XB-ART-57435Sci Rep January 1, 2020; 10 (1): 16446.
Caveolin 1 is required for axonal outgrowth of motor neurons and affects Xenopus neuromuscular development.
Caveolins are essential structural proteins driving the formation of caveolae, specialized invaginations of the plasma membrane. Loss of Caveolin-1 (Cav1) function in mice causes distinct neurological phenotypes leading to impaired motor control, however, the underlying developmental mechanisms are largely unknown. In this study we find that loss-of-function of Xenopus Cav1 results in a striking swimming defect characterized by paralysis of the morphants. High-resolution imaging of muscle cells revealed aberrant sarcomeric structures with disorganized actin fibers. As cav1 is expressed in motor neurons, but not in muscle cells, the muscular abnormalities are likely a consequence of neuronal defects. Indeed, targeting cav1 Morpholino oligonucleotides to neural tissue, but not muscle tissue, disrupts axonal outgrowth of motor neurons and causes swimming defects. Furthermore, inhibition of voltage-gated sodium channels mimicked the Cav1 loss-of-function phenotype. In addition, analyzing axonal morphology we detect that Cav1 loss-of-function causes excessive filopodia and lamellipodia formation. Using rescue experiments, we show that the Cav1 Y14 phosphorylation site is essential and identify a role of RhoA, Rac1, and Cdc42 signaling in this process. Taken together, these results suggest a previously unrecognized function of Cav1 in muscle development by supporting axonal outgrowth of motor neurons.
PubMed ID: 33020520
PMC ID: PMC7536398
Article link: Sci Rep
Genes referenced: cav1 cdc42 ncam1 rac1 rbm8a rho.2 rhoa ung
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|Figure 1. Loss-of-function of Cav1L affects sarcomeric organization and causes swimming defects. (A) Cav1L MO and Cav1L Spl-MO inhibit Xenopus Cav1L expression. Xenopus embryos were injected with 20 ng Morpholinos at the one-cell stage and Cav1α and GAPDH expression was analyzed by Western blotting at stage 27. The graph shows the relative Cav1α expression of four independent experiments, in relation to GAPDH expression and normalized to Co MO-injected embryos, data are mean ± s.e.m. ***p-value ≤ 0.001 (Student’s t-test). (B) Xenopus embryos were injected with 20 ng Morpholinos and mGFP RNA in one blastomere at the two-cell stage and analyzed at stage 42. Cav1L morphants show mild morphological abnormalities including a shortening of the anterior–posterior axis, craniofacial malformations and edema formation (arrowhead). (C,D) Tracks of the swimming movement (time frame 4 s) of controls (C) and morphant embryos (D). (E) Percentage of swimming defects of embryos injected unilaterally with Morpholinos (5–7.5 ng) alone or in combination with res-cav1L RNA (+ = 100 pg; + + = 200 pg, + + + = 300 pg), a Cav1L-construct lacking the Morpholino binding side, at the two-cell stage. Number of analyzed embryos is indicated for each column. Data from at least three experiments are presented as the mean ± s.e.m. * p-value ≤ 0.05; **p-value ≤ 0.01; ***p-value ≤ 0.001 (Student’s t-test). (F,G) Muscle morphology of stage 38 embryos, injected unilaterally with 20 ng MO at the two-cell stage; asterisks mark injected side. Phalloidin staining reveals sarcomeric actin; DAPI staining marks the nuclei. (F’,G’) Higher magnification of the boxed areas in (F,G). Controls show normal sarcomeric actin organization, while morphants display wavy and disorganized actin fibers. (H,I) Single somitic segment of a control embryo (H) or a Cav1L morphant (I), injected with 10 ng MO in both blastomeres at the two-cell stage, showing disrupted actin organization within muscle cells. (H’,I’) Transmission electron microscopy (TEM) picture of control muscle cells with normal sarcomeric organization (H’) and Cav1L-morphant muscle cells, which do not show the characteristic sarcomeric structure. Arrowhead marks detached actin bundles; L lipid droplet, Nc notochord.|
