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Pax6 is required for the normal development of the forebrain axonal connections

Lucy Jones1,*, Guillermina López-Bendito1,*, Peter Gruss2, Anastassia Stoykova2 and Zoltán Molnár1,{dagger}

1 Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK
2 Max-Planck Institute of Biophysical Chemistry, Department of Molecular Cell Biology, 37077 Gottingen, Germany



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  		Fig. 1. lacZ expression correctly reflects the activity of the endogenous Pax6 promoter in the proliferative neuroepithelium in the Pax6-/- mice. (A,B) Whole-mount in situ hybridization analysis for Pax6 (A) and ß-gal (B) staining of E12.5 wild-type (+/+) and heterozygous (+/-) transgenic embryos show similar distribution of the signal in the developing CNS and eye. (C-E) The in situ hybridization analysis with probes for Pax6 (C) or lacZ (D,E) genes in wild-type (C) and heterozygous (D) and homozygous (E) transgenic forebrains, reveals similar expression pattern confined to the proliferative neuroepithelium of the dorsal telencephalon. (F-H) At stage E15.5, similar expression is seen for Pax6 in differentiating nuclei of ventral thalamus by the in situ probes for Pax6 (F) or lacZ (G) in the wild-type and heterozygous embryos, respectively. (H) In homozygous embryos, the differentiation of the ventral thalamic nuclei as revealed by the lacZ expression pattern fails. dt, dorsal thalamus; vt, ventral thalamus; ctx, cortex; ge, ganglionic eminence; nc, nasal cavity; e, eye; T, telencephalon; M, mesencephalon; sc, spinal cord. Scale bars: 1 mm in A,B; 200 µm in C-E; 500 µm in F-H.

 


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  		Fig. 3. Alterations of the early thalamocortical projections in Pax6-/- mice. (A,B) After dorsal thalamic (dt) DiI placements in both hemispheres at E15.5, TCAs were observed extending through the internal capsule (A), reaching the intermediate zone of the dorsal cortex (ctx) (arrow in B). (C,D) Two coronal sections stained for ß-galactosidase in an E14.5 Pax6-/- mouse. Note the altered staining in ventral and lateral telencephalic areas compared with heterozygous brains shown in Fig.2A-C. (E,F) Dorsal thalamic DiI crystal placement in the Pax6-/- brains (asterisk in E) showing that the ventrolateral hypothalamus (ht) was invaded by TCAs (E; arrowheads in F). (G) High power image of the box indicated in F. TCAs were observed crossing to the contralateral ventral hypothalamus (double arrowheads in G). (H) Backlabelled cells were also observed in abnormal locations in the hypothalamus (arrows in H). (I) Dorsal thalamic DiI crystal placement revealed few backlabelled cells within the internal capsule. (J) High-power image from the region indicated with the box in I (arrows indicate labelled cells). (K,L) By E18.5, TCAs in the Pax6-/- emerged abnormally from the base of the ventral telencephalon (double arrowheads). GE, ganglionic eminences; pa, pallium; LGE, lateral ganglionic eminence; LV, lateral ventricle; PSPB, pallial/subpallial boundary. Scale bars: 400 µm in A,E; 100 µm in B,G,H,I,K,L; 200 µm in C,D,F; 20 µm in J.

 


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  		Fig. 2. The precise anatomical correlation between ß-galactosidase staining, which reflects Pax6 in situ hybridization pattern, and the trajectory of the developing TCAs were studied in E14.5 (A-F) and E15.5 (G-I) Pax6+/- brains. A single DiI crystal was placed into the dorsal thalamus (dt) (asterisk in F) of Pax6+/- brains after they had been stained with ß-galactosidase. From the E14.5 brain, three adjacent serial sections were examined with light microscopy for ß-galactosidase expression, which appears green-blue (A-C), and with two filters of a fluorescent microscope to reveal the DiI-labelled thalamic fibres (red) and the bisbenzimide counterstaining (blue) on superimposed images (D-F). (D-F) The TCAs crossed the diencephalic-telencephalic boundary (E) and extended through the internal capsule (D). By E15.5 (G-I), labelled cells were observed in the internal capsule (arrow in H). (I) On the superimposed image of G and H, note that the ß-galactosidase staining extends lateral to the group of backlabelled internal capsule cells (arrow in I). ctx, cerebral cortex; GE, ganglionic eminences; PSPB pallial/subpallial boundary. Scale bars: 200 µm in A-F; 100 µm in G-I.

