The origins of the Drosophila leg revealed by the cis-regulatory architecture of the Distalless gene

Abstract

Limb development requires the elaboration of a proximodistal (PD) axis, which forms orthogonally to previously defined dorsoventral (DV) and anteroposterior (AP) axes. In arthropods, the PD axis of the adult leg is subdivided into two broad domains, a proximal coxopodite and a distal telopodite. We show that the progressive subdivision of the PD axis into these two domains occurs during embryogenesis and is reflected in the cis-regulatory architecture of the Distalless (Dll) gene. Early Dll expression, governed by the Dll304 enhancer, is in cells that can give rise to both domains of the leg as well as to the entire dorsal (wing) appendage. A few hours after Dll304 is activated, the activity of this enhancer fades, and two later-acting enhancers assume control over Dll expression. The LT enhancer is expressed in cells that will give rise to the entire telopodite, and only the telopodite. By contrast, cells that activate the DKO enhancer will give rise to a leg-associated larval sensory structure known as the Keilin's organ (KO). Cells that activate neither LT nor DKO, but had activated Dll304, will give rise to the coxopodite. In addition, we describe the trans-acting signals controlling the LT and DKO enhancers, and show, surprisingly, that the coxopodite progenitors begin to proliferate approximately 24 hours earlier than the telopodite progenitors. Together, these findings provide a complete and high-resolution fate map of the Drosophila appendage primordia, linking the primary domains to specific cis-regulatory elements in Dll.

Fig. 1.

LT and DKO are active in mutually exclusive subsets of the limb primordia. Embryos are oriented anterior to the left and dorsal up. (A) The Dll 5′ cis-regulatory region. LT is in red, Dll304 in yellow, DKO in blue, and the Dll promoter in white. (B,C) Stage 11 (B) and stage 14 (C) embryos stained for Dll (red) and En (green). (C′) A magnification of the Dll-positive cells at stage 14. (D) A stage 11 embryo stained for Dll304 activity (red) and En (green). (E) A stage 14 embryo stained for LT activity (red) and En (green). (E′) A magnification of LT-positive cells at stage 14. LT is active in a subset of Dll-expressing cells (compare with C′). (F) Schematic representation of a leg primordium at stage 14. (G) Dll (green), Ct (blue) and LT activity (red) in a leg primordium at stage 14. LT activity is mutually exclusive with Ct expression. LT-positive and Ct-positive cells are subsets of the Dll expression domain. (H) Hth (green), Ct (blue) and LT activity (red) in a leg primordium at stage 14. LT-positive cells also express Hth (arrow). ct and hth are expressed in mutually exclusive domains. (I) esg-lacZ (green), Ct (blue) and LT activity (red) in a leg primordium at stage 14. esg and Ct are mutually exclusive and LT activity overlaps with a subset of the esg-expressing cells. The ventral esg-expressing, LT-negative cells (bracket) are the coxopodite progenitor cells. (J) LT (red) and DKO (green) activities are mutually exclusive within the Dll-positive cells of the leg primordium at stage 14.

Fig. 2.

Lineage analyses of genes active in the ventral limb primordia. All discs except for those in F were stained for the lineage marker (red), Dll (green, subset of telopodite), and Hth (blue, coxopodite); see Materials and methods for details. (A) The progeny of cells in which Dll304 was active contribute to both dorsal (wings and halteres) and ventral (legs, both coxopodite and telopodite) thoracic limbs. Although individual wing discs show labeling in only a subset of the disc, labeled cells can contribute to any part of the disc. (B) The progeny of cells in which LT was active generate the telopodite of the leg. Expression in the dorsal coxopodite (arrow) may be due to the imperfection of the LT-Gal4 driver. The arrowhead marks a clone in the trochanter region. (C,D) The progeny of cells that expressed tsh become more restricted over time. Restricting tsh-Gal4 activity to the beginning of second instar (48-72 hours AEL) results in the labeling of both the coxopodite and telopodite (C). Allowing tsh-Gal4 to be active beginning at third instar (72-96 hours AEL) results in the labeling of only the coxopodite (D). The asterisk in D indicates lacZ-positive adepithelial cells that are not part of the disc epithelium. (E) The progeny of esg-expressing cells adopt both wing and leg (coxopodite and telopodite) fates. (F) The progeny of the cells in which DKO was active (red) occasionally contribute to larval neurons that co-express Elav (blue, arrow). All lacZ-positive cells express elav but not all elav cells are lacZ positive (see inset).

Fig. 3.

