Abdul-Wajid S , Morales-Diaz H , Khairallah SM , Smith WC .

R01 HD038701 NICHD NIH HHS , HD038701 NICHD NIH HHS

Xla Wt + cacna1h MO (Fig 3. E-G)
Xla Wt + cacna1h MO (Fig 3. I, L)
Xla Wt + cacna1h MO (Fig 3. J, M)
Xla Wt + cacna1h MO (Fig 3. J, M)
Xla Wt + cacna1h MO (Fig 4 B, C)

	Figure 3. X. laevis CAV3.2 Morpholino 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 Experimental Procedures) 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 tail-bud stage embryos (stage 22) injected at the one-cell stage with a CTL-MO1 (C) or a CAV3.2-MO1 (D). Yellow arrowheads in (D) indicate anterior open neural tube. (E–G) Tail-bud 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 either with cellular matter erupting from the open neural tube (yellow arrowhead in F) or 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 approximately stage 24 based on somite development. All sections were also stained for nuclei (DAPI, blue).
	Figure 5. Calcium 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 shows 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+] is 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 and the length and amplitude of the transients, but not the frequency. The y axis in the amplitude graph shows relative fluorescent intensity (see Experimental Procedures). The frequency values are also described in Experimental Procedures. Control embryos were injected with a non-specific MO (CTL-MO2, CTL). Data represent quantification of three embryos from three independent experiments; error bars, SEM (∗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 image of Ca2+ transients in the C. savignyi MHB region. Representative images from two time points (neurula and mid tail-bud) of an embryo expressing GCaMP3 with the MHB region of interest (ROI) are 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 tail-bud; ETB-MTB, early to mid tail-bud.
	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 indicate 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 were 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) Percentage 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 indicate significance, p = 0.0006, Fisher’s 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.

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