Loss of cell adhesion causes hydrocephalus in nonmuscle myosin II-B-ablated and mutated mice

Abstract

Ablation of nonmuscle myosin (NM) II-B in mice during embryonic development leads to marked enlargement of the cerebral ventricles and destruction of brain tissue, due to hydrocephalus. We have identified a transient mesh-like structure present at the apical border of cells lining the spinal canal of mice during development. This structure, which only contains the II-B isoform of NM, also contains beta-catenin and N-cadherin, consistent with a role in cell adhesion. Ablation of NM II-B or replacement of NM II-B with decreased amounts of a mutant (R709C), motor-impaired NM II-B in mice results in collapse of the mesh-like structure and loss of cell adhesion. This permits the underlying neuroepithelial cells to invade the spinal canal and obstruct cerebral spinal fluid flow. These defects in the CNS of NM II-B-ablated mice seem to be the cause of hydrocephalus. Interestingly, the mesh-like structure and patency of the spinal canal can be restored by increasing expression of the motor-impaired NM II-B, which also rescues hydrocephalus. However, the mutant isoform cannot completely rescue neuronal cell migration. These studies show that the scaffolding properties of NM II-B play an important role in cell adhesion, thereby preventing hydrocephalus during mouse brain development.

Figure 1.

Evidence for hydrocephalus in B^CN/B^CN mice. H&E staining of embryonic mouse brain sections. (a and b) Sagittal sections of B⁺/B⁺ (a) and B^CN/B^CN (b) brains at E14.5 show mild dilatation of the cerebral ventricles (b) and abnormal protrusion of facial neurons (b, arrow) into the fourth ventricle of B^CN/B^CN mice. (c–f) Coronal sections of B⁺/B⁺ (c and e) and B^CN/B^CN (d and f) brains at P0 show significant dilatation of the cerebral ventricles as well as the aqueduct of Sylvius (AQ) in a B^CN/B^CN mouse compared with a B⁺/B⁺ mouse. LV, lateral ventricle; 3V, third ventricle.

Figure 2.

Blockage of the spinal canal in B⁻/B⁻ and B^CN/B^CN mice. H&E-stained images of embryonic mouse spinal cords in cross section. (a–c) At E11.5, the spinal canals of B⁺/B⁺ and B^CN/B^CN mice are patent with intact ventricular surfaces (a and b), whereas the ventricular surface of the spinal canal in B⁻/B⁻ mice is disrupted with underlying neuroepithelial cells invading the canal (c). (d–f) At E12.5, the B⁺/B⁺ canal remains patent (d), the B^CN/B^CN canal is partially blocked by protruding neuroepithelial cells (e), and the B⁻/B⁻ spinal canal is almost completely obstructed (f).

Figure 3.

Complete obstruction of the spinal canal in B^CN/B^CN mice. H&E-stained images of E14.5 mouse spinal cords in cross section. The spinal canal from B^CN/B^CN mice is completely obstructed (b); in comparison, the spinal canal of a B⁺/B⁺ littermate is patent (a). c and d show magnified images for B⁺/B⁺ and B^CN/B^CN mice, respectively.

Figure 4.

Immunofluorescence confocal images of mouse spinal cords in cross section at E12.5. (a–c, enlarged in d–f) Costaining of the antibodies for NMHC II-A (a and d, green), II-B (b and e, green), and II-C (c and f, green) with antibodies for N-cadherin (a–f, red) in B⁺/B⁺ mice shows that only NMHC II-B stains significantly at the ventricular surface (b and e, arrow) of the developing mouse spinal canal together with N-cadherin (b and e, yellow). NMHC II-A predominantly stains the vasculature (a and d, green, arrows), and NMHC II-C stains neural cells but not the apical border (c and f, green). The white brackets delineate the neuroepithelial cells (see text). (g–i) Distribution of NMHC II-A, II-B and II-C in B^CN/B^CN spinal cords further confirms the predominant expression of NMHC II-B (h) rather than II-A (g) and II-C (i) at the apical border of the ventricular surface. The white asterisks indicate neuroepithelial cells abnormally protruding into the spinal canal in a B^CN/B^CN mouse.

