Herrera-Rincon C et al. (2017), The brain is required for normal muscle and ner...

XB-ART-54057

Nat Commun 2017 Sep 25;81:587. doi: 10.1038/s41467-017-00597-2.

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The brain is required for normal muscle and nerve patterning during early Xenopus development.

Herrera-Rincon C , Pai VP , Moran KM , Lemire JM , Levin M .

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Possible roles of brain-derived signals in the regulation of embryogenesis are unknown. Here we use an amputation assay in Xenopus laevis to show that absence of brain alters subsequent muscle and peripheral nerve patterning during early development. The muscle phenotype can be rescued by an antagonist of muscarinic acetylcholine receptors. The observed defects occur at considerable distances from the head, suggesting that the brain provides long-range cues for other tissue systems during development. The presence of brain also protects embryos from otherwise-teratogenic agents. Overexpression of a hyperpolarization-activated cyclic nucleotide-gated ion channel rescues the muscle phenotype and the neural mispatterning that occur in brainless embryos, even when expressed far from the muscle or neural cells that mispattern. We identify a previously undescribed developmental role for the brain and reveal a non-local input into the control of early morphogenesis that is mediated by neurotransmitters and ion channel activity.Functions of the embryonic brain prior to regulating behavior are unclear. Here, the authors use an amputation assay in Xenopus laevis to demonstrate that removal of the brain early in development alters muscle and peripheral nerve patterning, which can be rescued by modulating bioelectric signals.

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Species referenced: Xenopus laevis
Genes referenced: ctrl hcn2 tub

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	Fig. 1. The absence of the early brain leads to abnormal muscle development and patterning. a After fertilization, the brain was removed from stage 25 embryos to generate BR− animals. Morphological evaluation of muscle phenotype was performed at early- (stages 30–41) and late- (42–48) stages. b Lateral views of stage 25 embryos before (left) and after (right) brain removal. The area occupied by the developing brain is marked with a white-dashed line. (left) rostral is left and dorsal is up. Scale bar, 250 μm. cg, cement gland; e, eye; fb, forebrain, hb, hindbrain, sm, somites. c–h The brain is required for normal muscle development and patterning, as shown after quantitative evaluation of collagen density (short arrows), length of myotome fibers (double-headed arrows), central body axis and myotome angle (overlaid dashed axis and arrowhead-like lines) at early c, d and late f–h stages. At the onset of development, BR− embryos possessed a lower collagen density in myotome fibers (magenta arrow in c compared to turquoise arrow in b), a significantly more open central angle along the rostra-caudal axis e and shorter somites than control (Ctrl) embryos. During development, defects in the organization of central body axis and muscle patterning were not corrected at any anatomical level in BR− (magenta dashed lines in g compared to turquoise dashed lines in f. The mean angle for BR− is significantly displaced to 180°, compared to those in Ctrl (H). c, d, f, g Photomicrographs taken under polarized light. Rostral is upper right and dorsal is up. Turquoise and magenta arrows indicate correct and incorrect anatomical pattern, respectively. Scale bar, 500 μm. e, h Graphic representation of the mean angle of myotome fibers at rostral, central and caudal levels (blue squares) of Ctrl (white) and BR− (gray) embryos. Data represent the mean and s.d. of three independent replicates (n = 75 animals per group). P values after t (equal variances, black labels) or Mann–Whitney (unequal variances, blue labels) tests are indicated as *P < 0.01, P < 0.05, ns no significant difference
