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Role of Pax3/7 in the tectum regionalization

Eiji Matsunaga1, Isato Araki1,* and Harukazu Nakamura1,2,{ddagger}

1 Department of Molecular Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan
2 Graduate School of Life Sciences, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan
* Present address: Max-Planck-Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr 108, D-01307 Dresden, Germany



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  		Fig. 1. Normal expression patterns of Pax3 and Pax7 in the prosencephalon and the mesencephalon. Whole-mount in situ hybridization for Pax3 (A-C) and Pax7 (D-F) at 10-somite stage (A,D), 18-somite stage (HH13) (B,E) and E2.5 (HH17) (C,F). Pax3 and Pax7 are expressed near the dorsal midline of the neural tube and in the migrating neural crest cells in the mesencephalic region at stage 10 (A,D). Expression of Pax3 and Pax7 commences around stage 13 in the alar plate of the mesencephalon (B,E). In the alar plate, Pax3 and Pax7 are expressed posterior to p1/p2 boundary at stage 17. Expression around the roof plate extended rostrally beyond the p1/p2 boundary (C,F). Pax7 is also expressed in anterior midline of the telencephalon, but Pax3 is not expressed there. Scale bars: 500 µm in F; 200 µm in D,E. di, diencephalon; mes, mesencephalon; met, metencephalon; p1, prosomere 1; p2, prosomere 2; pro, prosencephalon; rho, rhombencephalon; tel, telencephalon.

 


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  		Fig. 2. Pax3/7 misexpression changed the fate of the alar plate of the diencephalic P2 region to that of the mesencephalon. (A,B) Morphological change at 48 hours after electroporation of Pax7. Horizontal section. Hematoxylin and Eosin staining (A). X-gal staining of an embryo at 48 hours after co-electroporation of Pax7 and lacZ expression vector (B). White arrowheads show the ectopic swelling in the diencephalon. Note that the ectopic swelling is formed in the p2 region, although expression vectors were uniformly transfected from the telencephalon to the mesencephalon. (C-F) Morphological change at 5 days after electroporation of Pax3. View from the experimental side (C), dorsal view (D). Horizontal section at the plane indicated in C. Hematoxylin and Eosin staining (E). (F) A high power micrograph of boxed area in (E). Arrowheads on E indicate the ectopic swelling in the diencephalon. Note that ectopic swelling (ect. tec) has the laminar structure similar to the tectum proper (tec). Differentiation of laminar architecture in the ectopic swelling is behind of tectum proper. (G-J) In situ hybridization for Lim1 to show that pretectum is retained after Pax7 misexpression. View from the experimental side (G), view from the control side (H) and dorsal view (I) of an E4.0 embryo. (J) Horizontal section at the plane indicated in G. The control side is printed in reverse for the comparison. Lim1 expression in the pretectum is detected (between arrowheads on panel J). (K-R) Retinal fiber trajectory and histology of E13.5 embryos after Pax7 misexpression. Schematic drawing of retinal fiber trajectory (K), and lateral view (L). Horseradish peroxidase was injected into the left eye at E12.5 and the embryo was sacrificed at 24 hours later (E13.5, 12 days after Pax7 misexpression). Most of retinal fibers innervate the ectopic swelling (arrow), and some of them innervate the tectum proper (arrowhead). After observation of retinal fiber trajectory, the specimen was embedded in Technovit, and sagittal sections were stained with Hematoxylin and Eosin. Approximate planes of M,N are indicated in K. Panels P-R are higher magnifications indicated on M,N. The tectum proper (O) does not contain stratum opticum (SO) as is inferred from L, which may resulted in maldifferentiation of superficial layers (layer x is barely discernible). The ectopic tectum (P) contains SO, and layers vi, vii, viii, ix are discernible, though its differentiation is behind the normal tectum. Torus semicircularis at the proper site and ectopically differentiated in the diencephalon are shown in Q,R, respectively. We could identify pretectal nuclei (nucleus spiriformis lateralis (Spl) and medialis (Spm), and nucleus principalis precommissuralis (Ppc)). We adopted the criterion of the tectum layers of LaVail and Cowan (LaVail and Cowan, 1971). Scale bars: 2.0 mm in L; 1.0 mm in D,H,I; 500 µm in A,B,E,J,M,N; 200 µm in F,R; 100 µm in O. cont., control side; cer, cerebellum; exp., experimental side; tec, tectum; ect. tec, ectopic tectum; ts, torus semicircularis; ect. ts, ectopically differentiated torus semicircularis.

 


