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Leg Patterning Driven by Proximal-Distal Interactions and EGFR Signaling

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Science  12 Jul 2002:
Vol. 297, Issue 5579, pp. 256-259
DOI: 10.1126/science.1072311

Abstract

wingless and decapentaplegicsignaling establishes the proximal-distal axis of Drosophilalegs by activating the expression of genes such asDistalless and dachshund in broad proximal-distal domains during early leg development. However, here we show thatwingless and decapentaplegic are not required throughout all of proximal-distal development. The tarsus, which has been proposed to be an ancestral structure, is instead defined by the activity of Distalless, dachshund, and a distal gradient of epidermal growth factor receptor (EGFR)–Ras signaling. Our results uncover a mechanism for appendage patterning directed by genes expressed in proximal-distal domains and possibly conserved in other arthropods and vertebrates.

Animal appendages develop along a proximal-to-distal (PD) axis, from proximal body wall to distal tip. This axis is not inherited from the embryo and is established anew in each developing appendage. In Drosophila legs, the combination of a dorsal signal provided by the BMP4 homologdecapentaplegic (dpp) with a ventral signal provided by the Wnt homolog wingless(wg) establishes the PD axis (1), in addition to organizing the dorsal-ventral appendage pattern (2,3). Signaling from wg and dppactivates the expression of the genes Distalless(Dll) and dachshund (dac) early in leg development (4, 5). Dll encodes a homeodomain protein expressed and required in the distal half of the leg, from tibia to pretarsus, whereas dac encodes a nuclear protein expressed and required medially in the femur and tibia. However, the leg comprises 10 segments along the PD axis, whose specification involves further genes (6). Here we describe how, after 84 hours of development, PD patterning becomeswg- and dpp-independent. Instead, a mechanism mediated by genes expressed in PD domains, such as Dll,dac, and the epidermal growth factor receptor (EGFR) ligandvein, activates the expression of further genes and generates distal leg fates such as the tarsus. Previous data inDrosophila and on homologous genes in other arthropods and vertebrates suggest that this PD patterning mechanism might be conserved and ancestral.

We studied the timing of wg requirements for PD development with a temperature-sensitive mutant (7). Removal ofwg function before 72 hours after egg laying (hours AEL) produces truncated legs lacking the distal parts and showing ventral patterning defects (Fig. 1, A and B). However, shifts of animals from permissive to restrictive temperature at approximately 84 hours AEL produces legs with ventral patterning defects but with an otherwise intact PD organization (Fig. 1C).

Figure 1

wg and dpp signaling are not required for PD development and rn and babactivation after 84 hours AEL. (A) Wild-type leg showing femur (asterisk), five tarsal segments (yellow) with ventral apical bristles (arrowheads) and pretarsal claws at distal tip (arrow). (B) Leg from which wg protein was removed before 72 hours AEL, showing a truncated PD axis lacking tibia, tarsal segments, and claws (asterisk labels femur). (C) Leg after removal of wg from 84 hours AEL, showing a normal PD axis despite loss of ventral structures such as apical bristles. Claws are present (arrow) but ventral pretarsal components are missing. (D) Leg deprived of Mad function from 84 hours AEL. PD organization is intact, despite defects mirroring those of C). The pretarsus lacks claws (arrow) but retains ventral elements, and apical bristles remain while dorsal structures on the opposite side are missing. (E) 80 hours AEL leg disc showing no babexpression. (F) bab expression appears in the center of the disc as the central tarsal fold forms from 90 to 96 hours AEL and is quickly restricted to a ring. (G) babexpression at 120 hours AEL. (H) bab expression is still present after removal of wg function from 84 hours AEL, although the disc is ventrally defective.

To assess the temporal requirements for dpp signaling, we identified a temperature-sensitive allele of Mothers against decapentaplegic (Mad), the dpp signal transducer (7). Removal of Mad function before 72 hours AEL also produces truncations of the PD axis, whereas removal from 84 hours AEL until the end of development renders a normal PD organization despite dorsal patterning defects (Fig. 1D). Clones of null Mad mutant cells (7) show the same results: clones induced before 72 hours AEL show elimination of entire tarsal segments, whereas after 84 hours AEL, only dorsal pattern features are affected. These results show that the input fromdpp and wg signals into PD development ends by 84 hours AEL.