	Fig. 2. Scopolamine rescues the BR− muscle phenotype. a, b Quantification of the mean percentage of abnormal embryos and statistical comparisons among Ctrl and BR− populations under normal conditions and after drug treatment, at early- (a) and late- (b) stages after brain removal. Values are plotted as mean % ± s.d. (no-pooled data from, at least, three different replicates). c–f. Typical muscle phenotype for Ctrl (c) and BR− (d), and BR− after scopolamine (e) or carbachol treatment (f), as seen under polarized light. Rostral is upper right and dorsal is up. Turquoise, magenta and yellow arrows indicate correct, incorrect and aberrant formation, respectively. Scale bar, 100 μm. g, h Quantification of the mean length of myotome fibers and statistical comparisons among untreated Ctrl and untreated BR−, scopolamine-treated BR− and carbachol-treated BR− at early- (g, one-way ANOVA, P < 0.01) and late- (h, Kruskal–Wallis test, P < 0.01) stages after brain removal. No significant differences after a posteriori analysis were detected among the different Ctrl groups. Data represent the mean and s.d. of three independent replicates. i. Scopolamine exposure and rescue effects on BR− phenotype. (left) Graphical representation of the different exposure times to scopolamine in BR−, after brain removal (t = 0, magenta arrow; white band means no drug and blue bands means drug treatment) and for a 2-week (2w) period. First-week experimental group was exposed to scopolamine immediately after brain removal and consecutively for the first week. Second-week animals were exposed to the drug 1 week after the brain removal, for 1-week period. First- and second-week animals were exposed to scopolamine immediately after the brain removal and for the 2 next consecutive weeks. (right) Quantification of the mean percentage of embryos with abnormal phenotype within each BR− group. Values are plotted as mean % ± s.d. (no-pooled data from three different replicates). For all panels, number in bars indicates n or number of embryos analyzed for each group. P values after z-test a, b, i and post-hoc Bonferroni’s g or Dunn’s test h are indicated as *P < 0.01, P < 0.05, ns P > 0.05
	Fig. 3. Ectopic expression of HCN2 rescues the BR− muscle phenotype. a Embryos were microinjected (Inj) with HCN2 mRNA (wild-type channel, WT) either in the two cells (HCN2 WT-group, turquoise arrows) or in one cell (1/2 HCN2 WT, see b blue arrow) at the two-cell stage. Brain was removed at stage 25, and animals with and without brain (Ctrl and BR−, respectively) were analyzed for muscle structure and patterning at early- (stages 30–41) and late-stage (stages 42–48). b 1/2 HCN2-WT injection: embryos were microinjected with HCN2 and lacZ mRNA in one of the cells at two-cell stage (blue arrow). The injection side was confirmed by enzymatic detection of β-galactosidase, β-gal (dorsal view of one Ctrl animal is showed on the right). Rostral is up. Scale bar, 1 mm. c Quantification of the mean percentage of abnormal embryos (macroscopic phenotype) and statistical comparisons between uninjected BR− embryos (No Inj) and the different injected-BR− populations (Water, black arrows: water-injection in the two cells; HCN2: HCN2-WT mRNA injection in the two cells; 1/2 HCN2 + β-gal: co-injection of HCN2-WT and lacZ gene reporter in one LR side). Values are plotted as mean % ± s.d. (no-pooled data from two different replicates). d, e Typical muscle phenotype for uninjected BR− (d) and HCN2-WT injected BR− (e), as seen under polarized light. Rostral is upper right and dorsal is up. Scale bar, 100 μm. f, g Quantification of the mean length of myotome fibers and statistical comparisons among uninjected Ctrl and uninjected BR− (BR−), HCN2-WT injected BR- and 1/2 HCN2-WT (measured on uninjected contralateral side) at early- (f one-way ANOVA, P < 0.01) and late- (g one-way ANOVA, P < 0.01) stages after brain removal. No significant differences after a posteriori analysis were detected among the different Ctrl groups. Data represent the mean and s.d. of two independent replicates. For all panels, number in bars indicates n or number of animals for each group. P values after after z-test c or post-hoc Bonferroni’s test f, g are indicated as **P < 0.01, ns P > 0.05
