Axonal growth and guidance defects in Frizzled3 knock-out mice: a comparison of diffusion tensor magnetic resonance imaging, neurofilament staining, and genetically directed cell labeling

Abstract

Previous work has identified axonal outgrowth and/or guidance defects in the brain and spinal cord of prenatal Frizzled3 (Fz3)(-/-) mice. To systematically explore the axonal defects in Fz3(-/-) mice and to compare techniques for the global assessment of axon tracts in the developing mouse, we have analyzed wild-type and Fz3(-/-) brains using (1) diffusion tensor magnetic resonance imaging (muDTI), (2) neurofilament staining, and (3) two genetically directed neuronal labeling methods. Confirming and extending the previous work of Wang et al. (2002), we find that the following structures/tracts are absent or greatly reduced in the Fz3(-/-) brain: the anterior commissure, cerebral peduncle (corticospinal tract), corpus callosum, fornix, internal capsule (thalamocortical and corticothalamic tracts), stria medullaris, stria terminalis, and hippocampal commissure. An aberrant U-shaped fiber bundle immediately caudal to the optic tract connects the left and right sides of the Fz3(-/-) thalamus and likely represents a default pathway for thalamic axons that failed to enter the internal capsule. At embryonic day 18, labeling of cortical pyramidal cells with a yellow fluorescent protein reporter reveals widespread fragmentation of axons with no apparent loss of pyramidal cell bodies. Fragmentation likely represents one stage in the process that normally eliminates stalled or mistargeted axons. This work demonstrates the usefulness of muDTI and genetically directed neuronal labeling for the analysis of nervous system defects in the mouse.

Figure 1.

Surface morphology of WT and Fz3^–/– brains and cerebral cortices at E18 determined by MRI.A,B,I, Dorsal view; E, F, J, lateral view. C, D, Dorsal(left) and ventral(right) views of the cerebral cortex; G, H, lateral view of the cerebral cortex. I, J, A superposition of WT and Fz3^–/– surfaces.The vertical arrow inHindicates the more dorsal location of the lateral edge of the neocortex in the Fz3^–/– brain.

Figure 2.

Midsagittal μDTI images of WT and Fz3^–/– brains at E18. Anterior is to the left. The Fz3^–/– brain is missing the corpus callosum(cc), anterior commissure(ac), and hippocampal commissure(hc). The optic chiasm(ox) is present in both WT and Fz3^–/– brains. The region marked by an asterisk immediately posterior to the optic chiasm in the Fz3^–/– brain is the midsagittal part of a novel U-shaped fiber bundle within the ventral thalamus. The arrows indicate oriented cells in the wall of the third ventricle. The key in the top left illustrates the false color code for the directions of maximal diffusion with respect to the image plane: horizontal is represented by blue, vertical is represented by red, and perpendicular to the image plane is represented by green. An analogous false color code applies to each of the μDTI images in later figures. Scale bars, 1 mm.

Figure 3.

Comparison of neurofilament immunostaining and μDTI of WT and Fz3^– brains in horizontal section at E18. The midline is adjacent to the left border of each image. In the Fz3^–/– brain, the density of subcortical fibers and the thickness of the corpus callosum are markedly reduced, and the hippocampus and fimbria appear approximately normal but the number of fibers crossing the midline at the hippocampal commissure is reduced. The key in the bottom left of C illustrates the false color code for oriented diffusion for C and D. cc, Corpus callosum; CP, cortical plate; ec, external capsule; fi, fimbriae; H, hippocampus; hc, hippocampal commissure; ic internal capsule. Scale bars, 1 mm.

Figure 4.

Comparison of neurofilament (NF) immunostaining and μDTI of WT and Fz3^–/– brains in coronal section at E18. Sections are at the level of the optic chiasm (left member of each pair of images) and ∼300μm posterior (right member of each pair of images). The midline is adjacent to the left border of each image. In the Fz3^–/– brain, the internal capsule is missing, and a novel U-shaped fiber bundle marked by asterisks links the dorsolateral regions of the left and right sides of the thalamus. The key in the top right of H shows the false color code for oriented diffusion in E–H. cc, Corpus callosum; CP, cortical plate; ec, external capsule; fi, fimbria; H, hippocampus; ic, internal capsule; ot, optic tract; ox, optic chiasm; st, stria terminalis; sm, stria medullaris. Scale bars, 1 mm.

Figure 5.

Neurofilament (NF) immunostaining and μDTI of the ventral thalamus at E18 showing the trajectory of the U-shaped fiber bundle in the Fz3^–/– brain in horizontal section. A–F, Conventional MRI of the head with the anterior at the top. G–L, Neurofilament staining of the ventral thalamus and surrounding structures; M–R, μDTI of the ventral thalamus and surrounding structures. The locations of the individualμDTIimages(M–R) are shown by the rectangular insets inA–F. InG–R, the midline is adjacent to the left border of each image. The U-shaped fiber bundle, marked by an asterisk, courses caudal to the optic tract. The key in the bottom right of M shows the false color code for oriented diffusion in M–R. on, Optic nerve; ot, optic tract; ox, optic chiasm; V, trigeminal nucleus. Scale bars: A–F, 1 mm; M–R, 0.5 mm.