 


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  		Fig. 4. Defects in CFAs in the Pax6-/- brains revealed by tracing with carbocyanine dyes. (A-D) Early CFA were labelled from single DiI crystal placement in the dorsal cortex (ctx) (asterisks in A and C) of an E15.5 wild-type (A,B) and Pax6-/- brain (C,D). CFA in both phenotypes descend through the intermediate zone and turn towards the primitive internal capsule (arrows in B,D). (E) ß-galactosidase staining in a coronal section of an E18.5 Pax6+/- brain. (F) Trajectories of fibres labelled from cortical DiI placement (asterisk) in a similar section to that shown in E, of an E18.5 Pax6+/- brain. (G) ß-galactosidase staining is displaced ventrolaterally in the PSPB (arrows) in the Pax6-/- brain. Note the presence of an abnormal cellular mass (asterisk) protruding into the lateral ventricle (LV). (H) Same section as G showing the abnormal pathway of CFA in Pax6-/- brains. (J-M) Coronal sections from more rostral (J) to more caudal (L) levels, showing abnormal CFA in an E18.5 Pax6-/- brain. (M) High-power image from the region indicated by the box in K. Some fibres descend towards the ventral pallium and cross the PSPB at a more ventral position in large fascicles (arrows in J-L). (N) Numerous backlabelled cells are seen in the marginal zone (mz) of the paleocortex (plx) (arrowheads), suggesting that they develop abnormal projections to the tracing site. (O) Fibres of the corpus callosum (cc) labelled by cortical DiI crystal placements. (P-U) Adjacent serial sections from an E18.5 Pax6-/- brain in which double carbocyanine dyes were placed, DiA in the cortex and DiI in the dorsal thalamus. Note that the two sets of fibres failed to encounter each other at the PSPB (arrowheads indicate DiI-labelled TCAs, and arrows indicate DiA-labelled CFA in Q,S,U). (P) ß-Galactosidase staining of the section shown in Q; (R,T) The bizbenzimide staining of sections in S,U, respectively. GE, ganglionic eminence; cc, corpus callosum; se, septal eminence; hp, hippocampus; dt, dorsal thalamus. Scale bars: 200 µm in A,C,I; 100 µm in B,D,N,M,O; 400 µm in E-H,J-L,P,Q.

 


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  		Fig. 5. Immunolabeling for L1 (A-L) and TAG1 (M-P) reveals defects in TCAs and CFA in the Pax6-/-brains. (A,B) At E14.5 in wild-type brains, TCAs and CFA are both immunoreactive for L1 through the internal capsule into the intermediate zone of the cortex (ctx). (C,D) At E14.5 in the Pax6-/-, TCAs (arrows) and CFA (arrowheads) are both aberrant, thalamic axons fail to extend towards the cortex and CFA fail to turn towards the internal capsule. (E,I) At E18.5, L1-positive fibres are observed crossing the PSPB in wild-type brains (arrows in I). (F,G,J,K) L1 immunoreactive fibre trajectories follow aberrant pathways and cannot cross the PSPB (arrows) in the Pax6-/-. A cellular mass (asterisk) fills the lateral ventricle (LV). (H,L) Abnormal fasciculation of L1 immunopositive axons along the dorsal cortex of the Pax6-/-. (M,N) At E14.5 wild-type brains, TAG1 immunoreactive CFA extend to the PSPB (arrows). (O,P) TAG1 immunoreactive CFA in the Pax6-/- cortex (arrowheads) do not cross the PSPB. GE, ganglionic eminences; hp, hippocampus. Scale bars: 100 µm.

 


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  		Fig. 6. Morphological defects in the developing telencephalon of the Pax6-/- mice revealed with Nissl staining. (A-C) Nissl-stained coronal section of an E14.5 wild-type brain showing a normal cortical lamination (B) and the morphology of the PSPB (arrowheads in C). (D-F) In Pax6-/- brain, an expanded ventricular zone (vz) and a thinner cortical plate (cp) (arrows in E) was observed. Note the absence of cp in the lateral pallium (arrow in F). ctx, cerebral cortex; LV, lateral ventricle; mz, marginal zone. Scale bars: 200 µm in A,D; 100 µm in B,C,E,F.

