Wing tips: The wing disc as a platform for studying Hedgehog signaling
Introduction
Initial analyses of Hh-mediated developmental events were conducted in the embryo, where Hh ligands are necessary for larval segment polarity. Larvae with null hh mutations lose naked cuticle tissue. What remains is a shrunken body covered with disorganized denticles, which gave rise to the name “hedgehog” [1], [2]. This same phenotype occurs in embryos lacking function of wingless (wg) [2]. In fact, a primary function of Hh signaling in the embryo is to promote the expression of wingless (wg) in adjacent cells [3], [4], [5]. Wg is necessary to specify ectodermal cells that secrete larval cuticle [6]. Wg also feeds back to induce expression of engrailed (en), which drives expression of hh. This forms a positive feedback circuit that sustains wg and hh expression in neighboring cells [4], [7], [8], [9], [10].
Understanding steps in Hh signal transduction began with studies of embryos that implicated another segment polarity gene “patched” (ptch). Embryos with diminished Ptch expanded wg expression [7], [8]. This indicated that Ptch represses wg, yet paradoxically, Ptch and Wg were found to be transcribed in the same row of cells [11]. Thus, Ptch could only be a repressor of wg expression if it were functionally inactivated at a post-transcriptional level. It was hypothesized that Ptch, a transmembrane protein [12], [13], might be a receptor that is deactivated through the binding of an unknown ligand. The inhibition of the receptor would lead to wg induction. The hypothetical ligand was theorized correctly to be Hh [11]. That idea was based on knowledge that Hh is produced by cells adjacent to those that co-transcribe ptch and wg [9], [14], and that wg expression is dependent upon hh [4], [7], [8], [9], [10]. Consistent with the idea that Hh blocks Ptch’s repression of wg expression, hh; ptch double mutant embryos had the ptch phenotype of wg overexpression – proving that ptch is epistatic to and thus downstream of hh [11]. This model was proven through the subsequent demonstration that Hh is a secreted molecule [15] that can bind Ptch [16], [17], be sequestered by Ptch [18], and negatively influence Ptch function [19].
The study of Hh signaling in the embryo established a blueprint of genetic interactions that has been built upon and refined over the last two decades. The imaginal wing disc has often facilitated these studies. The wing disc is made up of about 50,000 cells that develop into the adult wing and into body wall structures to which the wing is attached (Fig. 1A and B). The ventral and dorsal most regions develop into the pleura and notum structures of the dorsal thoracic body wall where the wings are attached. More internal disc regions will become the ventral and dorsal wing hinge, and the centrally located wing pouch differentiates into the adult wing blade.
The wing disc is subdivided into anterior and posterior compartments that are separated by an anatomical boundary called the A/P border. These compartments are defined by lineage restrictions; clones of cells will expand to fill parts of a single compartment, but normally do not cross the A/P border [20]. Anterior structures such as wing veins 1–3, the anterior crossvein (ACV), and Costal (Co), triple row (Tr), and double row (Dr) bristles along the wing margin are established by patterning events in the anterior compartment of the wing disc. Posterior structures such as the posterior row (Pr) and alula hairs (Al), wing veins 4 and 5, and the posterior crossvein (PCV) are patterned in the posterior compartment of the wing disc [21], [22], [23] (Fig. 1A and B).
The wing disc consists of two juxtaposed layers of epithelial cells referred to as the “columnar layer” cells and the “peripodial layer” cells. The columnar and peripodial layers are separated by a lumen. The two cell layers are situated such that their apical surfaces are oriented toward the lumen and their basolateral surfaces are oriented away from the lumen [22] (Fig. 1A).
Hh patterns the entire wing through cellular events it induces in the imaginal wing disc [15], [24], [25]. Wing development is orchestrated by many molecular events, starting with the expression of hh exclusively in the posterior compartment in response to the Engrailed and Invected transcription factors (Fig. 1C) [9], [15], [26], [27]. Hh ligands are secreted directionally from the posterior compartment and reach ∼12 cell rows near the A/P border of the anterior compartment (Fig. 1C) [15], [24], [28]. Hh secretion depends upon a balance of Dispatched, Dlp, and Dally, which promote Hh release and flow through the tissue, and Ihog, and Boi, which restrain Hh release [28], [29], [30]. Mature Hh protein carries N-terminal palmitic acid and C-terminal cholesterol adducts [31], [32], [33] that can impact its movement through tissue and its strength as a signaling molecule [34]. Lipid modifications generally tether proteins to the cell membrane, so the flow of dually lipidated Hh protein through tissues may be facilitated by packaging into multimers [35], [36], [37], [38], exovesicles [39], and lipoproteins [40], [41], [42]. An exciting new model for how Hh moves through tissues came from the recent discovery that Hh navigates wing disc epithelia along the exterior of cellular extensions of the basolateral membrane called cytonemes that can span up to 70 μm in length [29], [30], [43], [44].
In most cases, Hh signaling controls tissue development by regulating post-translational modifications of the Cubitus Interruptus (Ci) transcription factor and, by extension, the genes Ci regulates [45], [46] (Fig. 2). In the absence of Hh ligands, the kinases Protein Kinase A (PKA), Casein Kinase 1 (CK1), and Glycogen Synthase Kinase 3 (GSK3) phosphorylate the C-terminus of full-length (155 kDa) Ci, which is termed CiF. The multi-site phosphorylation upon CiF create a site primed for ubiquitination by SCFSlimb, leading to partial proteosome processing and removal of Ci’s C-terminal trans-activation domain [47], [48], [49], [50], [51], [52], [53], [54], [55], [56]. The resulting 75 kDa Ci, with its N-terminal zinc-finger DNA binding domain intact, behaves as a transcriptional repressor termed CiR [56]. CiF is complexed alongside these kinases by Cos2, a kinesin-like protein that serves as a scaffold for Hh signaling components [57], [58], [59], [60], [61], [62], [63].
