T-type Calcium Channel Regulation of Neural Tube Closure and EphrinA/EPHA Expression - PubMed (original) (raw)

T-type Calcium Channel Regulation of Neural Tube Closure and EphrinA/EPHA Expression

Sarah Abdul-Wajid et al. Cell Rep. 2015.

Abstract

A major class of human birth defects arise from aberrations during neural tube closure (NTC). We report on a NTC signaling pathway requiring T-type calcium channels (TTCCs) that is conserved between primitive chordates (Ciona) and Xenopus. With loss of TTCCs, there is a failure to seal the anterior neural folds. Accompanying loss of TTCCs is an upregulation of EphrinA effectors. Ephrin signaling is known to be important in NTC, and ephrins can affect both cell adhesion and repulsion. In Ciona, ephrinA-d expression is downregulated at the end of neurulation, whereas, with loss of TTCC, ephrinA-d remains elevated. Accordingly, overexpression of ephrinA-d phenocopied TTCC loss of function, while overexpression of a dominant-negative Ephrin receptor was able to rescue NTC in a Ciona TTCC mutant. We hypothesize that signaling through TTCCs is necessary for proper anterior NTC through downregulation of ephrins, and possibly elimination of a repulsive signal.

Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

CAV3 is required for Ciona intestinalis neural tube closure. A and B. Representative C. intestinalis embryos expressing a control guide RNA (sgRNA) (A), or guide RNAs targeting exons 3 and 49 of the C. intestinalis CAV3 gene (ciCAV3 sgRNA). See also Figure S1. (B). Green fluorescence from a pan-neural ETR1 promoter driving nuclear GFP construct marks neural tube cells. C and D. Bug mutants differentiate and polarize the anterior neural tube. Bugeye (bug) and wild type siblings stained for the neural markers CRALBP, Opsin and Arrestin, as indicated. E and F. Immunostaining for the cell polarity marker aPKC in wild type and bug mutants. s.v. = sensory vesicle, n.d.= neurohypophyseal duct, v.g.= visceral ganglion. The staining intensity is consistent between bug and wild type, although the sensory vesicle in bug embryos is open (arrowheads in F). Views are dorsal. G and H. Phalloidin staining of bug and wild type sibling larvae. The most intense staining is in red. cnt= caudal neural tube, osp=oral siphon primordium. Scale bar = 24 μm.

Figure 2

Figure 2

Onset of open brain phenotype in Ciona. A and B. Mid-sagittal confocal sections of late tailbud (stage 25) bug and wild type (WT) sibling embryos showing immunostaining for CRALBP. Yellow arrowheads indicate the anterior neuropore. The pigmented cells of the sensory vesicle are indicated by red asterisks. C and C′. Still images from the start and end of Movie S1 of a bug embryo. Cells of the neural tube express GFP from the pan-neural ETR1 promoter. Magenta indicates FM464 stained membranes. Yellow arrowheads indicate sensory vesicle that, at the start of movie (C; t=0) is covered by non-neural tissue (i.e., epidermis), and then at four hours (C′; t=4h) is exposed on the surface. (A–C′: lateral view of head region with palps and anterior to the left). D. Treatment with mibefradil during neurulation phenocopies the bug phenotype. E. Treatment with nifedipine during neurulation stages causes an arrest in development. F. Mibefradil treatments. Dechorionated embryos at each developmental stage were subjected to control (water) or 1μM Mibefradil, washed after treatment, and then allowed to develop to swimming larvae stage. Percentage phenotype was calculated by counting the number of _bugeye_-like embryos over the total number of embryos treated. Error bars represent standard error of the mean from three independent experiments.

Figure 3

Figure 3

X. laevis CAV3.2 morpholino (MO) knockdown. A. Quantification of MO knockdown phenotype at stages 21 and 27. Two MOs targeting X. laevis CAV3.2 (CAV3.2-MO1 and CAV3.2-MO2) and two control MOs [mismatch (CTL-MO1) and standard control (CTL-MO2); see Material and Methods)] were tested. The quantities of MO injected per embryo are indicated below the number (N) of embryos scored for each dose of MO. B. Disruption of CAV3.2 transcript splicing by CAV-MO1. RT-PCR was used to detect both the correct and expected splice-disrupted transcripts in cDNA samples from three pooled CTL-MO and three CAV-MO1 injected embryos. Muscle actin (M. Actin) RT-PCR was used to control for RNA load, and RT-minus controls are also shown. C and D. Early tailbud stage embryos (stage 22) injected at the one-cell stage with a CTL-MO1 (C), or a CAV3.2-MO1 (D). Yellow arrowheads in B indicate anterior open neural tube. E–G. Tailbud stage (stage 26) CTL-MO1 (E) and CAV3.2-MO1 (F and G) injected embryos. Anterior neural tube defects were observed in the CAV3.2-MO1 injections with either cellular matter erupting from the open neural tube (yellow arrowhead in F), or a with a malformed head and deep dimple in the hindbrain (yellow arrowhead in G). See also Movie S2. H–J. NCAM staining (red) in anterior or posterior coronal sections of CTL-MO1 (H) and CAV3.2-MO1+2 (I and J) injected embryos. K–M. E-cadherin staining (yellow) in anterior and posterior coronal sections of CTL-MO1 (K) or CAV3.2-MO1+2 (L and M) injected embryos. MO injected embryos used for sections were stage matched at ~stage 24 based on somite development. All sections were also stained for nuclei (DAPI, blue).