|Figure 2. Cav1L is expressed in the notochord and the cardio-vasculature during Xenopus development Temporal and spatial cav1L expression analyzed by in situ hybridization. (A) Dorsal view of a stage 12.5 embryo, cav1L is detected in the notochord and two thin stripes on both sides at the dorsal midline. (B) Dorsal view of an embryo at stage 15. Cav1L expression is visible in the notochord and in a punctuated pattern in the skin. (C) Stage 18 embryo showing the same expression pattern as described in B. Cav1L is strongly expressed in the epidermis of a stage 20 embryo (D), stage 23 embryo (E), stage 26 embryo (F) and stage 28 embryo (G). (H) Cav1 expression is visible in the notochord, epidermis and aortic arches of a stage 37 embryo. (I) Cav1 is expressed in the notochord and the cardio-vasculature of the tail (dlav, pcv, isv) of a stage 41 embryo. (I’) Magnification of the embryo shown in I. Cav1 is expressed in the lung, lymph heart and aortic arches. Aa aortic arches, Dlav dorsal longitudinal anastomosis vessel, Isv/Isa intersomitic vessels/artery, lh lymph heart, lg l ung, nc notochord, Pcv posterior cardinal vein.|
|Figure 3. Cav1 is expressed in the nervous system during Xenopus development. (A) Immunostaining of a stage 35 embryo showing Cav1 protein expression in the notochord and the cranial nerves. (B) Dorsal view of a stage 35 embryos. Cav1 expression is visible in the cell bodies of the cranial nerve located in the brain. (C) Stage 42 embryo. (D) Higher magnification of a stage 42 embryo. Cav1 is expressed in the cranial nerves, aortic arches and the lymph heart. (D’) Image focusing on the Cav1 expression in the lung of the embryo shown in (F). (E) Ventral view of the embryo shown in (D). Cav1 staining is visible in the heart, aortic arches and cranial nerves. (F) Tail of a stage 42 embryos showing Cav1 expression in the cardio vasculature and neural tube. (G) Tail of a stage 42 embryo showing Cav1 expression in the notochord and motor neurons (H) Transverse section of the hindbrain of a stage 37 embryo showing Cav1 expression in red and DAPI staining in blue. Cav1 is expressed in neurons and the notochord. (I) Magnification of the epidermis of a stage 37 embryo. Cav1 is expressed in the deep layer of the epidermis. (J) Transverse section of the neural tube of a stage 37 embryo showing Cav1 expression in the neural tube and notochord. Aa aortic arches, Dlav dorsal longitudinal anastomosis vessel, ep epidermis, h heart, il inner layer of the epidermis, Isv/Isa intersomitic vessels/artery, lh lymph heart, lg lung, mn motor neurons, nc notochord, nt neural tube, Pcv posterior cardinal vain, so somites, sl sensory layer of the epidermis, VII facial nerve, Vop ophthalmic trigeminal ganglion, Vmd mandibular trigeminal ganglion, IX glossopharyngeal nerve, X vagus nerve.|
|Figure 4. Loss-of-function of Cav1L in neural tissue or Benzocaine-treatment causes muscular disorganization and swimming defects. (A,B) 10 ng MO alone or in combination with 100 pg cav1L-HA RNA were targeted to the somites (A) or the neural tube (B) at the 8-cell stage and the percentage of swimming defects (three independent experiments) were analyzed at stage 38. LacZ RNA injection was used to control for RNA toxicity or unspecific rescue effects. Data from at least three experiments are presented as the mean ± s.e.m.. ***p-value ≤ 0.001 (one-way ANOVA comparing the total number of swimming defects). (C–E) Muscle morphology of neural-injected embryos were analyzed by sectioning and Phalloidin staining. Asterisks indicate the injected side. (C) Normal actin organization in a control embryo. (D) Highly disrupted actin organization on the injected side of Cav1-morphant embryo. (E) Co-expression of res-cav1L RNA in neural tissue restores actin organization of Cav1L-morphant muscle cells. (C’,D’,E’) Higher magnification of the boxed areas shown in (C–E). (F) Embryos were injected unilaterally with 20 ng MO and cultivated until stage 26 when Benzocaine was added to the medium. Embryos were fixed at stage 38, sectioned longitudinally and stained with Phalloidin. Asterisks in (G–J) indicate the injected side. (G) Normal muscle morphology of a control embryo. (H) Loss-of-function of Cav1L disrupts somitic actin fiber organization. (I,J) Benzocaine-treated embryos. The absence of neural activity caused by Benzocaine-treatment results in disorganized actin fibers in controls (I) as well as Cav1L-morphant embryos (J). (G’,H’,I’,J’) Higher magnification of the boxed areas shown in (G–J).|
