Specification of individual adult motor neuron morphologies by combinatorial transcription factor codes

Abstract

How the highly stereotyped morphologies of individual neurons are genetically specified is not well understood. We identify six transcription factors (TFs) expressed in a combinatorial manner in seven post-mitotic adult leg motor neurons (MNs) that are derived from a single neuroblast in Drosophila. Unlike TFs expressed in mitotically active neuroblasts, these TFs do not regulate each other's expression. Removing the activity of a single TF resulted in specific morphological defects, including muscle targeting and dendritic arborization, and in a highly specific walking defect in adult flies. In contrast, when the expression of multiple TFs was modified, nearly complete transformations in MN morphologies were generated. These results show that the morphological characteristics of a single neuron are dictated by a combinatorial code of morphology TFs (mTFs). mTFs function at a previously unidentified regulatory tier downstream of factors acting in the NB but independently of factors that act in terminally differentiated neurons.

Figure 1. Organization of Lin B MNs

(A): Drawing of an adult fly showing the position of the CNS (grey brain and VNC) and leg MNs (green cell bodies in the VNC and axons in the legs). Red box indicates the VNC imaged in B. (B): Adult VNC expressing mCD8::GFP (blue/green, depending on intensity) under the control of VGlut-Gal4 and the neuropil marker Bruchpilot (BRP; grey) (Wagh et al., 2006). Arrowheads and asterisk indicate leg and wing MN cell bodies, respectively. (T1-T3, thoracic segments 1-3, ab: abdominal segments). (C,D): T1 leg expressing mCD8::GFP and the synaptic marker rab3::YFP (yellow) under the control of VGlut-Gal4 and Mhc (Myosin heavy chain)-RFP (D; red) (Gajewski and Schulz, 2010). (E-G): Lin B MARCM clone in the T1 segment of an adult VNC labeled with mCD8::GFP (green) and rab3::YFP (yellow) under the control of VGlut-Gal4), and BRP (grey). (F) Enlargement of boxed region in (E). (G) Same as (F), but with a transparent neuropil; arrowheads and arrows point to Lin B MN cell bodies and axons exiting the VNC, respectively. (H,I): Lateral view of a right T1 leg containing a Lin B MARCM clone stained for axons (green), rab3::YFP (yellow), and Mhc (Myosin heavy chain)-RFP (red). Axons of Co1-4 (blue arrow) and Fe1, Tr1, and Tr2 (white arrow) are indicated; white boxes magnify the trochanter. The five muscles (tirm, tilm, trlm, fedm, and ferm) innervated by Lin B MNs are labeled. See also Figure S1 and movies 1-3 for 3D images. Muscle nomenclature is from Soler et al., 2004. (J,K): Single Lin B MNs visualized using the MARCMbow technique (see Experimental Procedures and Figure S5). In the VNC (J), the seven Lin B MNs exhibit three characteristic morphologies: Fe-like (red), Tr-like (green), and Co-like (blue). In the adult leg (K), all seven Lin B MNs have distinct axon targeting properties. In this example, Tr2 and Fe1 were independently labeled (magenta and red, respectively) and all other Lin B MNs (Co1-4 and Tr1) were labeled in green. (L) Top: birth order of the seven Lin B MNs (Baek and Mann, 2009); middle: schematic of the three patterns of dendritic aborization (Co-like, blue; Tr-like, green; Fe-like, red); bottom: schematic summarizing muscle (red) targeting of the seven Lin B MNs.

