The Drosophila immunoglobulin gene turtle encodes guidance molecules involved in axon pathfinding

Abstract

Background: Neuronal growth cones follow specific pathways over long distances in order to reach their appropriate targets. Research over the past 15 years has yielded a large body of information concerning the molecules that regulate this process. Some of these molecules, such as the evolutionarily conserved netrin and slit proteins, are expressed in the embryonic midline, an area of extreme importance for early axon pathfinding decisions. A general model has emerged in which netrin attracts commissural axons towards the midline while slit forces them out. However, a large number of commissural axons successfully cross the midline even in the complete absence of netrin signaling, indicating the presence of a yet unidentified midline attractant.

Results: The evolutionarily conserved Ig proteins encoded by the turtle/Dasm1 genes are found in Drosophila, Caenorhabditis elegans, and mammals. In Drosophila the turtle gene encodes five proteins, two of which are diffusible, that are expressed in many areas, including the vicinity of the midline. Using both molecular null alleles and transgenic expression of the different isoforms, we show that the turtle encoded proteins function as non-cell autonomous axonal attractants that promote midline crossing via a netrin-independent mechanism. turtle mutants also have either stalled or missing axon projections, while overexpression of the different turtle isoforms produces invasive neurons and branching axons that do not respect the histological divisions of the nervous system.

Conclusion: Our findings indicate that the turtle proteins function as axon guidance cues that promote midline attraction, axon branching, and axonal invasiveness. The latter two capabilities are required by migrating axons to explore densely packed targets.

Figure 1

Genomic organization and in situ expression of the turtle gene. (A) Schematic diagram showing intron-exon structure of turtle transcripts, the proteins that they encode (color-coded to show only clearly defined protein domains shared between the different isoforms), the span of the tutl^ex383 and tutl⁴ deletion, and the two P-element insertions. (B) Western blot against the His-tagged diffusible GH015753-encoded isoform of turtle protein expressed in S2 cells. After transfer, both samples were examined on the filter by Ponceau S staining, and although there was abundant protein in both lanes, most of the His-tagged protein was found in the supernatant (2), not the cell pellet (1). Similar results were obtained using the His-tagged AT02763-encoded diffusible isoform of turtle protein (data not shown). (C) RT-PCR showing low expression of the AT0276 and HL01565 isoforms in stage 12/13 embryos compared to GH015753, LD2884, and GH08133 isoforms. (D, E) RNA in situ hybridization against the domains shared by all isoforms indicates that turtle is initially expressed close to the midline in embryonic stages 12 to 13 (D, arrow), with later expression spreading throughout the central nervous system (E, arrow). (F) RNA in situ hybridization of GH015753 (red), LD2884 (green), and GH08133 (blue) isoforms indicates overlapping nervous system expression in stage 13/14 embryos (arrow), with some expression of LD28224 and GH08133 in salivary gland (asterisk) and gut (arrowhead). (G-J) Staining with the neuronal marker anti-Elav (G, I, red) indicates that both GH015753 and LD2884 (G-I, green) are expressed in neuronal cells. Both isoforms also co-localized with some anti-Repo-positive glial cells (H, J, red). (K) RNA in situ hybridization in wandering third larval instar eye discs indicates that all isoforms are expressed in the morphogenic furrow (asterisk), with some cells at the tip of the eye disc expressing only GH015753 (arrow), which are surrounded by cells expressing only LD28224 (arrowhead). (L) RNA in situ hybridization in wandering third larval instar brains indicates strong, overlapping expression of the turtle isoforms throughout the optic lobe, with some cells in the optic lobe-retinal nerve junction expressing only the LD2884 isoform (arrow).

Figure 2

turtle is involved in promoting midline crossing. (A) Wild-type embryonic longitudinal connectives (arrow) and commissures (arrowhead) stained with BP102 antibody. (B) tutl^ex383 homozygous embryos have fragmented connectives and missing commissures (arrowhead and arrow, respectively). (C, D) Both Elav-Gal4 (C) and Sim-Gal4 (D) expression of the different turtle isoforms fully rescues the tutl^ex383 longitudinal connective (arrow) and commissure (arrowhead) defects. (E) No rescue of those phenotypes is observed with non-midline glia driver Repo-Gal4. (F) The ventral nerve cord in wild-type embryos has FasII-positive fascicles that are well-formed and do not cross the midline. (G) tutl^ex383 homozygous embryos have FasII-positive fascicles that are fragmented (arrowhead), with axons that cross the midline (arrow). (H, I) Elav-Gal4 (H) and Sim-Gal4 (I) expression of the different turtle isoforms fully rescues the tutl^ex383 FasII-positive fascicle defects, preventing fascicles from fragmenting or crossing the midline. (J) No rescue of longitudinal connectives (arrow) and commissures (arrowhead) phenotypes is observed with non-midline glia driver Repo-Gal4. (K-M) Df(2L)ed-dp/wild type (wt) embryos have normal commissures and longitudinal connective (K, M); however, embryos trans-heterozygous for the same genomic deficiency over the tutl^ex383 allele in Df(2L)ed-dp/tutl^ex383 have commissure and longitudinal (arrowhead and arrow, respectively) connective defects that are similar to those of tutl^ex383 homozygous embryos (L, M). (N) Sca-Gal4 pan-neuronal overexpression of only the diffusible turtle isoforms causes entire innermost FasII-positive fascicles to cross the midline (arrowhead. (O) Sim-Gal4 overexpression of the diffusible turtle isoforms produces similar defects (arrowhead) to Sca-Gal4, even though, in this case, turtle is expressed only in the midline cells and not in FasII-positive tracks.