Figure 4

Figure 4

Expression of C. savignyi CAV3. A–D. In-situ hybridization of CAV3 expression in wild-type C. savignyi embryos. Expression is first detectable at early tailbud stage (A; stage 19) in the midbrain-hindbrain (MHB) region and faintly in the caudal nerve cord (cnt). This expression intensified by mid tailbud and new expression in the forebrain (fb) is observed (B; stage 21). By late tailbud (C and D; stage 23/24) new expression is seen in a cluster of cells in the epidermis (arrowheads) (sv=sensory vesicle). E–G. Double fluorescent in situ for CAV3 and engrailed yields overlapping expression at the MHB (but not in the cnt). Embryos are stage 22.

Figure 5

Figure 5

Calcium (Ca2+) transients in the neural tubes of C.savignyi and X. laevis. A–A″. Xenopus embryo injected with GCaMP3 RNA shows Ca2+ transients during neurulation. Top left, a late neurula embryo expressing GCaMP3 in the closing neural tube (A). Low magnification view of X. laevis embryo showing typical area recorded for Ca2+ transients (yellow box). Anterior is up. A′, High magnification of the Z plane of the X. laevis anterior neural plate that was imaged. A″. GCaMP fluorescence for a single cell in the neural plate undergoing a Ca2+ transient ([Ca2+] indicated by color scale). See also Movies S3 and S4. B. CAV3.2 morpholino knockdown (CAV3.2-MO1, CAV) reduces the number of cells in the neural tube displaying Ca2+ transients, the length and amplitude of the transients, but not the frequency. The y-axis in the amplitude graph shows relative fluorescent intensity (see Materials and Methods). The frequency values are also described in Materials and Methods. Control embryos were injected with a non-specific MO (CTL-MO2, CTL). Data represent quantification of three embryos from three independent experiments and error bars represent standard error of the mean (*= p<0.05). C. Typical Ca2+ transients in CAV-MO knockdown and CTL-MO X. laevis neural tube cells. D. Ca2+ transients in C. savignyi embryos detected with GCaMP3 expressed from the pan-neural ETR1 promoter. Top panel shows one frame from a time-lapse movie. Lower panel is a close up of the midbrain hindbrain region (yellow box in top panel) showing a single Ca2+ transient with fluorescence intensity using the color scale from A″. See also Movie S5. E. Time-lapse of Ca2+ transients in the C. savignyi MHB region. Representative images from two time points (neurula and mid-tailbud) of an embryo expressing GCaMP3 with the MHB region of interest (ROI) outlined in yellow. Dashed white lines outline the embryo. Bottom panels show relative fluorescence intensity in the ROI over time. Neu-iTB = neurula to initial tailbud and ETB-MTB = early to mid tailbud.

Figure 6

Figure 6

T-type Ca2+ channel regulation of EphrinA signaling components. A. qRT-PCR assay for expression of the neural genes etr1, otx, six3/6, NCAM and ephrinA-d in bug and wild type (WT) larvae. ΔΔCT values were calculated by normalizing to the ubiquitously expressed gene RPS27A and then comparing to WT expression levels. The values for three independent biological repeats are indicated (open squares, etc.), as well as the average of the three (black bar). Red asterisks indicates significant difference between bug mutants and WT for given transcript; p≤0.05, T-test. RPS27a values represent ΔCT comparison to WT. B. C. savignyi EphrinA-d transcript levels quantified over developmental time. qRT-PCR results are represented as log2(X) changes in absolute cycle threshold values for each developmental time point sample. C. RT-PCR for expression of EPHA2, NCAM, M.Actin and histone 4(H4) in stage 24 X. laevis embryos injected with either CAV3.2 splice disrupting morpholinos (CAV-MO1+2, CAV), or a control MO (CTL-MO2, CTL). The values for three independent biological repeats are indicated (open squares, etc.), as well as the average of the three (black bar). EPHA2 values are significantly different between CAV and CTL embryos (Red asterisks, p=0.025, Standard T-test). D. Percent of open brain phenotype observed in WT embryos electroporated with either the EphrinA-d cDNA expression construct (WT+ EphrinAd), or H2B:GFP cDNA (WT+ control). Results from three independent trails are shown, as well as the average of the three (black bars). Red asterisks indicates significance, p=0.0006, Fisher exact test. E. EphrinA-d overexpression driven by pan-neural ETR promoter in C. intestinalis embryos phenocopies bug. Yellow arrowheads indicate the open brain. Two representative embryos are shown. The co-electroporated plasmid ETR>H2B:GFP labels the nervous system. F. Control embryo expressing only ETR>H2B:GFP.

Figure 7

Figure 7

Rescue of Ciona bugeye (bug) embryos with dominant negative EPH3 (dnEPH3). A. Embryos from heterozygous bug parents were electroporated with either dnEPH3 driven by the pan-neural promoter ETR1, or an empty ETR1 vector (control). The fraction of embryos with the bug phenotype (open brain) was determined for each group. Circles represent the percent of embryos showing the bug phenotype from four independent trials, and the black line is the average of the four trials (n is the combined number scored from the four trials). Red asterisks indicate significant P-value of 0.002 using a chi-squared test of deviation from the expected open brain frequency of 25%. B. Single tadpoles electroporated with dnEPH3 (lanes 1–12) and scored as having closed brains (CB) were genotyped with primers that distinguished the insertion-containing bug allele from the wild type allele. The bug and wild type (WT) lanes show the sizes the two alleles. Three of the twelve electroporated embryos (4–6, red) genotyped as homozygous for the bug allele despite having closed brains, and are considered as rescued. Representative images of wild type, bug, and rescued embryos are shown. See also Figure S2. C. Representative image of a partially rescued bug embryo.

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