the same sets of target sites in addition to those shown (Nellesen et al., 1999). The E(spl) genes themselves encode bHLH repressors (of those shown, mS, my, m5, ml, m8) or small proteins probably involved in N signaling (ma, m4). [(A) Adapted from Nellesen et al. (1999) Dev. Biol. 213, 33-58.] (B) Expression of individual lacz constructs driven by E(spl) c/s-regulatory elements in wild-type wing discs and in discs in which an activated form of N is expressed ubiquitously. Despite these similar inputs some E(spl) genes must respond to additional cues as well, since they are expressed in several different patterns (Nellesen et al., 1999; Ligoxygakis et al., 1999; Cooper et al., 2000). (Bl) m4.lacz, revealed by in situ hybridization against lacz mRNA; (B2) m4.lacz in ectopic N disc; (B3) my.lacz; (B4), my.lacz in ectopic N disc. [(B) From Nellesen et al. (1999) Dev. Biol. 213, 33-58.] (C) Lineage of mechanosensory bristle cell types. In each of the divisions indicated N signaling differentiates between alternative fates, in that the cell on the left as shown by the barred horizontal lines is prevented by the signal from executing the fate of its sister cell. Signaling is bidirectional, but the PIIB cell and the shaft and neuron cells do not respond because of the activity of N pathway antagonists, viz, Numb and Hairless (H); furthermore, the effectors of N signaling are different in the socket and sheath cells (Posakony, 1994; Frise et al., 1996; Nagel et al., 2000). The shading indicates levels of accumulation of the Pax2 transcription factor. (D) Pax2 expression in microchaetes of the notum, 32 h after puparium formation, identified immunocytologically (red, green in inset) together with an antigen expressed on the neuron and shaft cell (blue stain). The two nuclei containing Pax2 protein are the small sheath cell nucleus (arrowhead) and the shaft cell nucleus. The neuron is identified by the arrow. The inset shows a macrochaete: Pax2 is here indicated in the sheath cell in green; the neuron, identified by a neuron-specific antibody, in red. Pax2 is also present in the shaft cell, but not the socket cell (cf. C). (E) Pax2 function in cell type specification. (El) Scanning EM of wild-type macrochaete shaft surrounded with microchaetes; (E2) failure of shaft formation in a loss of function pax2 mutant, resulting in an empty socket; (E3) normal expression of pax2 gene in bristle lineage identified immunocytologically as above; (E4) pax2 expression after repression of N signaling (by overexpression of the H antagonist of N signaling, driven by a heat shock cis-regulatory element). Three cells rather than two now express the pax2 gene in each mechanosensory structure; these are the sheath, shaft, and what should have been the socket cells. (E5) Normal bristle shafts; (E6) bristles with double shafts, due to conversion of sheath cell to shaft fate after repression of N signaling. Other experiments show that this effect is indeed caused by the ectopic pax2 expression. [(C-E) From Kavaler et al. (1999) Development 126, 2261-2272 and The Company of Biologists Ltd.; in (C) information from Gho et al., 1999 has been added.]
descendants, but the gene is turned off in the prospective neuron and socket cells (Fig. 4.12C). Expression of Pax2 is seen cytologically in Fig. 4.12D, and the requirement for this factor in order for differentiation of the shaft to take place is dramatically illustrated in Fig. 4.12E1, E2 (Kavaler et al., 1999). Without pax2 gene expression a hole appears in place of the bristle shaft. Furthermore, pax2 gene is a direct target of N signal transduction (Kavaler et al., 1999)- If the N pathway is repressed, the Pax2 protein remains present in both of the PIIA daughter cells (Fig. 4.12E3, E4); the result is that both become shaft cells, producing a double shaft phenotype (Fig. 4.12E6).
A considerable number of differentiation gene batteries must be expressed in the development of the whole wing, including its veins, its intervein epidermis, its hinge structures, its other peripheral nervous system elements, and so forth; there are four cell types in the macrochaete alone. This is scarcely atypical. Formation of an adult body part always involves the (more or less exact) spatial installation of multiple gene batteries encoding effector proteins, superimposed on all the patterning mechanisms that have gone before.
Installation of Cell Type-specific Differentiation Programs in the
The transition between morphogenetic pattern formation processes and those detailed strategies used to call in cell type-specific differentiation programs can be seen anywhere one looks. The terminal differentiation programs themselves (the "routines" of the title of this section) are not our subject here (cf. Chapter 2); rather is their linkage into the prior spatial regulatory apparatus. Our final example in this chapter is the pituitary, in which many differentiation programs that are responsible for expression of this organ's endocrine genes have been defined (Simmons et al., 1990). Key aspects of the pattern formation process leading to the morphogenesis of the structure from which the pituitary develops, Rathke's pouch, are also known. Figure 4.13A1 shows a diagram of the formation of Rathke's pouch in the mouse, and some prominent signaling interactions involved in setting up its regional transcriptional states. Rathke's pouch arises at the interface of the dorsal oral ectoderm and the ventral diencephalon. A large set of genes encoding transcription factors is expressed in different regions of the pouch, as shown in Fig. 4.13A2 (Treier et al., 1998). Six different endocrine-secreting cell types arise, in specific spatial domains of the forming gland (Fig. 4.13A3). These are the corticotropes, which produce adrenocortico-stimulating hormone (ACTH); the melanotropes, which produce melanocyte-stimulating hormone (MSH); the thyrotropes, which produce thyroid-stimulating hormone (TSH); the somato-tropes, which secrete growth hormone (GH); the lactotropes, which produce prolactin (PRL); and the gonadotropes, which produce luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Each of these cell types arises within a domain defined earlier by a particular transcriptional state, as indicated in the diagram of Fig. 4.13B. As implied there, part of the mechanism we want to know about will emerge from detailed descriptions of the cz's-regulatory transactions which account for the activation of the genes encoding these transcription factors. There seem always to be lots of special little mechanistic devices utilized at this level of the developmental control apparatus, and a closer look at the specification of the cell types that arise in the ventral region of the forming pituitary after about day 13 provides an illustration.
The ventral region is where gonadotropes, thyrotropes, and somatotropes plus lactotropes differentiate, in three adjacent domains. The key upstream transcriptional regulators which are required to install these respective differentiation programs in their respective regions are the POU homeodomain factor Pitl and the Gata2 factor (Fig. 4.13B, C; see legend for details and references) The regulatory relations by which this occurs are indicated in Fig. 4.13C2, C3 (Dasen et al., 1999). Essentially, a ventral-to-dorsal concentration gradient of gata2 expression that is set up under the influence of a ventral source of BMP2, plus the interrelations between the gata2 and pitl genes and their products establish the three different regulatory domains. This works as follows: high levels of Gata2 factor repress the pitl gene, but Pitl, off the DNA, interacts with the Gata2 factor so as to interfere with its ability to serve as a transcriptional activator, except in cz's-regulatory elements where sites for both factors are contiguous. Given the constitution of the cz's-regulatory target sites in genes controlling the final differentiated functions of gonadotropes, thyrotropes, and somatotropes plus lactotropes, this suffices to establish the three ventral regulatory states (Fig. 4.13C; see legend). Note that in this case as in the preceding example of bristle cell-type specification, signaling plays an essential role, right to the end of the process. But if we ask where lies the spatial "intelligence" of the regulatory system by which the differentiation programs are installed, the answer is of course in the genomic cz's-regulatory elements. It is these elements which enable the genes of the different cell types to respond differentially to the Pitl and Gata2 concentrations with which they are confronted (Fig. 4.13C3); and it is the relevant cz's-regulatory modules of the pitl and gata2 genes which determine their own sites of expression.
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