Self-maintenance regulatory mechanisms maintain Dlland dac expression beyond this point (5,8). However, new genes with PD domains of expression and function become active. rotund (rn) encodes a zinc-finger nuclear protein expressed in a transient ring during 84 to 96 hours AEL (9) (fig. S1, A and B). Tarsal segment loss inrn mutants shows that this period is crucial for tarsal development, and a rn-lacZ reporter with persistentlacZ expression reveals that the ring ofrn-expressing cells gives rise to most of the tarsus (fig. S1C). The bric-a-brac (bab) (10) gene encodes a nuclear protein with a BTB/POZ domain required for appropriate tarsal differentiation, whose expression starts in the presumptive tarsus shortly after rn, from 90 hours AEL until the end of development (Fig. 1, E to G). At 120 hours AEL, after extensive cell proliferation, bab expression occupies a wider area, similar to rn-lacZ, corresponding to presumptive tarsal segments one to four (Fig. 1G). Because the onset ofrn and bab expression occurs after 84 hours AEL,wg and dpp signaling should neither be activatingrn and bab nor be directly required for their expression. As expected, removal of wg or dppsignaling in temperature-sensitive mutants or in clones ofMad cells from 84 hours AEL does not affectrn or bab expression (Figs. 1H and2A and fig. S1D).

Figure 2

.Dll and dacdefine tarsal development and gene expression. (A)bab expression (red label, yellow in overlap) is unaffected in clones of Mad cells (absence of green, arrow). (B) The ring of bab expression is included within the Dll domain (red) and appears yellow, but central and medial (arrow) Dll-expressing cells do not express bab. (C) Dll (red) anddac (green) expression overlap in the medial cells (yellow, arrow) of a 120 hours AEL leg disc. (D) rn(green) and dac (red) domains abut at 96 hours AEL. (E) rn expression is not activated inDll leg discs (arrow; compare with Fig. 1F). (F) Dll clones (loss of green) generated at 80 hours AEL. bab expression (yellow) is absent autonomously in Dll cells (arrow), even in single isolated ones (arrowheads). (G) Indac mutants, bab expression is de-repressed and coincides medially with Dll [arrow; staining as in (B)]. (H) bab expression (green) is eliminated (arrow) by ectopic expression of dac (red).

A different patterning mechanism must generate the domains of expression of rn and bab and must lead to tarsal growth and differentiation. The presumptive tarsus is included within the Dll-expression domain (Fig. 2B), and Dllfunction is required for bab and rn activation and tarsal development at 84 hours AEL, as shown by Dllmutants and Dll clones [Fig. 2, E and F; (7)]. However, neither theDll-expressing cells in the center of the disc (the presumptive distal tip), nor the most peripheralDll-expressing cells, express rn orbab (Fig. 2B). These peripheral cells appear to be medial cells expressing both Dll and dac (compare B and C of Fig. 2), and double-staining reveals that indeed the early rings of rn and bab expression are abutted bydac (Fig. 2D). These observations suggest that rnand bab expression, promoted by Dll, is repressed in medial Dll-expressing cells by dac and in the central cells by another factor. In clones ofdac cells induced at 80 hours AEL, we observe ectopic outgrowths and ectopic expression of bab in medial cells near the endogenous bab domain [fig. S1F (7)]. Staining of entiredac mutant discs reveals this effect clearly:bab expression now extends to all medialDll-expressing cells (Fig. 2G). This result agrees with ectopic expression of dac in the tarsal region, which represses bab and eliminates tarsal structures (Fig. 2H and fig. S1G).