Figure 6.

Three-dimensional reconstruction fromμDTI images of the major thalamic fiber tracts in WT and Fz3^–/– brains at E18. A–C, Surface morphology of the WT brain showing with red squares the regions and orientations of the images in D–F. Matched images from an Fz3^–/– brain are shown in G–I. In D–I, the optic tract is white; the cerebral peduncle and internal capsule (in WT; D–F) or the intrathalamic U-shaped fiber tract (in Fz3^–/–;G–I) are yellow; and the stria terminalis is blue. AG, Amygdala; BS, brainstem; CX, cortex; L, lateral geniculate nucleus; ox, optic chiasm; SP, septum; TH,thalamus.

Figure 7.

Fz3^–/– fiber tract defects at E14 visualized by neurofilament immunostaining and μDTI. A–L, Matched coronal sections beginning at the level of the optic chiasm (A, D, G, J) and ∼400μm(B, E, H, K) or ∼800μm(C, F, I, L) posterior to the optic chiasm. In A–L, the midline is adjacent to the left border of each image. The key in the bottom right of G illustrates the false color code for oriented diffusion for G–L. M–P, Conventional MRI of the head shown by the square insets the locations of the enlarged μDTI images below (Q–T). M, O, Q, S, Sagittal views; N, P, R, T, horizontal views. The key in the bottom right of Q–T shows the false color code for oriented diffusion for each of these panels. In the Fz3^–/– brain, the stria medullaris and the internal capsule are missing; the developing U-shaped fiber bundle is marked by an asterisk. fr, Fasciculus retroflexus; ic, internal capsule; sm, stria medullaris; ox, optic chiasm; V, trigeminal nucleus. Scale bars, 0.5 mm.

Figure 8.

Local disorganization of fibers in the Fz3^–/– brainstem in horizontal sections at E18. A, D, Conventional MRI of the head showing with rectangular insets the locations of the enlargedμDTI images (C, F) and the matched neurofilament-stained sections (B, E). The midline is adjacent to the left border inB,C,E, andF. The key in the bottom right ofCshows the false color code for oriented diffusion for each of these panels. The microscopic disorganization of the fiber bundles in the Fz3^–/– brainstem (E) are below the spatial resolution of μDTI (F). V, Trigeminal nerve; VIII, eighth nerve. Scale bars, 1 mm.

Figure 9.

Defects in Fz3^–/– fiber tracts visualized with sparse AP labeling of neurons in brains of WT and Fz3^–/– littermates at E18. Littermates carrying the ROSA26-creER and ZAP transgenes were exposed to a single maternal injection of 200 μg of 4HT on gestational days E12 (A, B), E13 (C–F), or E10 (G, H). A, B, Horizontal sections with the thalamus in the center. The fibers of the left and right internal capsules are seen in the WT brain but are absent from the Fz3^–/– brain. C–F, Coronal sections showing the smaller corpus callosum and the absence of the internal capsule in the Fz3^–/– brain. G, H, Two small clusters of labeled cortical neurons show extensive axonal projections (arrows) in WT but not Fz3^–/–. In C–F, the midline is adjacent to the left border of each image. cc, Corpus callosum;ic,internal capsule;TH,thalamus.

Figure 10.

Cortical pyramidal cells and fragmented intermediate zone axons in the Fz3^– brain visualized with a Thy1-YFP transgene at E18. Coronal sections from littermates carrying the Thy1-YFP transgene were imaged by confocal microscopy. The surface of the cortex is at the top, the pyramidal cell bodies (PCB) are in the center, and the intermediate zone (IZ) is near the bottom of the image. From an examination of multiple vibratome sections in different focal planes, it is clear that the fluorescent objects in the intermediate zone of the Fz3^–/– cortex are also discontinuous in the dimension perpendicular to the image shown here. Scale bars, 100μm.

Figure 11.

Intermittent defects in spinal cord morphology but nearly normal peripheral nerve development in E12 Fz3^–/– embryos. A, C, Whole-mount neurofilament staining shows that, except for delayed outgrowth of the femoral nerve (red arrows), the development of the Fz3^–/– peripheral nervous system is indistinguishable from WT. B, D, A series of discrete neurofilament-rich zones (arrows) are present along the length of the Fz3^–/– spinal cord. E–H, Cross-sections of WT and Fz3^–/– spinal cords stained with 4′,6′-diamidino-2-phenylindole (DAPI) or stained for neurofilament (NF). The Fz3^–/– spinal cord sections pass through one of the neurofilament-rich zones and show clusters of cells that likely represent ectopic neurons and their associated axons.