	Fig. 4. The brain can prevent drug-induced abnormalities of body patterning from occurring. a–d Lateral view of stage-45 tadpoles with brain (left column; Control, BR+) or without brain (right column; Brainless, BR−) after housing in normal conditions (top row; no drug treatment, Drug−) and after continuous treatment with 10 μm (RS)-(Tetrazol-5-yl)glycine (RS, an NMDA receptor agonist), respectively. Drug treatment in Control animals (c, Drug+ BR+) did not produce alterations in tail patterning (turquoise arrows in c similar to a), and there was no incidence of aberrant or hypercurved phenotypes. Drug treatment in BR− animals (d, Drug+ BR−) lead to a completely aberrant population, with highly curved phenotypes (different to those in BR− without drug treatment, yellow arrows in d compared to magenta arrows in b). Rostral is to the left and dorsal is up. Turquoise, magenta and yellow arrows indicate correct, incorrect and aberrant tail modules, respectively. Scale bar, 1 mm. e Analysis of the phenotype distributions within each experimental group showed that RS is able to induce a significantly aberrant body patterning (a ‘hypercurvature’ phenotype) only if the brain is absent. Data represent the pooled distribution of three replicates (n = 75 animals per group). P < 0.01 for X 2 (0.05, 6). f, g Evaluation under polarized light of drug-treated animals, with brain (f) and without brain (g), revealing clear muscle defects, both in structure and overall patterning, when the brain is not present (yellow arrows in g). This disorganization is not present in drug-treated control animals, exhibiting normal somites and myotome fibers (turquoise arrows in f, see Fig. 2c for similarity to Ctrl group). Turquoise, magenta and yellow arrows indicate correct, incorrect and aberrant muscle structure, respectively. Scale bar, 100 μm
	Fig. 5. The absence of a brain generates an abnormal neural network in the entire animal body. a, b Acetylated-tubulin (Tub) immunoexpression for Ctrl (a) and BR− (b) animals. There three types of nerve fibers: (i) commissural fibers (dorsoventral axis, long arrows); (ii) longitudinal fibers (anteroposterior axis, short arrow); and (iii) internal neuropil (no defined axis, unfilled triangles). Animals developed without a brain show normal commissural and longitudinal nerve fibers (turquoise long arrows in b), with some alterations (magenta long arrow), but a dense internal neuropil (yellow unfilled triangles). c, d Tub-immunoexpression for BR− treated with cholinergic drugs: scopolamine (c) and carbachol (d). Scopolamine treatment was not able to rescue the aberrant internal network (yellow unfilled triangles in c), and carbachol-treated animals exhibited a chaotic nerve patterning (magenta and yellow arrows in d). e Ectopic HCN2-WT expression (injected in both cells at two-cell stage) fixed the BR−–induced internal nerve branching. f Quantification of the mean OD of internal neuropil and statistical comparisons among untreated/uninjected Ctrl and untreated/uninjecetd BR− (BR−, without drug treatment nor ion channel misexpression), scopolamine-treated BR− (BR− + scopolamine), carbachol-treated BR− (BR− + carbachol), HCN2-WT both-sides injected BR− (BR− + HCN2 WT), and HCN2-WT LR side-injected BR− (BR− + 1/2 HCN2 WT) embryos (one-way ANOVA, P < 0.01). No significant differences after a posteriori analysis were detected among the different Ctrl groups. Data represent the mean OD units and s.d. of two independent replicates. Number in bars indicates n or number of animals analyzed for each group. P values after post-hoc Bonferroni’s test are indicated as *P < 0.01, P < 0.05, ns P > 0.05. g, h. Ectopic HCN2 expression in only one LR side fixes the BR−-induced internal nerve branching. Tub-immunoexpression on β-gal-reacted sections (dark deposits) in a 1/2 HCN2-WT BR−, showing both the contralateral uninjected side (g) and the injected (h) of the same embryo. Aberrant neural network was completely rescued (turquoise arrows), exhibiting a similar nerve pattern to the Ctrl group in both sides. a-e, h: Rostral is upper right and dorsal is up. g: Rostral is upper left and dorsal is up. Scale bar, 100 μm
