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doi: 10.1242/10.1242/dev.00232

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The role of Grg4 in tectal laminar formation

Sayaka Sugiyama* and Harukazu Nakamura{dagger}

Department of Molecular Neurobiology, Institute of Development, Aging and Cancer, and Graduate School of Life Sciences, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan
* Present address: Laboratory for Neuronal Circuit Development, Brain Science Institute, RIKEN, 351-0198 Japan



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  		Fig. 1. Laminar formation of the tectum. (A) Laminar formation by early and late migratory cells. (B) Two hours after electroporation with lacZ vector at E5. (Upper panel) Immunohistochemistry with anti-lacZ. (Lower panel) Immunohistochemistry with anti-Hu-C/D. Hu-positive early neurons accumulate in the upper layer of the ventricular layer before migrating into their destination, so this layer could be distinguished as the early neuronal layer (*). lacZ staining is observed in the neuroepithelial layer, not in the early neuronal layer at 2 hours after electroporation. NE, neuroepithelial layer. (C) Horizontal section of an E6 tectum that was labeled at E3. DAPI staining (left panel) reveals laminae I and II and the ventricular layer. In the ventricular layer, the early neuronal layer could be distinguished (*). Cells labeled at E3 migrate out of the neuroepithelium and make up lamina II and I by E6. (D,H) DAPI staining of E8 (D) and E12 (H) tecta to show laminar formation. (E-G) Distribution of lacZ-positive cells at E8. Electroporation with pMiwZ was carried out at E3 (E), E6 (F) and E5 (G). Left panel shows lacZ staining, and right panel shows the number of lacZ-positive cells in each lamina. (I,J) Distribution of lacZ-positive cells at E12. Electroporation with pMiwZ was carried out at E3 (I) and E6 (J). Left panel shows lacZ staining, and right panel shows the number of lacZ-positive cells in each lamina. As the cells labeled at E6 migrated into lamina IId (F), these cells (late migratory cells) may have divided the laminae that had been formed by early migratory cells (C). Thus, the laminae of early migratory cells are split into two (E): upper laminae (laminae IV-IIu) and deeper lamina (lamina I). Distribution of late migratory cells at E8 and E12 indicates that lamina IId at E8 is remodeled to laminae h and i/j of the SGFS (F,J). E5 is the transitory stage and the ventricular layer may be composed of a mixture of the progenitors of early and late migratory cells. Thus, the cells labeled at E5 showed ubiquitous distribution (G). Scale bars: 100 µm.

 


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  		Fig. 2. Expression of Grg1, Grg4 and En2 in developing chick tecta. (A) Approximate position of the figures. (B-I) In situ hybridization for Grg4: (B) E4 (stage 22), (C,D) late E5 (stage 28, C at the caudal and D at the rostral), (E,F) E6 (stage 29, E at the caudal and F at the middle, E shows immunohistochemistry with anti-Hu-C/D in red and in situ hybridization for Grg4 in green), (G) late E6 (stage 30), (H) E8 (stage 34) and (I) E14 (stage 40). I' is higher magnification of I. (J-M) In situ hybridization for Grg1: (J) E4 (stage 22), (K) late E6 (stage 30), (L) E8 (stage 34), (M) E14 (stage 40). (N,O) Immunohistochemistry with anti-En2 antibody at late E5 (stage 28; N, caudal; O, rostral). Grg4 is not expressed at late E3 (B). Its expression resumes at late E5 in a gradient along rostrocaudal axis; rostral high (D) and caudal low (C). Grg4 is expressed in the neuroepithelium (NE) and in the Hu-C/D-positive early neuronal layer (*). At late E6.5, Grg4 expression is observed in laminae II and I (F,G). Expression in the ventricular layer (VL) is still observed at stage 40 (I). Grg1 is continuously expressed in the ventricular layer to E14 (J-M). En2 is expressed at late E5 in an opposite gradient to Grg4: caudal high (N) and rostral low (O). Scale bars: 100 µm.

 