Figure 2. Combinatorial expression of TFs in Lin B

(A-C): L3 CNSs with Lin B MARCM clones in T1 (boxed) expressing mCD8::GFP under the control of VGlut-Gal4. (A1, B1, C1) Ventral views of maximal confocal projections immunostained with anti-Pb (blue) and anti-Ems (red) (A1), anti-Toy (blue) and anti-Pb (red) (B1) and anti-Pros (blue) and anti-Toy (red) (C1). (A2, B2, C2) show magnifications of the GFP+ clones in A1, B1, C1, respectively. (A3, B3, C3) show confocal sections of each clone from ventral (I) to dorsal (III). Note: One of the two Zfh2+ cells consistently expresses lower levels of this TF, as illustrated by the inset in C3 that shows a higher magnification and intensity of this cell and a non-expressing cell for comparison. Pros levels are also consistently higher in one of the two Pros-expressing cells (B3). (D-F): Summary of the post-mitotic TF combinatorial code in Lin B in the L3 CNS (D), late pupa (E), and adult (F); (see also Figure S2, S3 and S4). Note that although the current code does not discriminate between Fe1 and Tr2 or Co3 and Co4, differing TF levels (e.g. Pros in Co3 and Co4) may contribute to their distinct identities.

Figure 3. Pb shapes the dendritic arbors of Tr1, Tr2, and Fe1

(A, E): WT (A) and pb mutant (E) Lin B MARCM clones labeled with mCD8::GFP under the control of VGlut-Gal4 in the right T1 hemisegments of adult VNCs. Shown are single Lin B clones in which the ventral and dorsal hemispheres were pseudo-colored in orange and blue, respectively. A1 and E1 are ventral views; A2 and E2 are anterior views; A3 and E3 summarize the phenotypes. The circles and double arrows indicate the most affected region. MAV, medial-anterior-ventral; MAD, medial-anterior-dorsal; MPB, medial-posterior-dorsal. See movie 4 for 3D representation and Figure S4 for examples in T2 with the same pb allele and in T1 with a different pb allele. (B-D and F-H): WT (B-D) and pb mutant (F-H) Lin B MARCMbow clones in the T1 segment of adult VNCs in which Fe1 (B, F), Tr1 (C, G), or Tr2 (D, H) were individually labeled with mCherry (red). In each example, the top panels shows the entire Lin B (GFP+) MARCM clone in white and the individually labeled MN in red; the bottom panels show heat maps of ventral and dorsal hemispheres, illustrating the degree of overlap for independent samples (N>4) with the same individually labeled MN. See Figure S5 and movie 5 for 3D images of MARCMbow clones. B5, C5 and D5 (WT) and F5, G5 and H5 (pb mutant) show representative single MNs in which the ventral and dorsal hemispheres were pseudo-colored in orange and blue, respectively. Note that the dendritic arborizations of these three MNs are readily distinguished in the WT (compare B5, C5, D5) but appear nearly identical when mutant for pb (compare F5, G5, H5). (I-L): WT (I, K) and Pb overexpressing (J, L) Lin A MARCM clones labeled with mCD8::GFP under the control of VGlut-Gal4 in the right T1 hemisegments of adult VNCs. I and J show single Lin A clones in which the ventral and dorsal hemispheres were pseudo-colored in orange and blue, respectively. K and L summarize these phenotypes; arrowheads and curved lines indicate regions most affected when pb function is altered. The white dashed lines indicate the position of the VNC midline.

Figure 4. Single cell analysis of pb axon targeting phenotypes

(A, B): Axons of a WT MARCMbow Lin B clone (A) and schematic (B) in which Tr1 and Tr2 were individually labeled. Note the stereotyped separation of the Tr1+Tr2 and Fe1 axons in the magnified image of the trochanter; the 90° rotation shows the medial and lateral positions of the Tr1 (red arrow) and Tr2 (yellow arrow) terminal branches, respectively. (C-E): Two examples (C, D) and schematics (E) of class I pb mutant phenotypes. In the first example (C), Tr2 targeted the femur instead of the trochanter. In the second example, both Tr1 and Tr2 targeted the femur instead of the trochanter; note the presence of three femur-targeting axons in the magnified (boxed) regions, and the absence of any branching in the trochanter (circles). (F-H): Two examples (F, G) and schematics (H) of class II pb mutant phenotypes. In both examples, a single labeled MN targeted both the trochanter and the femur. In the first example (F) two MNs targeted the femur, and one of these also targeted the trochanter. In the second example (G), only one MN targeted the femur, and it also targeted the trochanter. Because Tr1 and Tr2 still targeted the trochanter, we can unambiguously determine that Fe1 is the bifurcating MN. (I, J): In class III pb mutant clones, two MNs target the trochanter, but their terminal branches do not resemble either Tr1 or Tr2. Compare with (A, B). Red arrows point to lateral and medial terminal branches of a single MN, a pattern never observed for wild type Tr1 or Tr2 (compare with panel A).