|Figure 5. Loss of Cav1L affects motor neuron morphology. (A–D) Embryos, injected unilaterally with 20 ng MO at the two-cell stage, were immunostained using the neuronal surface marker Ncam. (A) Motor neurons display a characteristic chevron-shaped pattern in controls; nt = neural tube. (B,C) Motor neuron outgrowth and pathfinding is severely impaired in Cav1L morphants. (A’,B’,C’) Higher magnification of the dashed areas shown in (A–C). (D) Percentages of motor neuron outgrowth defects of Morpholino-injected embryos (20 ng, + +) and embryos injected unilaterally with 10 ng MO (+) in combination with 200 pg cav1L-HA. Co-expression of cav1L-HA partially rescues motor neuron defects. Graph in (D) present data as the mean ± s.e.m. from at least three experiments. Number of analyzed embryos is indicated for each column. **p-value ≤ 0.01 ***p-value ≤ 0.001 (one-way ANOVA).|
|Figure 6. Cav1L knockdown affects axonal growth and morphology in vitro. (A–N) Loss-of-function of Cav1L increases filopodia as well as lamellipodia formation in cultured spinal cord neurons, isolated from embryos that were injected with either 12 ng (++) or 10 ng (+) Morpholino in both blastomeres at the two-cell stage. Actin staining is shown in (D) (Co MO), (E) (Cav1L MO) and (F) (Cav1L Spl-MO). (A,B) Control axons show typical actin-positive growth cones. (C–F) Filopodia- and lamellipodia-like actin-positive structures in Cav1L-morphant neurons. (G) Box plot showing the number of axons (n) per explant (N) (Tukey Box plot with whiskers with maximum 1.5 IQR (Mann–Whitney test)). Co-expression of 10 pg Cav1L Spl-MO in combination with 200 pg cav1L-HA RNA significantly improved axonal outgrowth. (I) Percentage of axons with increased filopodia-like structures. Co-expression of 200 pg cav1L-HA RNA significantly decreased the number of axons with filopodia. (J) Number of filopodia per 100 µm axon length. (K) Percentage of axons with lamellipodia. Co-expression of 200 pg cav1L-HA significantly decreased the number of axons with lamellipodia. (L) Percentage of lamellipodia area in relation to total axon area. Data in (I,K,L) are mean ± s.e.m. from at least three experiments (Student’s t-test). Number of analyzed axons (n) are indicated for each column. * p-value ≤ 0.05; **p-value ≤ 0.01; ***p-value ≤ 0.001.|
|Figure 7. Y14 phosphorylation of Cav1L is necessary for Rho GTPases dependent axonal outgrowth and pathfinding of motor neurons. (A,B) Co-injection of 10 pg ca Cdc42, dn RhoA or dn Rac1 with 10 ng Cav1L Spl-MO in neural tissue significantly improved swimming defect (A) and motor neuron outgrowth (B) caused by Cav1L loss-of-function. Significances of the rescue experiments were calculated in comparison to the Cav1L Spl-MO injected embryos. (C) Motor neuron outgrowth was analyzed by Ncam immunostaining, injected constructs are indicated. (D) Overexpression of 200 pg cav1L Y14A RNA in Cav1L-morphant (10 ng) embryos did not rescue swimming defects, while 200 pg of wild-type cav1L significantly improved the morphant phenotype, when injected into one blastomere of the two-cell stage. All graphs (B–D) present data from at least three experiments as the mean ± s.e.m. ***p-value ≤ 0.001 (one-way ANOVA comparing the total number of swimming defects). (E) Model of Cav1L function in motor neuron outgrowth. Tyrosine phosphorylated Cav1L (blue, with red Y14 phosphorylation) affects Rho GTPases. The morphology of a wild-type (WT) and Cav1L-morphant motor neuron is shown; microtubules (green) and actin bundles (red) are shown.|