Figure 3

The turtle gene products act as netrin-independent midline attractants. (A) Df(1)NP-5 homozygous embryos are missing the netA and netB genes and, consequently, exhibit gaps in their longitudinal connectives (arrow) and fragmented commissures (arrowhead), as revealed by BP102 staining. (B, C) Df(1)NP-5; tutl^ex383/+ embryos show an enhancement of both defects (B, arrow and arrowhead), while Df(1)NP-5; tutl^ex383 double mutants have an extreme reduction in commissures, with only one thin fragmented commissure formed throughout the length of the embryo (C). (D) fra^GA957 homozygous embryos exhibit gaps in their longitudinal connectives (arrow) and fragmented commissures (arrowhead) as revealed by BP102 staining. (E)fra^GA957, tutl^ex383/+ embryos show an enhancement of both defects. while fra^GA957,tutl^ex383 double mutants have an extreme reduction in commissures (arrowhead) (G) slit¹/+, robo⁵/+ embryos have a reduction in slit signaling that can produce a minor midline crossing defect revealed by FasII staining (arrowhead). (H, I) slit¹/+, robo⁵/+, tutl^ex383/+ triple heterozygotes do not show any enhancement nor suppression of the FasII axon crossing defect compared to slit¹/+, robo⁵/+ double heterozygotes alone. (J) abl¹ homozygous embryos do not show any defect in commissure or longitudinal connectives as revealed by BP102 staining (arrow and arrowhead). (K, L) tutl^ex383; abl¹/+ embryos show an enhancement of commissural defects (K), while tutl^ex383; abl¹ double mutants have an extreme reduction in commissure formation (L, arrow) and fragmented longitudinal connectives (L, arrowhead).

Figure 4

The turtle gene functions non-cell autonomously in promoting motor axon branching. (A) Wild-type embryo stained with FasII antibody, showing the ISNb nerve branch innervating the cleft between muscles 6 and 7 (lower black arrow), muscle 13, and the cleft between muscles 12 and 13 (upper black arrow), the ISNd nerve branch (white arrow), and the TN nerve (arrowhead). (B) tutl^ex383 mutants have ISNb nerves that frequently fail to project final branches (black arrows), and many segments lack an ISNd (white arrow) but still posses a normal TN nerve (arrowhead). (C) Elav-Gal4 expression of the diffusible turtle isoform fully rescues both ISNb and ISNd nerve branch defects in tutl^ex383 homozygous embryos (arrows and arrowhead). (D, E) Both pan-neuronal Sca-Gal4 (D) and pan-skeletal muscle 24B-Gal4 and G14-Gal4(E and D, respectively) overexpression of turtle isoforms cause ISNb nerves to either excessively branch (black arrows) Yesor stall, cause the TNs to send out ectopic branches (arrowheads), and produce missing ISNd nerves (white arrows). (G) Quantification of the motor nerve defects seen in 55 to 60 A2 to A7 embryonic hemisegments in turtle mutants (tutl^k14703, tutl¹⁰⁸⁰⁵, and tutl^ex383), in complementation testing (tutl^ex383/tutl¹⁰⁸⁰⁵), and in tutl^ex383 homozygotes with different turtle isoforms transgenically expressed using Elav-Gal4. Note that only the two diffusible isoforms rescue the mutant to near-wild type levels of motor axon pathfinding errors. The turtle gene functions non-cell autonomously in promoting retinal axon invasiveness and branching. (H) Adult wild-type head horizontal section showing retinal axons visualized with 24B10 antibody. Note normal optic chiasm (arrowhead) and regular array of R7 axon terminations (arrow). (I) tutl^k14703 adult mutants have gaps in the R7 termination line (arrow) and an irregular chiasm (arrowhead). (J) The retinal axon defects in tutl^ex383/tutl^k14703 are rescued when the diffusible turtle isoforms are transgenically expressed using the pan-neuronal driver Elav-Gal4. (K) Eye-specific GMR-Gal4 overexpression of the diffusible turtle isoforms in a wild-type background produces retinal axons that invade the cortex (asterisk), gaps in the R7 termination line (arrow), and some axons with extra branches (arrowhead). (L) tutl^k14703 EGUF/hid mutant eyes have normal optic and R7 projections (L).

Figure 5

Pan-neuronal Elav-Gal4 overexpression of the diffusible turtle isoform AT02763 promotes neuronal invasiveness. (A) Representation of the major histological divisions of the adult fly nervous system in the head (LA, lamella; LO, lobula; MD medulla). (B) Wild-type head horizontal section, stained with the nuclear marker DAPI (blue fluorescence), showing the peripheral location of the cortex (arrow) and the central location of DAPI-free neuropil (asterisk). (C) Adult horizontal section stained with the neuronal marker Elav (green fluorescence), showing that most of the cortex is composed of neuronal cells (arrow). (D) Pan-neuronal overexpression of the diffusible isoform AT02763 produces cells that invade the normally cell-free neuropil layer (arrowhead)s. (E) Most of these invasive cells are Elav-positive neurons (arrowhead).