	Fig. 6. Brain effects on muscle and nerve patterning are partially mediated via spinal cord and mimicked via the dorsal expression of HCN2. a-c Upper row, a Lateral view of a stage-33 embryo following spinal cord resection (SC−) at stage 25. Site of injury is indicated by magenta arrow. b, c Embryos were microinjected with HCN2 (wild-type channel) and lacZ mRNA either in the two ventral cells (b, blue arrows) or two dorsal cells (c, blue arrows) at the four-cell stage. Animals were evaluated at stages 42–48. HCN2-ventral embryos were β-galactosidase negative (β-gal−, white arrow) for brain (center image in b, dorsal view) and SC (right image in b, lateral view) and β-gal+ (blue arrow) for ventral myotomes (right image in b). HCN2-dorsal embryos were β-gal+ for brain (center image in c) and SC (right image in c) and β-gal− for ventral myotomes (right image in c). For lateral views, rostral is left and dorsal is up. Scale bar, 500 μm. Middle row, Typical muscle phenotype for SC− (left panel), HCN2-ventral injected BR− (center panel), and HCN2-dorsal injected BR− (right panel), as seen under polarized light. Muscle patterning (angle of the myotomes, magenta dashed arrowhead-like line) in SC− was altered compared to Ctrl. SC− presented a less severe phenotype than BR− displaying myofibers with normal structure (turquoise arrow) and some incorrect patterning (magenta arrow). BR− + HCN2 ventral embryos presented profound defects in muscle structure, both in angle (magenta dashed line) and in length/organization (magenta arrow) of the myotome fibers. Ectopic or aberrant patterning was also present (yellow arrow). BR− + HCN2 dorsal embryos presented an organized myotome, with normal myofiber structure and organization (turquoise dashed line and arrows). Lower row, typical nerve patterning (commissural fibers indicated by long turquoise arrow, longitudinal fibers indicated by head arrows, and internal neuropil indicated by unfilled triangles) for SC− (left panel), HCN2-ventral injected BR− (center panel) and HCN2-dorsal injected BR− (right panel), shown on anti-acetylated alpha-tubulin antibody staining. SC− exhibited some degree of organization for commissural and longitudinal fibers (turquoise arrows), but frequent errors were present (magenta arrows). Internal neuropil was, nevertheless, profoundly altered, displaying the typical BR−aberrant or ectopic nerve branching (yellow). Nerve patterning in BR− + HCN2 ventral was markedly altered for all the different fiber types. Conversely, HCN2-mRNA injections in dorsal cells lead to an entirely well-organized nerve phenotype, indistinguishable from controls. Rostral is upper right and dorsal is up. Scale bar, 100 μm. d-f. Quantification of the mean angle (d Kruskal–Wallis, P < 0.01) and length (e one-way ANOVA, P < 0.01) of central myotome fibers and Tub-positive internal neuropil (f one-way ANOVA, P < 0.01), along with statistical comparisons for each experimental group vs. Ctrl (P values above the bar). Data represent the mean OD units and s.d. of two independent replicates (n = 50 animals per group). P values after post-hoc analysis are indicated as *P < 0.01, P < 0.05, ns P > 0.05
	Fig. 7. Brain signaling for muscle and nerve development and patterning a. Schematic representation drawing of a Xenopus embryo, showing the main components of our experiments: brain (blue), spinal cord-peripheral nerves (pink) and somites-muscle (brown). Brain effects on nerve patterning could occur directly (2), by using efferent spinal pathway. Brain effects on muscle patterning could occur indirectly (3a), by acting on neurons, or directly (3b), by acting on muscle. b A spinal mechanism, for coding the information about patterning and morphogenesis, could occur via direct signaling from the brain to the neurons in the spinal cord (pink circle). According to our results (different treatments are indicated with purple labels), the effects of the peripheral innervation on muscle cells can be partially explained in terms of developmental bioelectricity or changes in Vmem excitability. We hypothesize that at these stages, the brain is in part controlling the bioelectric state of peripheral tissues, and a correct balance (turquoise triangle) of brain activity (long-range instructive cues or top-down perspective) and local signals (bottom-up perspective) is necessary for correct morphogenesis. Both an excess of tonic activity (induced after carbachol or RS treatment) and an excess of slow If gradients through membrane lead to mispatterning. The extra-spinal pathway by which the brain is acting on muscles can