Dual role for Hox genes and Hox co-factors in conferring leg motoneuron survival and identity in Drosophila

Abstract

Adult Drosophila walk using six multi-jointed legs, each controlled by ∼50 leg motoneurons (MNs). Although MNs have stereotyped morphologies, little is known about how they are specified. Here, we describe the function of Hox genes and homothorax (hth), which encodes a Hox co-factor, in Drosophila leg MN development. Removing either Hox or Hth function from a single neuroblast (NB) lineage results in MN apoptosis. A single Hox gene, Antennapedia (Antp), is primarily responsible for MN survival in all three thoracic segments. When cell death is blocked, partially penetrant axon branching errors are observed in Hox mutant MNs. When single MNs are mutant, errors in both dendritic and axon arborizations are observed. Our data also suggest that Antp levels in post-mitotic MNs are important for specifying their identities. Thus, in addition to being essential for survival, Hox and hth are required to specify accurate MN morphologies in a level-dependent manner.

Fig. 1.

LinA MNs during development. (A-C) LinA MARCM clones in T2, labeled with vGlut-Gal4 >CD8GFP. (A) Cell bodies and dendrites of LinA MNs at late L3, mid-pupa and adult stages. Leg neuropil region is shaded pink. (B) LinA MARCM clones stained for d-CSP2 (red, labels the neuropil) and Dll (blue, labels the leg imaginal discs). VNCs are outlined with blue dotted lines. Regions shown in C are marked with white boxes. The pupal stage leg is outlined in pink. (C) LinA MN axons in the periphery. Shown is the GFP channel from the regions boxed in B. Late L3 leg imaginal disc, pupa stage leg and adult leg are outlined in pink. The VNC at the L3 stage is outlined with blue dotted lines. (D) LinA MARCM clones stained with anti-Repo (red) to mark glia. The left-most image shows a low-magnification view of the VNC (as depicted in the schematic); the region boxed is shown at higher magnification in the images to the right. About 22 LinA-derived glia are visualized; these surround the neuropil in the adult (data not shown).

Fig. 2.

Hox expression patterns. (A) Schematic of LinA in the larval VNC. The LinA NB (light blue) is positioned ventrally in the VNC and its progeny (green) extend dorsally. Consequently, earlier born MNs are located more dorsally and later born MNs are located more ventrally. Planes 1 to 5 refer to the z-stack slices shown in B-D. (F) Schematic of LinA in a mid pupa (3-4 day APF) VNC. LinA MN cell bodies (green) are located ventrally. Planes 1 to 5 represent the z-stack slices in G-I. (B-D,G-I) LinA MARCM clones in late L3 (∼120 hour AEL; B-D) and at the mid-pupa stage (G-I) showing five z-stack slices from ventral (1) to dorsal (5). Magnified areas are indicated with the yellow arrows of clones in the low-magnification images on the left and regions examined are depicted in the schematics. (B) LinA clones in T1 (top row), T2 (middle row) and T3 (bottom row) stained for Antp (red). LinA clones in T2 and T3 were generated in a single sample. (C) Antp^-/- LinA clone in T3 segment during late larval stages stained for Antp (red); the absence of staining in the clone confirms the specificity of the signal seen in B. (D) LinA clones in T1 and T3 (in a single sample) stained for Ubx (red) and Scr (blue). Approximately three cells in the LinA clone in T3 express Ubx. (G) Antp (red) is expressed strongly in all T2 LinA MNs, but not in T1 or in T3. Antp levels do not correlate with their dorsal-ventral position at this stage. (H) LinA clones in T1 and T3 stained for Ubx (red) and Scr (blue). Ubx is expressed in all LinA MNs in T3. Most MNs express high levels of Ubx. Scr is not expressed in LinA MNs. (I) Ubx^-/- LinA clone (green) in T3 segment stained for Ubx; the lack of staining confirms the specificity of the signal seen in H. (E) Summary of Hox expression patterns in LinA MN progeny in late L3. (J) Summary of Hox expression patterns at the mid-pupa stage. In T2, but not T1 or T3, Antp is expressed in all LinA MNs, mostly at high levels. In T3, Ubx is expressed in all LinA MNs. Hth is expressed in all LinA MNs in all three thoracic segments in both larval and pupal stages but its levels vary (supplementary material Fig. S3A).

Fig. 3.