Figure 5. Specific walking defects of flies with pb mutant Lin B MARCM clones

(A, B) Examples of stance traces, which mark the footprint position relative to the center of the fly's body as the fly moves forward, for representative control (A, blue lines) and experimental (B, red lines) flies. Stance linearity is the average distance (double arrows) between the actual stance trace and a ‘smoothed’ trace (gray lines) generated using every five frames, and is thus a measure of how straight the fly's path is; flies that walk perfectly straight would have a stance linearity index of 0 (Mendes et al., 2013). Insets show portions of actual traces from left T2 legs. (C) Stance linearity values averaged for the three legs in tripod stances I (top) and II (bottom) and calculated for both control (blue) and experimental (red) flies that were binned into three speed cohorts. Stance linearity values only differed in the fast speed group, and the difference was larger for tripod stance I, which includes the leg innervated by mutant pb Lin B MNs (red arrow, see Methods) (****p = 0.0001 and **p = 0.004, 2-way ANOVA Sidak's multiple comparisons test). (D) No differences in tripod index or footprint clustering were observed between control and experimental animals. Tripod index is the fraction of video frames that an animal spends in a tripod configuration (see schematics in (C)). Footprint clustering is the standard deviation from the average of the anterior extreme positions (AEPs; see blue and red circles in A, B) for all steps in a single video (AEP STD). See Mendes et al., 2013 for details.

Figure 6. Testing the Lin B TF code: dendritic arborization

(A): TF color key. (B): Top, birth order, TF code, and schematic of dendritic arbors for the seven wild type Lin B MNs. Bottom left, WT Lin B MARCM clone in the T1 hemisegment of an adult VNC in which the ventral (orange) and dorsal (blue) hemispheres are pseudo-colored; bottom right, heat maps of ventral and dorsal hemispheres illustrating the degree of overlap for five independent samples. (C): From top to bottom: schematic of expected transformation in ems^RNAi, pb-/-, UAS-toy MARCM clones; UAS-toy; ems^RNAi; pb-/-; ems^RNAi, pb-/- and ems^RNAi, pb-/-, UAS-toy Lin B MARCM clones. (D): From top to bottom: schematic of expected transformation in zfh1-/-, pb-/- MARCM clones; zfh1-/- and zfh1-/-, pb-/- Lin B MARCM clones. (E): Top: schematic of expected transformation in zfh1-/-, pb-/-, UAS-zfh2 Lin B MARCM clones; bottom: zfh1-/-, pb-/-, UAS-zfh2 Lin B MARCM clone. (F): WT Co4 MN labeled with MARCMbow in which the ventral (orange) and dorsal (blue) hemispheres are pseudo-colored. Note the similarity to the arborization pattern at bottom of panels C, D and E.

Figure 7. Testing the Lin B TF code: axonal targeting

(A-H): Axon targeting phenotypes of WT (A); pb-/- (B); ems^RNAi, pb-/- (C); UAS-ems, UAS-pb (D); UAS-zfh1 (E); zfh1-/- (F); zfh1-/-, pb-/- (G); and zfh1-/-, pb-/-, UAS-zfh2 (H) Lin B MARCM clones. The cuticle is light grey and the axons green; insets show leg regions most affected, red arrowheads and arrows point to either the absence of or aberrant targeting, respectively.

Figure 8. TF control of individual neural identities

NBs express a series temporal TFs, represented here by different colored circles. The blue outlines indicate TFs that may be shared throughout the lineage. NBs divide asymmetrically to eventually give rise to post-mitotic neurons that have both unique mTF codes, which specify individual dendritic and axonal morphologies, and terminal selector TF codes, which specify other terminal characteristics such as choice of neurotransmitter. Once differentiated, terminal selector TFs maintain the characteristics of terminally differentiated neurons.