Regulation of LT by Wg and Dpp. (A) Cells that activate LT (red) at stage 14 in the limb primordia are close to cells expressing high levels of Wg (blue) and Dpp (visualized by pMad staining, green). (B) A wg^ts stage 14 embryo raised at the permissive temperature stained for Ct (blue), Dll (green) and LT activity (red). (B′) An enlargement of the leg primordium boxed in B. LT activity in the head segments (arrows) is not affected in the wg^ts embryos. (C) A wg^ts stage 14 embryo shifted to the restrictive temperature at 10-14 hours, after the initial activation of Dll and Dll304 (see methods), stained for Dll (green), Ct (blue), and LT activity (red). Dll expression is still observed, probably due to the activity of Dll304, but LT activity and Ct are not observed. (C′) An enlargement of the leg primordium boxed in C. (D-F) Dpp and Wg activate LT. prd-Gal4 is expressed in T2 but not T1 and T3. LT (red) and Dll (green) are activated dorsally by prd>arm^* in T2 (arrow; D). LT activity (red) and Dll levels are reduced via prd>Dad (arrow; E). LT activity (red) and Dll (green) are expanded ventrally via prd>Tkv^QD (arrow; F). Insets show single channels for Dll and LT activity.

Fig. 4.

Regulation of DKO and LT. (A) Wild-type stage 14 limb primordia stained for LT activity (red) and Ct (green). LT activity and Ct are present in mutually exclusive domains. (B) A stage 14 prd>btd; DfC(1)52 embryo stained for LT (red) and ct (green). In the absence of btd and Sp1 both LT and ct are not activated (in T1 and T2; asterisks). Resupplying Btd in T2 rescues LT and ct activity (arrow). (C) In a stage 14 Dll^SA1 mutant embryo LT activity (red) initiates, but decays over time. Unlike in wild type (A), the LT expression domain is a stripe. (D) Ectopic expression of Dll (green) and Btd using prd-Gal4 activates LT (red) in the abdomen (arrows). The inset shows ectopic activation of LT in abdominal segment 5. (E) A Df(1)sc-B57 mutant embryo, deleted for the ASC, stained for LT activity (red) and Ct (green). LT activity is expanded at the expense of the ct-expressing cells, which are lost. (F) Ectopic expression of the proneural gene asense (ase) in T2 represses LT activity (red) and expands the number of cells that express ct (green; arrow). (G) Wild-type limb primordia stained for Dll (blue), DKO activity (green) and Ct (red). DKO activity and Ct expression overlap within the Dll-positive domain. (H) Df(1)sc-B57 mutant embryo stained for DKO activity (green), Ct (red) and Dll (blue). DKO activity and Ct staining are absent in the leg primordia, but the number of cells expressing Dll is not changed.

Fig. 5.

Distinct cell proliferation dynamics in the coxopodite and telopodite. (A) Time course of limb primordia development, staining for LT activity (green) and esg-lacZ (red). At 12 hours AEL (stage 14) the number of LT-positive telopodite progenitors is approximately equal to the number of esg-positive, LT-negative coxopodite progenitors (∼15 cells, each). At 24-36 hours AEL (early first instar) the number of LT-positive cells and esg-positive, LT-negative cells remain about the same. At 48-60 hours AEL (early second instar) the number of esg-positive, LT-negative cells is far greater than the number of LT-positive cells. LT-positive (arrow) nuclei are noticeably larger than esg-positive, LT-negative nuclei. By 96-108 hours AEL (third instar) the progeny of the LT-expressing cells now populate the entire telopodite. (B) LT activity (green) compared to tsh expression (red) during development. At early second instar (48-60 hours AEL) the number of cells that are tsh-positive but LT-negative is greater than the number of cells where LT is active. At this time the LT-positive cells also express tsh (arrow). At the end of the second instar (60-72 hours AEL) the number LT-positive cells has increased. At this time the LT-positive cells no longer express tsh (arrow). (C) At 48-60 hours AEL, the ∼15 cells in which LT is active (green) exist as a ring that is coincident with the LT lineage labeling. At 60-72 hours AEL, the LT lineage-labeled cells, although greater in number, all actively express LT. Discs have been outlined with a dotted line. (D) Randomly generated neutral clones induced at 12-24 hours of development were quantified for their position and cell number 36 and 48 hours after clone induction. Examples for each of these experiments are shown in Fig. S4 in the supplementary material. (E) Third instar discs containing neutral clones stained for Hth (red), Dll (green) and β-gal (blue, the clone marker). The subset of randomly marked neutral clones induced between 12 and 24 hours of development that reach the border do not cross between the coxopodite and the telopodite (n=25).

Fig. 6.

The fate map of the thoracic limb primordia. At stage 11 the cells of the limb primordia are multipotent and can contribute to the dorsal appendage (e.g. wing), coxopodite, telopodite, and Keilin's Organ (KO). Also shown are the genetic inputs that control the early Dll304 enhancer at this stage. Approximately 4 hours later (stage 14) the cells in the limb primordia are restricted in their potential. Cells that activate LT (red) give rise only to the telopodite. LT activation requires Wg and Dpp and is restricted to the thorax by Btd and Dll. LT is also repressed by the proneural genes of the ASC in the KO primordia. Cells that activate DKO (blue) are fated to form the KO. The DKO enhancer is controlled by Wg, Dpp, ASC, Btd and Dll. By stage 14 the coxopodite precursor cells (green) do not express Dll and are primarily located ventral to the Dll-positive cells. At the end of larval development the LT lineage gives rise to the entire telopodite (Dll and Dac, in red), while the coxopodite is marked by the restricted expression of tsh and hth (in green).