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

Nat Commun. September 25, 2017; 8 (1): 587.

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

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Abstract

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.

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. 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. 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

Adapted with permission from Springer Nature: Herera-Rincon et al. (2017). The brain is required for normal muscle and nerve patterning during early Xenopus development. Nat Commun. September 25, 2017; 8 (1): 587. doi: 10.1038/s41467-017-00597-2. Copyright 2017.

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