be mimicked pharmacologically, with pharmacological agents targeting bioelectricity (i.e., scopolamine). We hypothesize that scopolamine is acting at presynaptic/synaptic level, blocking the inhibitory ACh actions (via mAChRs) on slow ion flows, and leading the Vmem to appropriate values for muscle patterning. c, d. Schematic representation of neuromuscular specificity in normal development (c, with brain) and in absence of the brain (d, BR−). Our results suggest that ectopic branching detected in the absence of a brain is not due to deficits in early pruning or target retrograde signaling. Pathfinding behavior at the onset of Xenopus development starts at the spinal cord level, as early patterned electrical gradients in SC cells is required for the correct axon guidance. The different treatments applied in our experiments (purple labels and circles) are placed on the cellular/subcellular domains where they are probably acting

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	Fig. 1. The absence of the early brain leads to abnormal muscle development and patterning. a After fertilization, the brain was removed from stage 25 embryos to generate BR− animals. Morphological evaluation of muscle phenotype was performed at early- (stages 30–41) and late- (42–48) stages. b Lateral views of stage 25 embryos before (left) and after (right) brain removal. The area occupied by the developing brain is marked with a white-dashed line. (left) rostral is left and dorsal is up. Scale bar, 250 μm. cg, cement gland; e, eye; fb, forebrain, hb, hindbrain, sm, somites. c–h The brain is required for normal muscle development and patterning, as shown after quantitative evaluation of collagen density (short arrows), length of myotome fibers (double-headed arrows), central body axis and myotome angle (overlaid dashed axis and arrowhead-like lines) at early c, d and late f–h stages. At the onset of development, BR− embryos possessed a lower collagen density in myotome fibers (magenta arrow in c compared to turquoise arrow in b), a significantly more open central angle along the rostra-caudal axis e and shorter somites than control (Ctrl) embryos. During development, defects in the organization of central body axis and muscle patterning were not corrected at any anatomical level in BR− (magenta dashed lines in g compared to turquoise dashed lines in f. The mean angle for BR− is significantly displaced to 180°, compared to those in Ctrl (H). c, d, f, g Photomicrographs taken under polarized light. Rostral is upper right and dorsal is up. Turquoise and magenta arrows indicate correct and incorrect anatomical pattern, respectively. Scale bar, 500 μm. e, h Graphic representation of the mean angle of myotome fibers at rostral, central and caudal levels (blue squares) of Ctrl (white) and BR− (gray) embryos. Data represent the mean and s.d. of three independent replicates (n = 75 animals per group). P values after t (equal variances, black labels) or Mann–Whitney (unequal variances, blue labels) tests are indicated as *P < 0.01, P < 0.05, ns no significant difference
	Fig. 2. Scopolamine rescues the BR− muscle phenotype. a, b Quantification of the mean percentage of abnormal embryos and statistical comparisons among Ctrl and BR− populations under normal conditions and after drug treatment, at early- (a) and late- (b) stages after brain removal. Values are plotted as mean % ± s.d. (no-pooled data from, at least, three different replicates). c–f. Typical muscle phenotype for Ctrl (c) and BR− (d), and BR− after scopolamine (e) or carbachol treatment (f), as seen under polarized light. Rostral is upper right and dorsal is up. Turquoise, magenta and yellow arrows indicate correct, incorrect and aberrant formation, respectively. Scale bar, 100 μm. g, h Quantification of the mean length of myotome fibers and statistical comparisons among untreated Ctrl and untreated BR−, scopolamine-treated BR− and carbachol-treated BR− at early- (g, one-way ANOVA, P < 0.01) and late- (h, Kruskal–Wallis test, P < 0.01) stages after brain removal. No significant differences after a posteriori analysis were detected among the different Ctrl groups. Data represent the mean and s.d. of three independent replicates. i. Scopolamine exposure and rescue