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  		Fig. 3. Effects of Pax7 on expression of tectum related genes. (A-H) Cell autonomous repression of Pax6 by Pax7. In situ hybridization for Pax6 (blue), the control (A), and the experimental embryos (C) at 24 hours after electroporation. Immunostaining for Pax7 (brown) was also carried out on the same embryo (B). Repression of Pax6 is rarely detected in the p1 region (C). (D-H) Double immunocytochemical staining for Pax6 (green) and Pax7 (red) 24 hours after Pax7 electroporation. (F,G) High power magnifications of boxed areas in D,E, respectively. (H) A combined image. Pax6 is repressed in the cells that express Pax7 (arrowheads, F-H), indicating that Pax6 is repressed by Pax7 in a cell autonomously. (I-N) Induction of Fgf8 by Pax7. In situ hybridization for Fgf8 (blue, I,K), and immunostaining for Pax7 (brown) was added on the same embryo (J) at 24 hours after electroporation. (L,M) Higher power micrographs of the area indicated in J,K, respectively. (N) A micrograph of the same field as M, showing only Pax7 expression. This figure was taken after staining for Fgf8 was washed away. These figures indicate that induction of Fgf8 expression by Pax7 is in a cell autonomous manner. The arrows in L-N indicate the same position. (O-S) En2 induction via Fgf8 after Pax7 misexpression. In situ hybridization for Fgf8 (blue, O,P), and immunostaining for En2 (brown) on the same embryo (Q) at 48 hours after electroporation. (R,S) High power micrographs of boxed area in P,Q, respectively. The arrows in R,S indicate ectopic expression of Fgf8. Arrowheads in S indicate ectopic expression of En2. En2 induction was always detected around Fgf8-expressing cells. (T-X) En2 induction after Pax3 misexpression. In situ hybridization for Pax3 (blue, T,U), and immunostaining for En2 (brown) on the same embryo (V). (W,X) High power micrograph of boxed area in U,V. En2 induction was detected adjacent to the Pax3-misexpressing cells, which indicate that En2 was indicated in a same manner as after Pax7 misexpression. A,I,O,T are printed in reverse for the comparison. Scale bars: 500 µm in C,K,Q,V; 200 µm in E; 25 µm in H,N,S,X.

 


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  		Fig. 4. Interrelationship between Pax3 and Pax7. (A-I) In situ hybridization for Pax3 (blue) (A,C,E,F,H,I) and in situ hybridization for Pax3 and immunohistochemistry for Pax7 (brown) (B,D,G) 24 hours (A-E) and 48 hours (F-I) after electroporation. View from the control side (A,F), view from the experimental side (B,C,G,H), dorsal view (I). The control side is printed in reverse (A,F). High power magnification of boxed area in B,C (D,E). Corresponding arrow in D,E indicates the same position. Arrows in G indicate exogenous expression of Pax7. Arrows in I indicate repression of endogenous Pax3 expression by Pax7. Arrowheads in I indicate the line corresponding to p1/p2 boundary. (J-Q) Serial sections of the embryo at 96 hours after electroporation. (J-M) An embryo in which the swelling in the diencephalon is small; (N-Q) an embryo in which the swelling had enlarged. (J,N) In situ hybridization for Pax3. (L,P,Q) Double staining by immunohistochemistry for Pax6 (green) and for En2 (L,P, red), for Pax7 (Q, red). (M) Immunohistochemistry for HA-tag (green) and for Pax7 (red). (K,O) High power magnifications indicated in J,N. Areas surrounded by arrowheads on panels K and O indicate ectopic swelling, where Pax3 expression was induced. Arrows in K-M,O-Q indicate the approximate position of the boundary between the ectopic swelling and the pretectum. Exogenous Pax7 expression which is identified by HA-tag had disappeared at 96 hours after electroporation (M). Green cells in M are mesenchymal cells (stained for HA). En2 is expressed in almost entire region of the ectopic swelling with rostral-high and caudal-low level when ectopic swelling is small (L). En2 expression is almost absent by the time the ectopic swelling enlarged, leaving at the rostral tip of the swelling (P). Pax7 was induced in the ectopic swelling, making a sharp boundary with Pax6 (Q). This boundary is consistent with the Pax6/En2 boundary (P). Scale bars: 500 µm in H,I,J,N; 250 µm in C; 200 µm in M,Q; 50 µm in E. ptc, pretectum.

 


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  		Fig. 5. Pax3/7 misexpression caused fate change of the tegmentum to the tectum. (A-C,D-F) Morphology after Pax7 and Pax3 misexpression at E6.5 and E7.5, respectively. View from the control side (A,D), and view from the experimental side (B,E). A,D are printed in reverse for comparison. (C,F) Horizontal section (the plane is indicated in A,D) stained with Hematoxylin and Eosin. Arrows indicate the boundary between the tectum and tegmentum. The arrowhead indicates the ventral midline. The figures indicate that the tectal structure expanded ventrally after Pax7 and Pax3 misexpression. (G-I) Pax3 induction in the ventral mesencephalon after Pax7 misexpression. In situ hybridization for Pax3 (H), and addition of immunohistochemical staining for Pax 7 (I) on an E2.5 embryo at 24 hours after electroporation. View from the control side, printed in reverse (G). View from the experimental side (H,I). (H',I') High power micrographs of boxed areas in H,I, respectively. The arrow in H', I' indicates the same position. (K-N) Ventral view of E3.5 embryos after Pax7 misexpression. In situ hybridization for Pax3 (blue, K) and addition of immunohistochemical staining for Pax7 (brown, L). Pax3 is induced in the tegmentum region (K,L). Arrows in K,L indicate oculomotor nerve roots. Note that the distance from the ventral midline to the oculomotor nerve root is the same on the control and the experimental side, but the distance from the ventral midline to the ventral limit of the Pax3-expressing domain is narrower on the experimental side than on the experimental side (K,L). (M,N) In situ hybridization for Lim1 (blue) and addition of immunohistochemical staining for Pax7 (brown) on an E4.0 embryo, viewed ventrally, at 60 hours after electroporation (M). Lateral band of Lim1 expression in the tegmentum has disappeared on the experimental side (M). In situ hybridization for Pax6 (blue) and immunohistochemical staining for Pax7 (brown) after Pax7 misexpression (N). Pax6 expression arch was also repressed after Pax7 misexpression (N). In K-N, right-hand side is the control side. Scale bars: 1.0 mm in A,D; 500 µm in C,F,L-N); 200 µm in G; 50 µm in H'. di, diencephalon; L, lateral stripe of Lim1 expression; M, medial stripe of Lim1 expression; met, metencephalon; tgm, tegmentum.

 

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