Secretory pathway calcium ATPase 1 (SPCA1) controls mouse neural tube closure by regulating cytoskeletal dynamics

Abstract

Neural tube closure relies on the apical constriction of neuroepithelial cells. Research in frog and fly embryos has found links between the levels of intracellular calcium, actomyosin dynamics and apical constriction. However, genetic evidence for a role of calcium in apical constriction during mammalian neurulation is still lacking. Secretory pathway calcium ATPase (SPCA1) regulates calcium homeostasis by pumping cytosolic calcium into the Golgi apparatus. Loss of function in Spca1 causes cranial exencephaly and spinal cord defects in mice, phenotypes previously ascribed to apoptosis. However, our characterization of a novel allele of Spca1 revealed that neurulation defects in Spca1 mutants are not due to cell death, but rather to a failure of neuroepithelial cells to apically constrict. We show that SPCA1 influences cell contractility by regulating myosin II localization. Furthermore, we found that loss of Spca1 disrupts actin dynamics and the localization of the actin remodeling protein cofilin 1. Taken together, our results provide evidence that SPCA1 promotes neurulation by regulating the cytoskeletal dynamics that promote apical constriction and identify cofilin 1 as a downstream effector of SPCA1 function.

Keywords: Apical constriction; Atp2c1; Ca2+; Cofilin 1; Mouse; Neural tube closure; Non-muscle myosin II.

Fig. 1.

The 1D mutation causes exencephaly and spinal cord defects. (A,B) Lateral (left) and dorsal (right) views of E9.5 wild-type and 1D mutant embryos obtained from micro computed tomography (microCT) scans. Arrowheads in B demarcate the extent of exencephaly in the 1D mutant. Lines indicate the level of cross-sections shown in C-F. (C-F) Cross sections through the hindbrain (C,D) and spinal cord (E,F) regions in E9.5 wild type and 1D mutants. Black arrowheads point to the medial hinge point. Unfilled arrowhead in F points to the surface epithelia covering the abnormally shaped neuroepithelia in 1D embryos. Scale bars: 300 µm (A,B); 100 µm (C-F).

Fig. 2.

1D mutants contain a non-conservative mutation in SPCA1. (A,B) Sanger sequencing traces for the Spca1 coding region in wild type and 1D mutants. 1D embryos contain a T-to-A nucleotide difference (red and green shaded peaks), which is predicted to cause a Val-to-Glu amino acid change at position 183 (highlighted in yellow). Residue numbers are based on NP_778190 sequence. Note that the Spca1 sequence shown contains a known silent polymorphism between the FvB (wild-type) and C57J/B6 (1D mutant) strains (C-T SNP, asterisks). (C,D) Protein sequence alignments showing the conserved nature of the mouse SPCA1 V183 residue across species (C) and in comparison with other mouse Ca²⁺ ATPases (D). Conservation is indicated for identical residues (asterisks), for residues with strong similar properties (double dot) and for residues with weakly similar properties (dot). SPCA2, PMCA1, PMCA2, SERCA1 and SERCA2 are also known as ATP2C2, ATP2B1, ATP2B2, ATP2A1 and ATP2A2, respectively. (E-H) Lateral views of E9.5 wild-type (E), Spca1^1D homozygotes (F), Spca1^1D/KO trans heterozygote (G) and homozygote Spca1^KO (H) embryos. Scale bars: 500 µm.

Fig. 3.

Effects of the 1D mutation on the localization and function of SPCA1. (A-F) Immunohistochemistry with SPCA1 (A,C,E, green) and GM130 (GOLGA2) (B,D,F, magenta) antibodies in wild-type (A,B), Spca1^KO (C,D) and Spca1^1D (E,F) MEFs. Scale bars: 20 µm. (G-J) Calcium clearance assays in wild-type (black, n=9), Spca1^KO (blue, n=6) and Spca1^1D (yellow, n=9) MEFs in the absence (G,H) or presence (I,J) of drug inhibitors. Drug treatments were as follows: wild-type MEFs treated with 100 nM thapsigargin (Th; SERCA inhibitor, orange, n=8), Spca1^1D MEFs treated with 100 nM thapsigargin (green, n=7) and Spca1^1D MEFs treated with sodium orthovanadate (violet, n=4). At time 0, cells were spiked with 2 mM Ca²⁺ (black dashed lines). Resting cytosolic calcium levels 10 min after introduction of calcium (gray dashed lines) are quantified in H and J. Asterisks in G and I indicate measurements in resting conditions before the addition of Ca²⁺. Error bars indicate s.e.m. ns, not significant; *P<0.05, **P<0.01.

Fig. 4.

Apoptosis in Spca1^1D mutants. (A,B) MicroCT scans of E8.75 wild-type and Spca1^1D embryos (dorsal views). White arrowhead indicates midbrain area, unfilled arrowhead hindbrain area. (C-H) Immunodetection of activated caspase 3 (αCasp3) in wild-type and Spca1^1D embryos at E8.75 (C,D), E9.5 (E,F) and E10.5 (G,H). (I) Plot represents average apoptotic index (activated caspase 3 foci/total number of nuclei) in wild-type and Spca1^1D embryos at E8.75, E9.5 and E10.5 (n=10 sections from three biological replicates for each stage and genotype). HM, head mesenchyme; NT, neural tube. Error bars indicate s.e.m. Scale bars: 300 µm (A,B); 100 µm (C-H).

Fig. 5.

Analysis of apical constriction in Spca1^1D mutants. (A,B) Phalloidin staining of F-actin in the anterior spinal cord region of whole-mount wild-type and Spca1^1D embryos (en face view). (A′,B′) Semi-automated cell outlines generated in ImageJ and color coded based on size of apical area (see C,D). Scale bar: 20 µm. (C,D) Distribution of cell apical areas in wild-type and Spca1^1D neuroepithelia. (E) Box plot showing the apical area of cells in wild-type and Spca1^1D neuroepithelia. Data in C-E corresponds to 834 cells from four wild-type embryos and 966 cells from four Spca1^1D mutant embryos. The line represents the median, the box indicates the distance between the first and third quartile (interquartile range; IQR), whiskers represent ±1.5*IQR and points represent all individual measurements. *P<0.001.

Fig. 6.

Myosin localization and phosphorylation in Spca1^1D mutants. (A-B′) Immunodetection of NMHCIIB and F-actin in E9.5 wild-type (A,A′) and Spca1^1D (B,B′) embryos. Scale bar: 5 µm. (C) Quantification of junctional (JN) and medial (ME) NMHCIIB in E9.5 wild-type (white) and Spca1^1D (gray) embryos (n>1000 cells from four embryos for each genotype). The line represents the median, the box indicates the distance between the first and third quartile (interquartile range; IQR), whiskers represent ±1.5 *IQR and points indicate outliers.

Fig. 7.

Cofilin 1 localization and actin dynamics in Spca1^1D mutants. (A-B′) Immunodetection of CFL1 (magenta) and F-actin (green) in transverse sections of the E9.5 wild-type and Spca1^1D neuroepithelia. Scale bar: 20 µm. (A″-B‴) Magnified views of CFL1 and F-actin localization in the boxed areas in A′ and B′. Scale bar: 5 µm. (C-D′) En face views of CFL1 (magenta) and F-actin (green) localization in E9.5 wild-type and Spca1^1D embryos. Scale bar: 5 µm. (E) Western blot of F- and G-actin fractions isolated from wild-type and Spca1^1D embryos (right) and quantification of F/G actin ratios (left, n=5). Error bars indicate s.e.m. *P<0.05.