The conversion of CiF to CiR leads to loss of target gene induction [56], but this process is reversed when Hh binds to Ptch. Hh inhibits Ptch, activating signal transduction by relieving the negative influence of Ptch upon the 7-pass transmembrane protein Smoothened (Smo) [19], [64], [65]. Exactly how the Hh/Ptch interaction leads to Smo activation is unclear. At the molecular level, the inactivation of Ptch causes phosphorylation of Smo’s C-terminal tail by PKA and CK1, which leads to C-terminal tail dimerization. At the cellular level, Hh causes Ptch to move from the cell surface to the interior, while Smo accumulates on the cell surface [66], [67], [68], [69], [70], [71]. Activated Smo has an enhanced interaction with Cos2 and Fu that promotes Fu dimerization and activation [72], [73], [74]. Active Fu promotes Hh signaling by disrupting the interaction of Cos2 with Ci, accompanied by phosphorylation of Ser572 on Cos2, thereby preventing CiF phosphorylation by PKA, GSK3, and CK1 and its proteolysis to CiR [61], [72], [73], [74], [75], [76]. Active Fu also disrupts Su(fu)’s cytoplasmic sequestration of CiF, enabling CiF to move to the nucleus and induce target genes [72], [77], [78], [79], [80], [81]. Fu may even promote CiF activity independent of repressing Su(fu) and Cos2, because constitutively active Fu can more fully activate Ci targets in su(fu); slmb mutant clones where CiF cannot be processed to CiR nor sequestered in the cytoplasm [74].
The exposure of cells to normal levels of Hh in the wing disc’s anterior is critical for normal anterior wing development. Hh promotes three general activities to ensure precision in its concentration through the wing disc. First, Hh regulates gene expression by stabilizing CiF in the anterior [45], [46]. Then, through Ci regulation of target genes, Hh establishes cell-to-cell contact affinities that prevent anterior cells from intermixing with posterior cells [45], [82], [83]. This promotes the formation of the A/P border boundary between anterior and posterior cells. The defined A/P border creates a spatially precise starting point for Hh ligand release from posterior cells. This establishes consistent Hh doses in anterior cells immediately adjacent to the A/P border. Lastly, in anterior cells abutting the A/P border, Hh ligands induce transcription of the Hh receptor patched via Ci [15], [45]. Along with co-receptors Boi and Ihog, this heightened level of Ptch causes Hh ligands to become partially sequestered at the A/P border [18], [84], [85], [86]. This generates a gradient of Hh morphogen that spans at least ∼12 cell rows (Fig. 1B) [15], [24], [28].
Section snippets
Hh target genes, signal transducers, and wing anatomy as readouts of Hh signaling
The wing disc and wing have been key to assembling Hh signal transduction events, and these tissues provided great insight into how Hh and other morphogens control tissue development. In this section we describe the wing disc properties of Hh target gene induction and detail characteristics of Ci and Smo in the wing disc. We also describe Hh driven wing-patterning events. Experimental manipulations that led to a deviation of these Hh-driven processes from their wild-type patterns has provided
Methods to genetically manipulate the wing disc to study Hh signaling: Mosaics and region-specific Gal4 drivers
Work from numerous laboratories has generated a remarkably diverse tool kit that enables the genetic manipulation of precise or desirably variable regions of the wing disc. This set of tools is based on the binary Gal4/UAS system [127] and various approaches to create patches of cells, or “clones”, genetically unique from the surrounding tissue’s genotype. The versatility and unique properties of these tools have been a primary factor leading to the creation of the imaginal wing disc as a model
Culturing larvae for Gal4/UAS and mosaic analyses
Variations in food freshness, amounts, nutrient level, and larval density can lead to variable lengths of larval development. Inconsistent larval development duration can have an impact on phenotypic differences, if, for example, a tissue is exposed to RNAi-mediated knockdown for a greater period of time in one animal versus another. We set up crosses and culturing conditions in a manner that enables developmental timing consistency between different crosses. Thus, we use standard food that is
Acknowledgements
We thank Kaye Suyama for guiding T.A.H. in studies of the imaginal wing disc and for providing Fig. 3A. T.A.H. was a Robert Black fellow of the Damon Runyon Cancer Research Foundation (DRG-2045-10) and is currently supported by a Postdoctoral Fellowship (PF-13-191-01-DDC) from the American Cancer Society.
References (152)
- et al.
Cell
(1989) - et al.
Cell
(1994) - et al.
Cell
(1996) - et al.
Cell
(1992) - et al.
Dev. Cell
(2010) - et al.
Dev. Biol.
(2013) - et al.
Cell
(1996) - et al.
Trends Cell Biol.
(2007) - et al.
J. Biol. Chem.
(2006) - et al.
Dev. Cell
(2007)
Cell
Trends Genet.
Cell
Dev. Cell
Curr. Biol.
Dev. Cell
Cell
Cell
Dev. Biol.
Dev. Cell
Curr. Biol.
Cell
Cell
Cell
Dev. Cell
Dev. Biol.
Mech. Dev.
Cell
Cell
Curr. Biol.
Genetics
Nature
Development
Development
Nature
Development
Development
Nature
Genes Dev.
Nature
Nature
Nature
Development
Nature
Nature
Nature
Nat. New Biol.
J. Exp. Zool.
The metamorphic development of the adult epidermis
Imaginal disc development
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