Figure 5.

Transitory presence of a mesh-like structure at the border of cells lining the spinal canal between E8.5 to E14.5. (a–e) H&E-stained cross sections of representative spinal cords between E8.5 and E14.5 show the changes in the neuroepithelial cell layer (brackets). By E14.5, the spinal canal is lined by a single layer of differentiated ependymal cells. (f–j) Immunofluorescence confocal images of the neuroepithelial cells lining the spinal canal (indicated by dash-lined box in c), and stained for N-cadherin, show a mesh-like adhesion structure that is most obvious at the apical border between E9.5 and E12.5 (g–i). This structure is not obvious at E8.5, and it is markedly reduced at E14.5 (f and j). (k–t) Immunofluorescence confocal images of the neuroepithelial cells lining the spinal canal stained for NMHC II-B (k–o) and β-catenin (p–t) show staining patterns at the apical border of the spinal canal similar to N-cadherin.

Figure 6.

Immunofluorescence confocal images of the neuroepithelial cells lining the spinal canal stained for NMHC II-B (green) and β-catenin (red) in B⁺/B⁺, B⁻/B⁻, and B^CN/B^CN mice. (a–c) The mesh-like cell adhesion structure in the B⁺/B⁺ spinal canal at E11.5. NMHC II-B (a, green) and β-catenin (b, red) are present in the structure and colocalize (c, yellow) at the apical border. (d–f) Disrupted adhesion structure in B^CN/B^CN mice (e) compared with the normal structure found in a B⁺/B⁺ littermate at E12.5 (d). At E11.5, the adhesion structure remains in B^CN/B^CN mice (f), but individual neuroepithelial cells are beginning to protrude through the adhesion complex (f, arrow). (g–i) Disrupted adhesion structures lining the B⁻/B⁻ spinal canal at E11.5 (h and i) compared with that in B⁺/B⁺ canal (g). The adhesion structure is completely discontinuous in some regions (h) or collapsed in other regions (i) in B⁻/B⁻ mice at E11.5.

Figure 7.

Restoration of an intact ventricular surface in the spinal canal in B^C/B^C mice. (a and b) Cross sections of the spinal cord from B⁺/B⁺ (a) and B^C/B^C (b) mice stained with H&E show an unobstructed, patent spinal canal in both the B⁺/B⁺ and B^C/B^C mice at E14.5. (c–h) Immunofluorescence confocal images of E11.5 spinal cords from B⁺/B⁺ and B^C/B^C mice stained with β-catenin (c and d, red), NMHC II-B (e and f, green), and N-cadherin (g and h, red). The mutant NMHC II-B as well as adhesion molecules β-catenin and N-cadherin are normally localized and the mesh-like structure is restored to the apical border of the cells lining the spinal canal in B^C/B^C mice (d, f, and h).

Figure 8.

Localization of aPKCλ and NMHC II-B to the ventricular surface of the spinal cord. Immunofluorescence confocal images of the spinal cord stained for aPKCλ (c) and NMHC II-B (d) show that both are enriched at the ventricular surface lining the spinal canal in B⁺/B⁺ mice at E11.5. No significant pMLC20 (a, top) or myosin light chain kinase (b, MLCK) is detected in B⁺/B⁺ mice at the ventricular surface. No pMLC20 is detected in the B^C/B^C ventricular surface (a, bottom). pMLC20 and MLCK are detected in the vasculature of the spinal cord (a and b, arrows).

Figure 9.

Abnormal migration of pontine neurons in B^C/B^C mice. H&E-stained images of sagittal brain sections from B⁺/B⁺ and B^C/B^C mice at E16.5. (a and b) In B⁺/B⁺ mice, although some of the pontine neurons were still located in their migrating trajectory (a, arrows), many of them have arrived at their final destination (b, arrow). (c and d) In B^C/B^C mice by E16.5, many of the pontine neurons have accumulated along their migrating path near to where they were generated (c, arrows), and none of the pontine neurons had reached their destination (d, arrow).