Crossing the border: molecular control of motor axon exit

Abstract

Living organisms heavily rely on the function of motor circuits for their survival and for adapting to ever-changing environments. Unique among central nervous system (CNS) neurons, motor neurons (MNs) project their axons out of the CNS. Once in the periphery, motor axons navigate along highly stereotyped trajectories, often at considerable distances from their cell bodies, to innervate appropriate muscle targets. A key decision made by pathfinding motor axons is whether to exit the CNS through dorsal or ventral motor exit points (MEPs). In contrast to the major advances made in understanding the mechanisms that regulate the specification of MN subtypes and the innervation of limb muscles, remarkably little is known about how MN axons project out of the CNS. Nevertheless, a limited number of studies, mainly in Drosophila, have identified transcription factors, and in some cases candidate downstream effector molecules, that are required for motor axons to exit the spinal cord. Notably, specialized neural crest cell derivatives, referred to as Boundary Cap (BC) cells, pre-figure and demarcate MEPs in vertebrates. Surprisingly, however, BC cells are not required for MN axon exit, but rather restrict MN cell bodies from ectopically migrating along their axons out of the CNS. Here, we describe the small set of studies that have addressed motor axon exit in Drosophila and vertebrates, and discuss our fragmentary knowledge of the mechanisms, which guide motor axons out of the CNS.

Keywords: Nkx2.9; Robo; Slit; axon pathfinding; dorsally-exiting motor neuron; motor axon exit; motor exit point; spinal cord; ventrally-exiting motor neuron.

Figure 1

Motor neuron subtypes and the projections of their axons out of the CNS. (A) Schematic of motor neuron nuclei in the developing brainstem (rhombomere (r), r1 to r7) and spinal cord. vMNs are indicated in red on the left, whereas dMNs are indicated in blue on the right of the schematic. Trigeminal (V) motor nuclei are shown in purple. Each cranial motor nuclei is numbered in roman numerals, e.g., CN XI. Abbreviations: fp, floor plate; sMN, spinal motor neuron; (B) Axonal projections of vMNs in the hindbrain (VI, XII) and spinal cord (sMN) are shown in red; (C) Axonal projections of dMNs (VII, IX, X, XI) and trigeminal (V) dMNs are shown in blue and purple, respectively. Note that axons extending from trigeminal dMNs avoid sensory ganglia (white ovals), while axons of other dMN invade these ganglia [6].

Figure 2

Cadherin 7-expressing BC cells are located at both the lateral and ventral exit points in chick embryos. Cervical spinal cord-containing transverse cryosections derived from E4.5 chick embryos were doubled-labeled with either anti-Ben and anti-Laminin (Left) or anti-Cadherin 7 and anti-NF (Middle, Right), and the appropriate secondary antibodies. (Left) BEN is expressed by both SACMN and vMN at this developmental stage, the FP and the SAN, which is positioned outside and adjacent to the spinal cord. Anti-Laminin labeling demarcates the margin of the spinal cord. (Middle) Cadherin 7-expressing BC cells are located at both the LEP and VEP. In this panel, the SAN is labeled by anti-NF. (Right) A higher magnification view of the boxed area in the middle panel. SACMN, spinal accessory motor neurons; SAN, spinal accessory nerve; vMN, ventral motor neurons; FP, floor plate; LEP, lateral exit point; VEP, ventral exit point; BC cells, boundary cap cells. Scale bar in middle panel, 100μm, applies to the left and middle panels. Scale bar in right panel, 50 μm.

Figure 3

Electron micrograph of a section of neural tube derived from an E11 rat embryo at a presumptive motor exit point. A transverse section of the neural tube shows a thin layer of uninterrupted basal lamina that overlies glial end-feet, which form a conspicuous gap at a presumptive motor exit point. Black arrow, presumptive motor exit point; black asterisk, space found within presumptive white matter. Scale bar: 2 μm [42].

Figure 4

diwanka is required for motor axon exit in zebrafish embryos. (A–D): Wild-type axonal projections of CaP (A), MiP (B), and RoP (C) primary MNs labeled with fixable dyes as previously described [12]. White arrows indicate the lower half of the spinal cord, which is out of the focal plane, whereas black arrows indicate the choice point, the distal end of a common path followed by CaP, MiP, and RoP motor growth cones prior to their divergence into ventral, dorsal and medial myotomal regions, respectively (D); (E–H): In diwanka mutants, CaP (E) and MiP (F) axons extend wild-type projections within the spinal cord, however they exhibit abnormal projections along the common path (black arrow). Notably, the majority of RoP axons fail to exit the spinal cord in diwanka mutants and instead extend their axons caudally within the CNS (black arrowhead points to a RoP growth cone) (G,H) [12].

Figure 5

Zfh1 is required for a subset of motor axons to emerge from the spinal cord. (A,B) Flat-mounted zebrafish embryos were labeled with either anti-FASIII (A,B) or anti-myc (C,D) to visualize the axonal projections of an early-developing intersegmental nerve (ISN) called ISNb. Note, ISNb axons selectively express the isl-tau:myc transgene and montages were created by combining images taken from multiple focal planes to visualize the extent of the ISNb nerve. In stage 16 (A) and 15 (C) wild type embryos, zfh1-expressing RP MNs (white asterisk) form an ISNb nerve that extends out and beyond the lateral edge of the CNS (dashed white line). White arrowheads correspond to the terminal end of an ISNb axon. In contrast, a subset of RP MN axons fails to exit the CNS in zfh1 mutants (B,D, top), while others extend appropriately to their peripheral muscle field (B,D, bottom) [20].

Figure 6

Eve is required for motor axon exit in Drosophila and expression of Unc-5 in eve mosaic mutants restores motor axon exit defects. (A–C) Stage-16 RN2Gal4::CD8GFP embryos of various genotypes, including eve/+ (A), mosaic eve mutant (B), and Unc-5-expressing mosaic eve mutant siblings (RN2Gal4, UAS-HAUnc-5) (C) were labeled with anti-GFP to visualize axons of somatic MNs, aCC and RP2. In eve/+ embryos, aCC and RP2 MNs extend wild type axonal projections to peripheral muscle fields (white arrowheads) (A). In contrast, the majority of aCC and RP2 motor axons fail to leave the CNS (89%, n = 80 hemisegments) and instead, inappropriately extend their axons longitudinally within the CNS (white arrows) (B). However, a small subset of motor axons does manage to exit the CNS (open white arrowhead). Notably, expression of Unc-5 in eve mosaic mutants partially rescues the aberrant motor axon exit phenotype (60% hemisegments, n = 100, white arrowheads) (C). In all panels, anterior is at the top of the micrograph [10].

Figure 7

SACMN extend dorsally directed axons towards and through the LEP. Schematic illustrating the axonal trajectory of SACMN and the SAN in the developing vertebrate spinal cord. BEN is selectively expressed on SACMN cell bodies and their axons, as well as on the SAN. D, dorsal, V, ventral, A, anterior, P, posterior, LEP-Lateral Exit Point; SAN-Spinal Accessory Nerve.