effects on BR− phenotype. (left) Graphical representation of the different exposure times to scopolamine in BR−, after brain removal (t = 0, magenta arrow; white band means no drug and blue bands means drug treatment) and for a 2-week (2w) period. First-week experimental group was exposed to scopolamine immediately after brain removal and consecutively for the first week. Second-week animals were exposed to the drug 1 week after the brain removal, for 1-week period. First- and second-week animals were exposed to scopolamine immediately after the brain removal and for the 2 next consecutive weeks. (right) Quantification of the mean percentage of embryos with abnormal phenotype within each BR− group. Values are plotted as mean % ± s.d. (no-pooled data from three different replicates). For all panels, number in bars indicates n or number of embryos analyzed for each group. P values after z-test a, b, i and post-hoc Bonferroni’s g or Dunn’s test h are indicated as *P < 0.01, P < 0.05, ns P > 0.05
	Fig. 3. Ectopic expression of HCN2 rescues the BR− muscle phenotype. a Embryos were microinjected (Inj) with HCN2 mRNA (wild-type channel, WT) either in the two cells (HCN2 WT-group, turquoise arrows) or in one cell (1/2 HCN2 WT, see b blue arrow) at the two-cell stage. Brain was removed at stage 25, and animals with and without brain (Ctrl and BR−, respectively) were analyzed for muscle structure and patterning at early- (stages 30–41) and late-stage (stages 42–48). b 1/2 HCN2-WT injection: embryos were microinjected with HCN2 and lacZ mRNA in one of the cells at two-cell stage (blue arrow). The injection side was confirmed by enzymatic detection of β-galactosidase, β-gal (dorsal view of one Ctrl animal is showed on the right). Rostral is up. Scale bar, 1 mm. c Quantification of the mean percentage of abnormal embryos (macroscopic phenotype) and statistical comparisons between uninjected BR− embryos (No Inj) and the different injected-BR− populations (Water, black arrows: water-injection in the two cells; HCN2: HCN2-WT mRNA injection in the two cells; 1/2 HCN2 + β-gal: co-injection of HCN2-WT and lacZ gene reporter in one LR side). Values are plotted as mean % ± s.d. (no-pooled data from two different replicates). d, e Typical muscle phenotype for uninjected BR− (d) and HCN2-WT injected BR− (e), as seen under polarized light. Rostral is upper right and dorsal is up. Scale bar, 100 μm. f, g Quantification of the mean length of myotome fibers and statistical comparisons among uninjected Ctrl and uninjected BR− (BR−), HCN2-WT injected BR- and 1/2 HCN2-WT (measured on uninjected contralateral side) at early- (f one-way ANOVA, P < 0.01) and late- (g one-way ANOVA, P < 0.01) stages after brain removal. No significant differences after a posteriori analysis were detected among the different Ctrl groups. Data represent the mean and s.d. of two independent replicates. For all panels, number in bars indicates n or number of animals for each group. P values after after z-test c or post-hoc Bonferroni’s test f, g are indicated as **P < 0.01, ns P > 0.05
	Fig. 4. The brain can prevent drug-induced abnormalities of body patterning from occurring. a–d Lateral view of stage-45 tadpoles with brain (left column; Control, BR+) or without brain (right column; Brainless, BR−) after housing in normal conditions (top row; no drug treatment, Drug−) and after continuous treatment with 10 μm (RS)-(Tetrazol-5-yl)glycine (RS, an NMDA receptor agonist), respectively. Drug treatment in Control animals (c, Drug+ BR+) did not produce alterations in tail patterning (turquoise arrows in c similar to a), and there was no incidence of aberrant or hypercurved phenotypes. Drug treatment in BR− animals (d, Drug+ BR−) lead to a completely aberrant population, with highly curved phenotypes (different to those in BR− without drug treatment, yellow arrows in d compared to magenta arrows in b). Rostral is to the left and dorsal is up. Turquoise, magenta and yellow arrows indicate correct, incorrect and aberrant tail modules, respectively. Scale bar, 1 mm. e Analysis of the phenotype distributions within each experimental group showed that RS is able to induce a significantly aberrant body patterning (a ‘hypercurvature’ phenotype) only if the brain