The vein (vn) gene is expressed at the very center of the leg disc shortly after 72 hours AEL (Fig. 3A). The onset of vnexpression requires both wg and dpp signaling andDll, but by 84 hours AEL, when rn andbab are activated, vn is no longer dependent onwg and dpp (fig. S1H). vn encodes a neuregulin-like secreted protein that is the only ligand of EGFR (11) active here. We detect activation of the EGFR-Ras signal transducer mitogen-activated protein kinase (MAPK) (12) in the center of the leg disc from 80 hours AEL to 96 hours AEL, in the region devoid of rn and babexpression (Fig. 3B). Secretion of the vn protein could lead to the EGFR-mediated repression of rn andbab several cell diameters away from the center (Fig. 3, A to C; fig. S1E). Blocking vn signaling with ectopic expression of the secreted EGFR antagonist argos throughout the Dll domain produces mutant phenotypes only near thevn-expressing cells, and these phenotypes are similar to those of vn, MAPK, and Ras mutant conditions: the pretarsus is missing at the tip of the leg, and tarsus five is missing or abnormal (fig. S1, I and J). bab expression invades the center of the disc, which loses the expression of the distal tip markerdlim1 (13) (Fig. 3, F and G). Using both a temperature-sensitive allele of EGFR and a dominant-negative version of the EGFR protein, we deprived leg discs of EGFR function during 80 to 96 hours AEL (7) and observed similar results: loss of distalmost structures and bab expression covering the disc center (Fig. 3, D and H), which has also lost the expression of the pretarsal markers dlim1 and aristaless(al) (1), as well as the tarsus five markerBar (14) (fig. S1K). Reciprocally, in legs expressing ectopically an activated version of EGFR, tarsal structures and the expression of bab are lost, whereas ectopic expression of dlim1, al, andBar is observed (Fig. 3, E and I). Hence, a central gradient of EGFR-Ras signaling promotes simultaneously and independently [fig. S1L (7)] three effects: activation of aland dlim1 at the very center, activation of Barin nearby cells, and repression of bab from the whole area. Regarding tarsal segments 1 to 4, vn acts as the repressory element working in parallel to dac to restrict the activation of rn and bab to the middle of theDll domain. Tarsal segments 1 to 4 are thus defined in thoseDll-expressing cells not simultaneously exposed to either the dac protein or EGFR-Ras signaling triggered byvn (Fig. 4 and fig. S1E).

Figure 3

EGFR-Ras signaling prevents babexpression and promotes distal leg development. (A)vn transcript and (B) distribution of activated MAPK in the center of 80 hours AEL leg discs. These patterns remain until 96 hours AEL. (C) bab (red) andvn (green) expression at 96 hours AEL. vn is expressed in the center of the disc where bab is absent. Note the gap between the two domains (arrow), revealing the range ofvn action. (D) Legs deprived of EGFR from 80 hours AEL have four tarsi, ending in a joint instead of pretarsus (labels as in Fig. 1A). (E) Ectopic EGFR activation generates ectopic expression (arrow) of al (green) andBar (purple; white overlap). (F toI) bab (red) and dlim1 (green) expression in 120 hours AEL discs from wild type (F), DllGal4 UAS-argos (G), dominant-negative EGFR (H), and ectopically activated EGFR (I). dlim1 expression is lost from the leg tip, which is invaded by bab expression in (G) and (H), whereas in (I) bab is lost (arrow) and dlim1 is activated ectopically (arrowhead).

Figure 4

Proximal-distal leg development inDrosophila. Before 72 hours AEL, the combination ofwg and dpp signaling establishes the expression of Dll (black) and dac (green) in the developing disc. After 84 hours AEL, PD development becomes wg- anddpp-independent and is driven by PD interactions.rn and bab expression (yellow) is inserted in theDll-expressing cells in between vn at the disc center (red), and the medial ring of dac expression (green).dac promotes femur and tibia development, whereasvn promotes distal development through the activation ofal, dlim1, and Bar. From 96 to 120 hours AEL, the leg pattern is completed by definition of further PD regions and intercalation of joints between segments. During pupal metamorphosis the disc everts and the fly leg differentiates.

Thus, interactions between genes and signals expressed in PD domains constitute a patterning mechanism for the development of new PD fates. We suspect that further PD interactions exist in Drosophilalegs, for example, during trochanter development proximal to thedac domain (7).

The Drosophila tarsus develops in the absence of homeotic genes, suggesting that it is an ancestral ground-state limb structure (15). Because tarsal development is driven by PD interactions, altogether these results might suggest that these interactions are an ancestral process, possibly to be found in other animals. In primitive insect limbs, a transition in dppexpression (16, 17) signals awg- and dpp-independent patterning phase, whereas the conserved expression of Dll, dac, andal suggests a conservation of their functional roles (18). In vertebrate limbs, expression and requirements ofDll, dac, and al homologs are similar to those of insects (19–21), and experimental embryology has shown that insect and vertebrate limbs react to PD axis alterations in the same way (22).

Further work is required to clarify these issues and to obtain a complete understanding of PD patterning in animal appendages. Our results show the importance of an element whose role had long been suspected (1, 22), that is, the existence of PD interactions that complete the initial PD organization generated by dorsal-ventral and anterior-posterior patterning cascades.

  • * To whom correspondence should be addressed. E-mail: J.P.Couso{at}biols.susx.ac.uk

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