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

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Science  28 Jul 2017:
Vol. 357, Issue 6349, pp. 400-404
DOI: 10.1126/science.aan3721
  • 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 (B) P11 control (PlexA1fl/fl, n = 7) and (C) PlexA1 mutant (PlexA1fl/fl;Emx1-Cre, n = 8) 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. *P < 0.05, **P < 0.01. (E to P) Cervical spinal cord sections from wild-type [P10, (E) and (F); P38, (G) and (H)], PlexA1fl/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 PlexA1fl/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 (B) control (PlexA1fl/fl) and (C) mutant (PlexA1fl/fl;Emx1-Cre) mice. Dotted line indicates the onset of the EMG response in the mutant; arrows indicate EMG onsets. (D and E) Frequency distributions of contralateral PStF values from (D) control (PlexA1fl/fl, n = 6; wild-type, n = 3) and (E) PlexA1fl/fl;Emx1-Cre (n = 8) mice. (F) PStF latencies after motor cortex stimulation in PlexA1fl/fl (41 stimulation sites; median delay = 11.34 ms) and PlexA1fl/fl;Emx1-Cre (37 stimulation sites; median delay = 13.88 ms) mice. ***P < 0.001. (G) Cumulative frequency distribution histogram. gray line, PlexA1fl/fl;Emx1-Cre mice; black line, PlexA1fl/fl mice. ***P < 0.001. (H) Schematic diagram of DC electrical stimulation-evoked muscle PStF. (I) Section from a DC stimulation experiment, showing the three 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); and 3, stimulation at the level of the central canal (K and L) PStF traces derived from average EMG responses evoked by ventral DC sites in (K) PlexA1fl/fl and (L) PlexA1fl/fl; Emx1-Cre mice. Dotted line indicates the onset of the EMG response in the mutant; arrows indicate EMG onsets. (M) PStF latencies in PlexA1fl/fl (44 sites from 8 animals; median delay = 4.88 ms) and PlexA1fl/fl;Emx1-Cre (36 sites from 6 animals; median delay = 3.66 ms) mice. ***P < 0.001. (N) Cumulative frequency distribution plot of PStF latencies from PlexA1fl/fl;Emx1-Cre (gray line) and PlexA1fl/fl mice (black line). Arrows indicate the onsets of EMG responses [(B), (C), (K), and (L)]. ***P < 0.001.

  • 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 to D) Results of the capellini handling test in 2-month-old control (PlexA1fl/+;AAV1-Cre, n = 10) and PlexA1fl/fl; AAV1-Cre (n = 10) mice during 4 testing days. Rate of adjustment is the average number of paw adjustments per piece/eating time. **P < 0.01, ***P < 0.001. (E to G) A mouse during the grasping test. Food pellets are indicated by black arrows. (H and I) Grasping success and consumption time in 3-month-old control (PlexA1fl/+;AAV1-Cre, n = 3; and PlexA1fl/fl;AAV1-td, n = 4) and PlexA1fl/fl;AAV1-Cre (n = 8) mice over 4 testing days. **P < 0.01. (J and K) Quantification and frequency distributions of pellet consumption time by control (145 trials) and PlexA1fl/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) ZFPM2 (FOG2) (layer 6 marker) (15) and PLEXA1 in the human motor cortex at 20 pcw assessed by means of in situ hybridization. (B) Sequence conservation of putative PlexA1 cis-regulatory elements. Multispecies DNA sequence alignment showing examples 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 cotransfection with a human FEZF2-expressing vector. Error bars represent the SEM of triplicate experiments. *P < 0.05. (D to 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 and G) High magnification view of the boxed areas in (D) and (F), respectively. (H to L) Summary of the axonal trajectories and CS connectivity within the cervical spinal cord in wild-type and PlexA1fl/fl;Emx1-Cre mice, and in humans.

Supplementary Materials

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

    Zirong Gu, John Kalamboglas, Shin Yoshioka, Wenqi Han, Zhuo Li, Yuka Imamura Kawasawa, Sirisha Pochareddy, Zhen Li, Fuchen Liu, Xuming Xu, Sagara Wijeratne, Masaki Ueno, Emily Blatz, Joseph Salomone, Atsushi Kumanogoh, Mladen-Roko Rasin, Brian Gebelein, Matthew T. Weirauch, Nenad Sestan, John H. Martin,* Yutaka Yoshida

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S29
    • References

    Images, Video, and Other Media

    Movie S1
    Movie of a typical adult control mouse (PlexA1fl/+; AAV1-Cre) performing the capellini handling test
    Movie S2
    Slow motion version of an adult control mouse (PlexA1fl/+; AAV1-Cre) in the capellini handling test.
    Movie S3
    Movie of a typical adult PlexA1 mutant mouse (PlexA1fl/fl; AAV1-Cre) performing the capellini handling test.
    Movie S4
    Slow motion version of an adult PlexA1 mutant mouse (PlexA1fl/fl; AAV1-Cre) in the capellini handling test.
    Movie S5
    Slow motion movie of an adult PlexA1fl/fl; AAV1-td mouse in the grasping test.
    Movie S6
    Slow motion movie of an adult PlexA1fl/fl; AAV1-Cre mouse in the grasping test.

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