is absent. Data represent the pooled distribution of three replicates (n = 75 animals per group). P < 0.01 for X 2 (0.05, 6). f, g Evaluation under polarized light of drug-treated animals, with brain (f) and without brain (g), revealing clear muscle defects, both in structure and overall patterning, when the brain is not present (yellow arrows in g). This disorganization is not present in drug-treated control animals, exhibiting normal somites and myotome fibers (turquoise arrows in f, see Fig. 2c for similarity to Ctrl group). Turquoise, magenta and yellow arrows indicate correct, incorrect and aberrant muscle structure, respectively. Scale bar, 100 μm
	Fig. 5. The absence of a brain generates an abnormal neural network in the entire animal body. a, b Acetylated-tubulin (Tub) immunoexpression for Ctrl (a) and BR− (b) animals. There three types of nerve fibers: (i) commissural fibers (dorsoventral axis, long arrows); (ii) longitudinal fibers (anteroposterior axis, short arrow); and (iii) internal neuropil (no defined axis, unfilled triangles). Animals developed without a brain show normal commissural and longitudinal nerve fibers (turquoise long arrows in b), with some alterations (magenta long arrow), but a dense internal neuropil (yellow unfilled triangles). c, d Tub-immunoexpression for BR− treated with cholinergic drugs: scopolamine (c) and carbachol (d). Scopolamine treatment was not able to rescue the aberrant internal network (yellow unfilled triangles in c), and carbachol-treated animals exhibited a chaotic nerve patterning (magenta and yellow arrows in d). e Ectopic HCN2-WT expression (injected in both cells at two-cell stage) fixed the BR−–induced internal nerve branching. f Quantification of the mean OD of internal neuropil and statistical comparisons among untreated/uninjected Ctrl and untreated/uninjecetd BR− (BR−, without drug treatment nor ion channel misexpression), scopolamine-treated BR− (BR− + scopolamine), carbachol-treated BR− (BR− + carbachol), HCN2-WT both-sides injected BR− (BR− + HCN2 WT), and HCN2-WT LR side-injected BR− (BR− + 1/2 HCN2 WT) embryos (one-way ANOVA, P < 0.01). No significant differences after a posteriori analysis were detected among the different Ctrl groups. Data represent the mean OD units and s.d. of two independent replicates. Number in bars indicates n or number of animals analyzed for each group. P values after post-hoc Bonferroni’s test are indicated as *P < 0.01, P < 0.05, ns P > 0.05. g, h. Ectopic HCN2 expression in only one LR side fixes the BR−-induced internal nerve branching. Tub-immunoexpression on β-gal-reacted sections (dark deposits) in a 1/2 HCN2-WT BR−, showing both the contralateral uninjected side (g) and the injected (h) of the same embryo. Aberrant neural network was completely rescued (turquoise arrows), exhibiting a similar nerve pattern to the Ctrl group in both sides. a-e, h: Rostral is upper right and dorsal is up. g: Rostral is upper left and dorsal is up. Scale bar, 100 μm
	Fig. 6. Brain effects on muscle and nerve patterning are partially mediated via spinal cord and mimicked via the dorsal expression of HCN2. a-c Upper row, a Lateral view of a stage-33 embryo following spinal cord resection (SC−) at stage 25. Site of injury is indicated by magenta arrow. b, c Embryos were microinjected with HCN2 (wild-type channel) and lacZ mRNA either in the two ventral cells (b, blue arrows) or two dorsal cells (c, blue arrows) at the four-cell stage. Animals were evaluated at stages 42–48. HCN2-ventral embryos were β-galactosidase negative (β-gal−, white arrow) for brain (center image in b, dorsal view) and SC (right image in b, lateral view) and β-gal+ (blue arrow) for ventral myotomes (right image in b). HCN2-dorsal embryos were β-gal+ for brain (center image in c) and SC (right image in c) and β-gal− for ventral myotomes (right image in c). For lateral views, rostral is left and dorsal is up. Scale bar, 500 μm. Middle row, Typical muscle phenotype for SC− (left panel), HCN2-ventral injected BR− (center panel), and HCN2-dorsal injected BR− (right panel), as seen under polarized light. Muscle patterning (angle of the myotomes, magenta dashed arrowhead-like line) in SC− was altered compared to Ctrl. SC− presented a less severe phenotype