A dynamic network of morphogens and transcription factors patterns the fly leg

Abstract

Animal appendages require a proximodistal (PD) axis, which forms orthogonally from the two main body axes, anteroposterior and dorsoventral. In this review, we discuss recent advances that begin to provide insights into the molecular mechanisms controlling PD axis formation in the Drosophila leg. In this case, two morphogens, Wingless (Wg) and Decapentaplegic (Dpp), initiate a genetic cascade that, together with growth of the leg imaginal disc, establishes the PD axis. The analysis of cis-regulatory modules (CRMs) that control the expression of genes at different positions along the PD axis has been particularly valuable in dissecting this complex process. From these experiments, it appears that only one concentration of Wg and Dpp are required to initiate PD axis formation by inducing the expression of Distal-less (Dll), a homeodomain-encoding gene that is required for leg development. Once Dll is turned on, it activates the medially expressed gene dachshund (dac). Cross-regulation between Dll and dac, together with cell proliferation in the growing leg imaginal disc, results in the formation of a rudimentary PD axis. Wg and Dpp also initiate the expression of ligands for the EGFR pathway, which in turn induces the expression of a series of target genes that pattern the distal-most portion of the leg.

Figure 7.1

Overview of fly leg development. On the left shows the relationship between En, Hh, wg, and dpp and the definition of the telopodite (Hh, Wg, and Dpp-dependent domain) and the coxopodite (Hh, Wg, and Dpp-independent domain). On the right shows the relationship between the three primary PD gene expression domains established by Hth, Dac, and Dll.

Figure 7.2

Wg+Dpp initiate the PD axis. (A) Meinhardt’s “three-sector” model for induction of the PD axis. (B) Hh, from the P compartment, induces Wg (yellow) in the anterior ventral (AV) domain and Dpp (blue) in the anterior dorsal (AD) domain; note that Wg- and Dpp-expressing cells are only adjacent in the center of the wild-type disc. (C–E) An ectopic source of Dpp (C) in the ventral domain induces an ectopic PD axis visualized in the disc (D) and in the adult appendage (E; from Campbell et al., 1993).

Figure 7.3

Dll and dac CRMs. (A,B) Schematic of the Dll (A) and dac (B) genomic regions showing the positions of identified CRMs (colored boxes) and transcription units (large arrows). The expression patterns driven by individual CRMs is indicated and compared to the intact genes. All CRMs are mentioned in the text except for DllMX, DllWM, dac3EE, and dac5EE, which are not active during leg development, or DllBR, which is active very late in leg development (Galindo et al., 2011; Pappu et al., 2005). A Dll rescue transgene (“312 rescue”) and a small Dll deficiency (Dll^R28) both result in a nearly complete PD axis, with defects primarily in the tarsal segments. dac⁷, which removes dacRE, is a deficiency that eliminates both dac expression and the medial Dac domain in the leg.

Figure 7.4

Embryonic appendage fate map. (A) Cells expressing Dll at stage 11 via the 304 CRM can give rise to the entire adult thorax, while those expressing Dll at stage 14 give rise to either the KO or telopodite, depending whether DKO or LT is driving expression, respectively. (B) Genes expressed in the progenitors to the telopodite, KO, and coxopodite. (C) Fate map and regulatory network defining the activity of Dll CRMs. The blue cells, expressing esg but not Dll, are fated to become coxopodite.

Figure 7.5

Gradient versus cascade models. (A) The gradient model, highlighting that, depending on a cell’s position in the disc, Dll and dac CRMs must interpret very different ratios of Dpp:Wg signaling. (B) The cascade model, in which Wg+ Dpp are only required to initiate PD axis formation by activating Dll and ligands for the EGFR pathway. Dll in turn activates dac, and both Dll and dac maintain their expression in a Wg+Dpp-independent manner. EGFR activity maintains dac repression, while Wg+Dpp repress dac in the center of the leg disc early in leg development.

Figure 7.6

Summary of the dynamic network establishing the PD axis. The relationship between Dpp, Wg, and Brk triggers the expression of Dll and represses dac in the center of the disc. As the disc grows, Dll activates dac in cells that escape repression by Wg+Dpp. These initial domains are likely maintained by a combination of autoregulation, cross-regulation, and transcriptional memory systems.

Figure 7.7

EGFR signaling patterns the tarsal segments. After the initial PD domains are established, EGFR ligands are produced at the center of the disc and activate a series of secondary PD targets in the progenitors to the tarsal segments.