Mena and vasodilator-stimulated phosphoprotein are required for multiple actin-dependent processes that shape the vertebrate nervous system

Abstract

Ena/vasodilator-stimulated phosphoprotein (VASP) proteins regulate the geometry of the actin cytoskeleton, thereby influencing cell morphology and motility. Analysis of invertebrate mutants implicates Ena/VASP function in several actin-dependent processes such as axon and dendritic guidance, cell migration, and dorsal closure. In vertebrates, genetic analysis of Ena/VASP function is hindered by the broad and overlapping expression of the three highly related family members Mena (Mammalian enabled), VASP, and EVL (Ena-VASP like). Mice deficient in either Mena or VASP exhibit subtle defects in forebrain commissure formation and platelet aggregation, respectively. In this study, we investigated the consequence of deleting both Mena and VASP. Mena-/-VASP-/- double mutants die perinatally and display defects in neurulation, craniofacial structures, and the formation of several fiber tracts in the CNS and peripheral nervous system.

Figure 1.

Developmental expression of Mena and VASP protein. A-F, At E10.5, Mena (A, C, E) and VASP (B, D, F) proteins are broadly distributed. In sagittal embryo sections, Mena (A) and VASP (B) are detected in several structures, including the neuroepithelium (NE), underlying mesoderm (ME), overlying ectoderm (E), branchial arch (BA), and heart (H). Frontal sections through the cephalic neural tube show that Mena (C) and VASP (D) are expressed throughout the neuroepithelium but concentrated at the apical surface (A-D, arrowheads) and cells in the MHP. VASP is also detected in blood vessels (BV) within and surrounding the neural tube. VASP (F) is also highly expressed in the intersomitic vessels (ISV) running between the dorsal root ganglia, which also express VASP (F, arrowheads) and Mena (E, arrowheads). In the E17 brain, Mena (G, I, K) and VASP (H, J, L) are label axons traveling in the CC, fornix (Fo), and AC. Mena and VASP expression in the midline region (magnification of boxed areas in G and H) are shown in I and J, respectively. Mena and VASP label the corpus callosum as well as the GW. Within the hippocampus, Mena (K) and VASP (L) are enriched in the CA1 region and fimbria (Fi), whereas Mena but not VASP is enriched in the dentate gyrus (DG). Scale bar: (in B) A, B, 500 μm; C, D, I-L, 120 μm; E, F, 100 μm; G, H, 300 μm.

Figure 2.

Neurulation and craniofacial defects. A, SEM of E9.5 cephalic neural tubes of control (top) and M^-/-V^-/- (bottom) littermates. The neural tube is closed in the control but is open in the mutant. B, E13.5 exencephalic mutants exhibit mild (middle) to severe (right) facial clefts compared with control (left) littermates. C-F, Phalloidin-stained frontal sections through the cephalic neural tube of E10.5 controls (C, E) and M^-/-V^-/- (D, F) littermates exhibiting severe (D) and mild (F) neural tube defects. C, D, In contrast to the control (C), the mutant neural folds are splayed apart, and an accumulation of actin is found at the base of the mutant neural tube (D, boxed area enlarged in inset). E, F, The dorsal neural folds are not fused properly in the mutant (F, boxed area enlarged in inset) compared with the control (E, boxed area enlarged in inset). Exencephalic (D) and nonexencephalic (F) mutant neuroepithelia contain several invaginations (asterisks) that are not observed in controls (C, E). Scale bar: (in F) C-F, 250 μm; insets, 100 μm.

Figure 3.

Spinal nerve defects. A-D, Whole-mount anti-neurofilament immunohistochemistry of E10.5 control (A, C) and M^-/-V^-/- (B, D) littermates. Boxed areas in A and B are enlarged in C and D, respectively. Spinal nerves extend and converge to form the branchial plexus (BP) in control animals (A, C). In M^-/-V^-/- mutants, spinal nerves are misrouted and/or have failed to extend (D, arrows). Cranial ganglia, cranial nerves (arrowheads), and dorsal root ganglia (asterisks indicate corresponding DRGs) appear to develop normally in both control (A) and mutant (B) embryos. Note the open neural tube in the mutant (B, open arrows). Scale bar: (in D) A, B, 250 μm; C, D, 100 μm.

Figure 4.