Hox and hth are required to generate the correct number of LinA progeny. (A-F) LinA MARCM clones labeled with tub-Gal4; vGlut-Gal4 >CD8GFP (green, outlined with light blue dotted lines), stained for Elav (red, marks mature neurons) and Pros (blue, marks young neurons). Magnified areas are indicated by boxes and yellow arrows and the position of clones in the VNC are shown by the red boxes in the schematics on the left. (A) WT, (B) WT +p35, (C) Scr^-/-Antp^-/-Ubx^-/-, (D) Scr^-/-Antp^-/-Ubx^-/- +p35, (E) hth^P2, (F) hth^P2 +p35. (G,H) Quantification of neurons (G; Elav+) and glia (H; identified by position and the lack of Elav staining; see Fig. 1D) in individual LinA clones at late L3 (∼120 hour AEL). Each point represents the cell number for a single MARCM clone. Ectopic p35 expression rescued the number of neurons (G) but not the number of glia (H). Compared with wild-type LinA clones, about ten additional neurons were generated when p35 was expressed, suggesting that apoptosis normally eliminates these cells (Truman et al., 2010). Elav- (non-glia) cell numbers were not significantly changed in mutant LinA clones [median values: WT (8); WT +p35 (3); Scr^-/-Antp^-/-Ubx^-/- (4); Scr^-/-Antp^-/-Ubx^-/- +p35 (8); hth^P2 (4); hth^P2 +p35 (2)]. Black bars represent median values. (See also supplementary material Fig. S4A.)

Fig. 4.

Axon targeting and dendritic defects in Hox and hth mutant LinA MNs. All panels show axons in adult T2 legs and dendrites in the VNC of LinA MARCM clones. In the VNC, the leg neuropil and midline are marked by blue dotted circles and red dotted lines, respectively. See supplementary material Fig. S5A,B for examples in T1 and T3, and supplementary material Fig. S5D for a summary. (A) LinA MNs projecting to T2 legs. Axon targeting defects are marked with red lines. Wild-type and Hox mutant LinA clones have thin dendrites that cross the midline (blue arrows); these are missing in hth LinA clones (red arrow). (B) LinA MNs expressing p35 projecting to T2 legs. Ectopic expression of p35 partially rescues the axon targeting phenotypes for both Hox and hth mutants. hth mutant LinA clones lack dendrites crossing the midline (red arrow).

Fig. 5.

Aberrant axon branching and dendritic morphologies in individual Hox or hth mutant MNs. Examples of single-cell MARCM clones. For each clone, both axon and dendrites are shown. The neuropil and midline are marked by blue dotted circles and red dotted lines, respectively. (A) Removing Hox and hth functions results in distinct phenotypes. Dendritic and axon defects are indicated by blue arrowheads and red lines, respectively. Coxa- and femur-targeting MNs are from LinB; the tibia-targeting MN is from LinA. (B,C) Examples of mutant single MNs in which there is a mismatch between axon and dendrite morphologies. The left-hand images show wild-type MNs, each outlined in red or blue. The right-hand images show Hox (B) or hth (C) mutant MNs that share either the axon or dendrite morphologies with one of the two wild-type MNs as indicated. (See also supplementary material Table S1.)

Fig. 6.

The levels of Antp are instructive for MN targeting. (A) Antp levels are highest in late-born LinA progeny and lowest in early-born LinA progeny, and early-born MNs target proximal regions of the leg segments, whereas late-born MNs target distal regions (Baek and Mann, 2009). (B) Examples in T2 (left) and T3 (right) of LinA MNs expressing either GFP (top row) or Antp (bottom row) via the vGlut-Gal4 driver. Pink and blue boxes highlight the proximal femur (PF) and distal femur (DF), which show a decrease and increase in branching, respectively. Defective midline-crossing dendrites are marked by light-blue arrowheads. (See also supplementary material Fig. S6.)

Fig. 7.

No compensatory targeting in legs with Hox or hth mutant LinA clones. (A) Experimental design. In this experiment, Hox or hth mutant (M^*) Lin A MARCM clones (labeled by vGlut-Gal4; UAS-CD8GFP) were generated in a background in which all MNs were labeled by vGlut-LexA; LexO-rab3YFP. (B,C) Examples of Hox mutant clones (B) and hth clones (C) in LinA and LinB. For comparison, the wild-type (WT) contralateral legs from the same animals are shown on the right. Targeting defects are indicated by red lines and are still observed when all MNs are visualized (YFP+GFP).