Chapter seven - A Dynamic Network of Morphogens and Transcription Factors Patterns the Fly Leg

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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.

Introduction

Animal appendages are external projections from the body wall that are used for very diverse functions including locomotion, grooming, and feeding. In the thorax of diptera, such as the fruit fly Drosophila melanogaster, there are dorsal appendages required for flight—a pair of wings in the second thoracic (T2) segment and a pair of halteres in T3—and three pairs of legs used for walking and grooming. The fly leg, the subject of this review, is composed of 10 morphologically unique segments: coxa, trochanter, femur, tibia, tarsal segments 1–5, and the claw. Together, these segments comprise the proximodistal (PD) axis, in which the proximal coxa is closest to the body and the claw is furthest from the body (Fig. 7.1).

Unlike the two other primary body axes (anteroposterior, AP; dorsoventral, DV), for each appendage, the PD axis is established during embryogenesis de novo. In contrast, at all stages of development, even in the unfertilized egg, rudimentary AP and DV axes exist. Thus, in this respect, the PD axis is unique among the main body axes. This topic, how so-called secondary developmental fields are established from preexisting developmental information, has been debated for decades both from theoretical perspectives and by classical developmental biologists (reviewed by Baker, 2011). Data generated over the past several years have provided novel mechanistic and molecular insights that build upon these earlier studies, providing interesting connections between cell division, secreted morphogens, and the use of dedicated cis-regulatory modules (CRMs) for transcriptional regulation of genes expressed along the PD axis. It is the goal of this review to summarize our current understanding of the intimate interplay between these components, orchestrated over developmental time, which establishes, elaborates, and fine-tunes the leg's PD axis.

Section snippets

The Molecular Players in PD Axis Formation

As for much of the adult fly, fly legs are derived from imaginal discs, elliptical sheets of epithelia that are highly folded by the end of larval development. The fate map of the leg disc is such that cells at its center will give rise to distal-most structures, while cells further away from the center generate more proximal structures (Fig. 7.1). Imaginal discs do not only give rise to appendages: cells at the periphery of the leg disc, for example, generate the ventral portion of the adult

The Initial Establishment of the PD Axis is Encoded in the cis-Regulatory Architecture of Dll

A common principle that has emerged from studying transcriptional regulatory mechanisms in many developmental genes is that dedicated CRMs drive small subsets of complex expression patterns (reviewed in Maeda and Karch, 2011). Dissection of the Dll locus revealed a similar level of CRM dedication, but with the additional finding that distinct Dll CRMs control Dll expression in cells that have different degrees of developmental potential (Fig. 7.3). The discovery of these CRMs has allowed

The Role of Sp1 in Distinguishing Ventral Appendage from Dorsal Appendage Fates

Although the above focus on Dll regulation reveals how the initial PD axis and leg fate map are established, several questions remain concerning this early stage of leg development. Some of these questions are answered by two paralogous genes, buttonhead (btd) and Sp1 (Estella and Mann, 2010, Estella et al., 2003, Wimmer et al., 1996). Both genes encode Sp family zinc-finger transcription factors that share a similar expression pattern throughout development. Despite their similar expression

Elaboration of the PD Axis: The Role of brk

By the end of embryogenesis, a rudimentary PD axis of the leg is apparent in the expression patterns of Dll (via DllLT, DllDKO, and DllLP), hth, and tsh. How are these initial patterns elaborated upon to create the mature PD axis present in the third larval instar stage? The separation between these two time points is huge both in terms of time (96 h) and tissue growth (from ∼ 60 cells to ∼ 10,000 cells). Also, at the end of embryogenesis, dac has not yet been activated but begins to be expressed

Elaboration of the PD Axis: The Role of a Transcription Factor Cascade and Cross-regulation

In addition to positing that Wg and Dpp are critical for initiating the PD axis, Lecuit and Cohen proposed a gradient model to account for the PD axis expression patterns of dac and Dll. According to this model, the expression of dac and Dll along the PD axis depends on the levels of Wg and Dpp a cell perceives: high concentrations of both Wg and Dpp activate Dll and repress dac in the center of the leg disc; intermediate levels activate dac but not Dll in medial regions of the disc; and low

Patterning the DV Axis

If gradients of Wg and Dpp are not used to establish the PD axis, what purpose might they serve? The posterior expression of the homeodomain transcription factors engrailed (en) and invected (inv) divides the leg into anterior and posterior compartments, which have distinct cell lineages (Morata and Lawrence, 1975). In contrast to strict lineage restrictions along the AP axis, the distinction between dorsal and ventral fates is controlled by the secreted molecules Wg and Dpp in a

EGFR Signaling Patterns the Tarsus

While Wg and Dpp play an important role in initiating the PD axis, by the early third instar, Wg and Dpp are no longer required for the PD axis and the role of further elaborating this axis is handed off to the EGFR signaling pathway (Campbell, 2002, Galindo et al., 2002; Fig. 7.7). Shifts of the temperature-sensitive mutant Egfr[ts] (Egfr[tsla]/Egfr[null]) to the restrictive temperature in the beginning of the third larval stage lead to development of legs without pretarsus and one or more

Leg Segmentation and Growth

The process of leg segmentation, or forming the joints that separate each of the leg segments, is one critical downstream consequence of PD gene expression (Rauskolb, 2001). Several genes and pathways required for forming the joints have been defined (Bishop et al., 1999, Chu et al., 2002, Ciechanska et al., 2007, de Celis Ibeas and Bray, 2003, de Celis et al., 1998, Galindo et al., 2005, Greenberg and Hatini, 2009, Greenberg and Hatini, 2011, Hao et al., 2003, Kerber et al., 2001, Mishra et

Concluding Remarks

The above review reveals that a molecular framework of PD axis formation is now emerging. Yet, many questions remain. For one, the initial stages of dorsal and ventral primordia establishment are not well understood. How, for example, does Sp1 block dorsal appendage fates? Second, although graded levels of Wg and Dpp activities may not be relevant to elaborating the PD axis, it remains an open question whether a gradient of EGFR signaling is used to turn on its targets in the tarsus. Third, it

Acknowledgments

We are grateful to members of the Mann, Struhl, and Johnston labs for comments, and funding from the NIH (GM058575). R. V. is a CDP Fellow of the Leukemia and Lymphoma Society.

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