Control of species-dependent cortico-motoneuronal connections underlying manual dexterity

Abstract

Superior manual dexterity in higher primates emerged together with the appearance of cortico-motoneuronal (CM) connections during the evolution of the mammalian corticospinal (CS) system. Previously thought to be specific to higher primates, we identified transient CM connections in early postnatal mice, which are eventually eliminated by Sema6D-PlexA1 signaling. PlexA1 mutant mice maintain CM connections into adulthood and exhibit superior manual dexterity as compared with that of controls. Last, differing PlexA1 expression in layer 5 of the motor cortex, which is strong in wild-type mice but weak in humans, may be explained by FEZF2-mediated cis-regulatory elements that are found only in higher primates. Thus, species-dependent regulation of PlexA1 expression may have been crucial in the evolution of mammalian CS systems that improved fine motor control in higher primates.

Fig. 1. Elimination of CM connections and the vlCST by Sema6D-PlexA1 signaling in early postnatal mice

(A) Experimental scheme for identifying CM connections. Rab-mCherry and AAV6-G were co-injected into forelimb muscles of P3 mice. (B and C) Coronal brain sections from P11 control (PlexA1^fl/fl, n=7; B) and PlexA1 mutant (PlexA1^fl/fl; Emx1-Cre, n=8; C) mice showing labeling of CM cells 8 days after Rab-mCherry virus injections into forelimb muscles. Scale bar, 500 µm. (D) Quantification of CM cells. (E–P) Cervical spinal cord sections from wild-type (P10, E and F; P38, G and H), PlexA1^fl/fl (P10, I and J; P38, K and L), and Sema6D^−/− (P10, M and N; P38, O and P) mice. Scale bars, 100 µm.

Fig. 2. Functional CM connections in adult PlexA1^fl/fl; Emx1-Cre mice

(A) Schematic diagram of experiment to examine PStF in adult mice (3 months or older). EMG recordings from contralateral flexor carpi radialis and biceps brachii muscles. (B and C) Contralateral PStF from control (B, PlexA1^fl/fl) and mutant (C, PlexA1^fl/fl; Emx1-Cre) mice. (D and E) Frequency distributions of contralateral PStF values from control (D, PlexA1^fl/fl, n=6; wild-type, n=3) and PlexA1^fl/fl; Emx1-Cre (E, n=8) mice. (F) PStF latencies following motor cortex stimulation in PlexA1^fl/fl (41 stimulation sites, median delay=11.34 ms) and PlexA1^fl/fl; Emx1-Cre (37 stimulation sites, median delay=13.88 ms) mice. (G) Cumulative frequency distribution histogram. PlexA1^fl/fl; Emx1-Cre (gray line), PlexA1^fl/fl mice (black line). (H) Schematic diagram of DC electrical stimulation-evoked muscle PStF. (I) Section from a DC stimulation experiment, showing the 3 electrode locations used to evoke M1 antidromic responses. (J) Electrical stimulation of the ventral DC evokes an antidromic field potential in the motor cortex. 1: Dorsal DC stimulation, where afferent fibers are located; 2: Ventral DC stimulation (where CST fibers are located); 3: stimulation of the central canal. (K–L) PStF traces derived from average EMG responses evoked by ventral DC sites in PlexA1^fl/fl (K) and PlexA1^fl/fl; Emx1-Cre (L) mice. (M) PStF latencies in PlexA1^fl/fl (44 sites from 8 animals, median delay=4.88 ms) and PlexA1^fl/fl; Emx1-Cre (36 sites from 6 animals, median delay=3.66 ms) mice. (N) Cumulative frequency distribution plot of PStF latencies from PlexA1^fl/fl; Emx1-Cre (gray line), PlexA1^fl/fl mice (black line). Arrows indicate the onsets of EMG responses (B, C, K, and L).

Fig. 3. Adult PlexA1 mutant mice outperform controls in dexterous manipulation tasks

(A) Mouse performing the capellini handling test. Guiding and grasping hands are indicated by green and red arrows, respectively. (B–D) Results of the capellini handling test in 2 month-old control (PlexA1^fl/+; AAV1-Cre, n=10) and PlexA1^fl/fl; AAV1-Cre (n=10) mice during 4 testing days. Rate of adjustment = average number of paw adjustments per piece/eating time. (E–G) A mouse during the grasping test. Food pellets are indicated by black arrows. (H–I) Grasping success and consumption time in 3 month-old control (PlexA1^fl/+; AAV1-Cre, n=3 and PlexA1^fl/fl; AAV1-td, n=4) and PlexA1^fl/fl; AAV1-Cre (n=8) mice over 4 testing days. (J–K) Quantification and frequency distributions of pellet consumption time by control (145 trials) and PlexA1^fl/fl; AAV1-Cre (251 trials) mice.

Fig. 4. Expression of PLEXA1 in layer 5 of the motor cortex in mice and humans, and FEZF2-mediated repression

(A) FOG2 (layer 6 marker) and PLEXA1 in the human motor cortex at 20 pcw assessed by in situ hybridization. (B) Sequence conservation of putative PlexA1 cis-regulatory elements. Multi-species DNA sequence alignment showing one example of FEZF2 CTNCANCN binding sites (top, blue bar and box) and CGCCGC binding sites (bottom, red bar and box). The motifs are highlighted by gray-filled boxes. (C) Luciferase activity of reporter constructs in a 293T cell line (expressing endogenous human Fezf2) with or without co-transfection with a human FEZF2-expressing vector. Error bars represent the SEM of triplicate experiments. (D–G) Analysis of transgenic mice using a 5 Kb region upstream from the first exon of the human PLEXA1 gene or mutated human FEZF2 binding sequences. (E–G) High magnification view of the boxed areas in D and F. (H–L) Summary of the axonal trajectories and CS connectivity within the cervical spinal cord in wild-type and PlexA1^fl/fl; Emx1-Cre mice, and in humans.