than BR− displaying myofibers with normal structure (turquoise arrow) and some incorrect patterning (magenta arrow). BR− + HCN2 ventral embryos presented profound defects in muscle structure, both in angle (magenta dashed line) and in length/organization (magenta arrow) of the myotome fibers. Ectopic or aberrant patterning was also present (yellow arrow). BR− + HCN2 dorsal embryos presented an organized myotome, with normal myofiber structure and organization (turquoise dashed line and arrows). Lower row, typical nerve patterning (commissural fibers indicated by long turquoise arrow, longitudinal fibers indicated by head arrows, and internal neuropil indicated by unfilled triangles) for SC− (left panel), HCN2-ventral injected BR− (center panel) and HCN2-dorsal injected BR− (right panel), shown on anti-acetylated alpha-tubulin antibody staining. SC− exhibited some degree of organization for commissural and longitudinal fibers (turquoise arrows), but frequent errors were present (magenta arrows). Internal neuropil was, nevertheless, profoundly altered, displaying the typical BR−aberrant or ectopic nerve branching (yellow). Nerve patterning in BR− + HCN2 ventral was markedly altered for all the different fiber types. Conversely, HCN2-mRNA injections in dorsal cells lead to an entirely well-organized nerve phenotype, indistinguishable from controls. Rostral is upper right and dorsal is up. Scale bar, 100 μm. d-f. Quantification of the mean angle (d Kruskal–Wallis, P < 0.01) and length (e one-way ANOVA, P < 0.01) of central myotome fibers and Tub-positive internal neuropil (f one-way ANOVA, P < 0.01), along with statistical comparisons for each experimental group vs. Ctrl (P values above the bar). Data represent the mean OD units and s.d. of two independent replicates (n = 50 animals per group). P values after post-hoc analysis are indicated as *P < 0.01, P < 0.05, ns P > 0.05
	Fig. 7. Brain signaling for muscle and nerve development and patterning a. Schematic representation drawing of a Xenopus embryo, showing the main components of our experiments: brain (blue), spinal cord-peripheral nerves (pink) and somites-muscle (brown). Brain effects on nerve patterning could occur directly (2), by using efferent spinal pathway. Brain effects on muscle patterning could occur indirectly (3a), by acting on neurons, or directly (3b), by acting on muscle. b A spinal mechanism, for coding the information about patterning and morphogenesis, could occur via direct signaling from the brain to the neurons in the spinal cord (pink circle). According to our results (different treatments are indicated with purple labels), the effects of the peripheral innervation on muscle cells can be partially explained in terms of developmental bioelectricity or changes in Vmem excitability. We hypothesize that at these stages, the brain is in part controlling the bioelectric state of peripheral tissues, and a correct balance (turquoise triangle) of brain activity (long-range instructive cues or top-down perspective) and local signals (bottom-up perspective) is necessary for correct morphogenesis. Both an excess of tonic activity (induced after carbachol or RS treatment) and an excess of slow If gradients through membrane lead to mispatterning. The extra-spinal pathway by which the brain is acting on muscles can be mimicked pharmacologically, with pharmacological agents targeting bioelectricity (i.e., scopolamine). We hypothesize that scopolamine is acting at presynaptic/synaptic level, blocking the inhibitory ACh actions (via mAChRs) on slow ion flows, and leading the Vmem to appropriate values for muscle patterning. c, d. Schematic representation of neuromuscular specificity in normal development (c, with brain) and in absence of the brain (d, BR−). Our results suggest that ectopic branching detected in the absence of a brain is not due to deficits in early pruning or target retrograde signaling. Pathfinding behavior at the onset of Xenopus development starts at the spinal cord level, as early patterned electrical gradients in SC cells is required for the correct axon guidance. The different treatments applied in our experiments (purple labels and circles) are placed on the cellular/subcellular domains where they are probably acting