Forebrain commissure defects. Silver-stained horizontal sections of adult brains. A, Serial sections through control M^+/-V^+/- brain indicate the location of the CC, DHC, VHC, and AC as they appear along the dorsoventral (left to right) axis. B, Sections through the M^-/-V^+/- brain reveal the complete absence of the CC and DHC. In dorsal sections, misrouted callosal axons form dense Probst bundles (arrow 1) lateral to the midline. Although the rostral portion of the VHC (seen in dorsal sections) bundles lateral to the midline and does not cross, the caudal portion of the VHC (seen in ventral sections) is formed and contacted by ectopically projecting callosal fibers (arrow 2). The anterior commissure is defective with only a few fibers crossing midplane (arrow 3) and severely misrouted fibers within the temporal lobe of the AC (arrow 4). C, Sections through the M^-/-V^-/- brain revealed the complete absence of all four commissures: CC, DHC, VHC, and AC. In dorsal sections, misrouted callosal axons form dense Probst bundles (arrow 1), whereas further ventrally, callosal fibers ectopically contact fibers of the aberrant VHC (arrow 2). Scale bar, 2 mm.

Figure 5.

Corpus callosum development. A, B, DiI-labeled callosal fibers (red) in coronal sections counterstained with DAPI (blue). A, At E16.5, callosal fibers in control and M^-/- brains have arrived at similar positions near the midline region but have not yet crossed. B, At E17.5, callosal fibers in control brains (left panel) have crossed the midline and project into the contralateral cortex. Callosal fibers (CC) cross at a level dorsal to the GW, shown in rostral (top panel) and caudal (bottom panel) sections of the same brain. However, in M^-/- brains (right panel), callosal fibers travel further ventrally, do not cross the midline, and form Probst bundles (PB) adjacent to the midline. In caudal sections of the same brain, mutant callosal fibers contact the VHC. C, DiI-labeled callosal fibers (red) and DiA-labeled hippocampal commissure fibers (green) in horizontal sections of E17.5 brains. In control brains, the callosal and hippocampal commissure fibers appear as distinct tracts. In mutant brains, the callosal fibers (red) travel in a greater caudoventral direction to reach the VHC (green). Scale bar (in A), 400 μm.

Figure 6.

Midline development. A, B, H/E-stained coronal sections of E16.5 brains. Cerebral hemispheres are not fused properly in the mutant M^-/-V^+/- brain (A) compared with the control brain (B). The walls of the SMTM bend medially and fuse with the pial membrane (pm) in control brains. However, in mutant brains, the walls of the SMTM bend medially, but the hemispheres do not properly fuse because of the ventrally penetrating pial membrane (B, arrow). In the mutant diencephalon, a gap is present where normally the walls of the thalamus meet, forming the interthalamic adhesion (B, asterisk). C-F, Midline glia are examined in control and mutant brains by anti-GFAP immunohistochemistry. C, D, At E16.5, GW development is similar in control (C) and mutant (D) brains. Glial processes of the GW extend from the lateral ventricles toward the midline. E, F, In the E17.5 control brains (E), the GW and IG glia are well developed. The CC forms dorsal to the GW. In the E17.5 mutant brains (F), the GW is less pronounced. Misrouted callosal axons form Probst bundles (PB) positioned ventral to the GW. Displaced IG glia, which are normally located at the midline where the hemispheres fused, appear to extend GFAP-positive processes into the PB. Scale bars: 100 μm; insets, 600 μm.

Figure 7.

Role of Mena in optic chiasm development. A-C, Expression of Mena protein in the E14.5 mouse visual system. A, Mena protein is expressed throughout the developing retina but is most abundant in the RGC layer. The highest levels are found on RGC axons, which are positive for neurofilament (NF). B, High-magnification images of a coronal section of retina, demonstrating that Mena is primarily highly expressed in the islet-positive (Isl1/2) RGC layer of the retina. C, In this midchiasm frontal section, NF-positive RGC axons at the chiasm midline express Mena as well. Mena is also found at lower levels throughout the ventral diencephalon, in both the neurons and GLAST-positive midline glia (arrow) of the chiasm region. D-I, Whole-mount ventral views of the optic chiasm in control (D, F, H) and Mena^-/- littermates (E, G, I) as revealed by unilateral anterograde labeling with DiI. In control embryos, the axons in the chiasm region are generally tightly fasciculated, particularly at older ages (H). The majority of axons cross the midline (D, E, asterisks) to project to the contralateral side of the brain, although a minority project ipsilaterally (D, F, H, arrows). In Mena^-/- mutants, many axons become defasciculated at the midline and stray posteriorly (E, G, arrowheads), giving the appearance of a double optic tract. This phenotype is fully penetrant, but exhibits variable expressivity and is generally most severe at younger ages (E, G). Dorsal is at the top in A-C. Anterior is at the top in D-I. Scale bars: A, C-I, 100 μm; B, 25 μm.