 


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  		Fig. 7. Ultrastructure of the PSPB in wild-type and Pax6-/- brains at E15.5 (A-J) and E18.5 (K-U). Electron microscopy was performed in coronal sections of wild-type (E-G,O-Q) and Pax6-/- (H-J,R-U) brains. Boxes on the camera lucida drawings (A,C), in the light microscopy images of resin-embedded sections (K,M) and in the Toluidine Blue-stained semithin sections (B,D,L,N) indicate the areas examined. (E-G,O-Q) At E15.5 and E18.5, wild-type axons (ax) are observed at the level of the intermediate zone (iz) of the cortex (ctx) (Q) and crossing the PSPB (E,O). (F,G) Higher magnifications of wild-type PSPB cells. (P) High-power view showing the ultrastructure of the ventricular zone (vz) in wild-type brains. (H-J,R-U). At both ages, the lateral telencephalon of the Pax6-/- brain was abundant in axon bundles (J,R) and less of them are seen crossing the PSPB (H,S) (compare with E or O). The ultrastructure of the PSPB is characterized by densely packed cells in the Pax6-/- brains (H,S). This ultrastructural abnormality was more evident at E18.5 than at early stages (asterisk in S). (T) Clusters of abnormal cells with loss of organelles on the cytoplasm are observed at the vz in Pax6-/- brains at both ages. str, striatum; dt, dorsal thalamus; ht, hypothalamus. Scale bars: 400 µm in A,C,K,M; 2 µm in E-F,J; 3 µm in H; 1 µm in G,I; 5 µm in O,Q-S,U; 2 µm in P; 1 µm in T.

 


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  		Fig. 8. In situ hybridization for Sema3c (A-C) and Sema5a (D-G) in wild-type (A,D,E), heterozygous (B) and Pax6-/- (C,F,G) forebrains at E14.5. (A,B) In wild-type and heterozygous brains, Sema3c expression is seen within the subventricular (svz) and intermediate zone (iz) of the entire pallium, with a most prominent signal detected within svz/iz of the lateral and ventral pallium (arrows). The signal had sharp ending at the PSPB. (C) In the Pax6-/- forebrain, the prominent expression of Sema3c in the svz/iz of the ventrolateral pallium is not detectable, while the expression in the dorsal pallium is shifted to the outermost surface of the developing cortical plate. (D-G) The normal strong expression of Sema5a in the ventricular zone (vz) of the entire pallium (D,E) is substantially diminished in the neuroepithelium of the Pax6-/- brains (F,G). The arrowhead in G indicates to the strongly affected expression of Sema5a in the vz of the ventrolateral pallium of the Pax6-/- brains, when compared with the expression in wild-type (arrow in E). GE, ganglionic eminences; se, septal eminence. Scale bars: 200 µm in A-C,G,I; 100 µm in D-F,H,J.

 


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  		Fig. 9. Schematic diagram illustrating the relationship between the developing reciprocal TCAs and CFA and ß-galactosidase stained early subdivisions of the embryonic forebrain in Pax6+/- (upper row) and in Pax6-/- (lower row) mice at E14.5 and E18.5. Blue represents the ß-galactosidase staining, which reflects the Pax6 promoter activity. Red dots represent cells with dorsal thalamic projections. In wild-type and heterozygote mice at E14.5, early thalamic and corticofugal fibres synchronously approach the internal capsule. The growing CFAs (dark blue) pause at the border of the Pax6-positive compartment before they continue their journey towards the diencephalon and encounter the growing TCAs (green). By E18.5, both sets of axons have almost reached their destination and the Pax6 expression is gradually reduced in the PSPB and it is limited to the cortical intermediate zone, but continues to be present in the ventral pallium. In the Pax6-/- mice, the cells with dorsal thalamic projection are displaced into the ventral IC and some to hypothalamus. TCAs descend into the hypothalamus and a subset of fibres cross the telencephalic-diencephalic junction at altered ventral or dorsal sites. The majority of the CFAs fail to turn into the internal capsule and continue to descend into the ventral pallium. The two sets of fibres fail to interact at the PSPB.

 

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