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  		Fig. 3. Effects of Grg4 misexpression on tectal laminar formation. (A-G) Tecta at E12. (A) Low-power micrograph of an RCAS-AP-misexpressing tectum, which served as the control. Immunohistochemistry is with anti-gag antibody. The transfection sites are indicated by brackets. (B,B') RCAS-Grg4-misexpressing tectum. In situ hybridization for Grg4 (B) and DAPI staining (B'). (C,D) DAPI staining of the control (C) and Grg4-misexpressing (D) tecta. (E) Higher magnification of Grg4 expressing site in E. (B,B',D,E,G) are from adjacent sections of the same tectum. On B and B', the site that corresponds to D and E is indicated by arrows. (F,G) Immunohistochemistry with anti-neurofilament antibody on the control (F) and Grg4-misexpressing tecta (G). (H-T) E14 tecta. Immunohistochemistry with anti-PV (H,I), anti-Glutamate (K,L) and anti-NgCAM antibodies (N,O). In situ hybridization for ER81 (Q,R) and for Lim1 (S,T). Merged figures of Grg4 and ER81 (R') and Grg4 and Lim1 (T') at the site indicated by brackets on R and T, respectively. Grg4 misexpression was assessed immunohistochemically with antibody against the HA tag (J,M,P,R',T'). Control (H,K,N,Q,S) and Grg4-misexpressing site (I,J,L,M,O,P,R,T). L and M are the same field of the same section. Grg4 expressing site is indicated in L. In Grg4-misexpressing regions, lamina g seemed to be reduced (D), but lamina i/j was increased (D,G). Arrowheads in G indicate radial neuronal fibers. Expression of PV (H) indicates that lamina g is specifically disrupted by Grg4 misexpression (I). Cells that express glutamate in SGC and SAC are reduced at the Grg4-misexxpressing site (right hand side of L, where Grg4 misexpressing site is indicated as Grg4). ER81 and Lim1, which are normally expressed in lamina i/j, are expressed in deeper layers in the Grg4-misexpressing region (Q-T). Scale bars: 100 µm for A-T; 20 µm for G'.

 


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  		Fig. 4. Clonal analysis of the migration of Grg4-expressing cells. (A) Electroporation with RCAS-AP on virus-resistant embryos at E2 that were fixed at E8. (Left column) AP immunocytochemistry. (Right column) Number of AP-positive cells in each lamina. (B) Electroporation with RCAS-Grg4 on virus-resistant embryos at E2 that were fixed at E8. (Left column) In situ hybridization for Grg4. (Right column) Number of Grg4-expressing cells in each lamina. In virus-resistant embryos, misexpression is limited to the descendents of originally transfected cells. AP-positive cells are present throughout all laminae (A), but Grg4-expressing cells are found mainly in lamina IId (B). Scale bars: 100 µm.

 


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  		Fig. 5. Grg4 instructs the tectal postmitotic cells to take a late migratory pathway. (A) Control E8 tecta in which RCAS-AP was electroporated at E2. (B) Grg4 misexpression by RCAS-Grg4. (C) Application of morpholino antisense oligonucleotide for Grg4 at E5. (D) Grg4-5' misexpression by RCAS-Grg4-5'. (E) En2 misexpression by RCAS-En2 infection. (F) Relative cell numbers of Sox2- and Sox14-positive cells. In A-E: (a) DAPI staining, (b) in situ hybridization for Sox2, (c) in situ hybridization for Sox14, (d) in situ hybridization for ER81. (A, part e; B, part e) Immunostaining for PV. (B, part f) Low-power micrograph to show Grg4 misexpression. (C, part e) immunostaining for FITC to show FITC-conjugated morpholino. (D, part e) Immunostaining for HA Tag. Immunostaining for En2. In the control (A), Sox2 is expressed in lamina IIu and the ventricular layer (VL). Sox14 is expressed in laminae IV and IId. ER81 is expressed specifically in lamina IId. PV is expressed in laminae III-II at E8. In the Grg4-misexpressing region, reduction of lamina I, IIu and IV is assessed from the expression of Sox2, PV and Sox14 (B, part a-c; B, part e). Treatment with morpholino antisense oligonucleotide for Grg4 exerted reverse effects: expansion of lamina IIu (C, part b), reduction of lamina IId (C, parts c,d). Grg4-5' acted as a dominant-negative form for Grg4; expansion of lamina I and IIu (D, part b), reduction of lamina IId (D, parts c,d). En2 misexpression also resulted in the reverse effects to Grg4: expansion of lamina IIu (E, part b), reduction of lamina IId (E, parts c,d). For quantification, percent of Sox2- and Sox14-positive cells around the lamina II in total cells of mantle layer was calculated (F). The number of cells in a rectangle, one side of which is 100 µm and parallel to the ventricular surface, was counted on four samples from two embryos each. The graph shows that Grg4 forces tectal postmitotic cells to follow a late migratory pathway. Error bars represent s.e.m. Asterisks indicate significant differences from the control (single asterisk, P<0.05; double asterisks, P<0.005). AP, control AP-transfected tecta; Grg4, Grg4 misexpressing tecta; Mor, tecta treated with morpholino antisense oligonucleotide. Scale bars: 100 µm.

 


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  		Fig. 6. Grg1 restricts cells to the neuroepithelial layer. (A) DAPI staining (a), and in situ hybridization for Sox2 (b), Sox14 (c) and ER81 (d) in Grg1-misexpressing E8 tecta visualized using by RCAS-Grg1. (B-D) In situ hybridization for Cash1 in E8 tecta that were transfected with RCAS-AP (B), RCAS-Grg1 (C) or RCAS-Grg4 (D). (E) Immunostaining for Grg4-HA in green and Hu-C/D in red. (F,G) Immunostaining for HA in green and BrdU in red in E8 tecta that were transfected with RCAS-AP (F) or RCAS-Grg4 (G). Cash1 is expressed in the ventricular layer (VL, B). In the Grg1-misexpressing region, Cash1 expression is repressed (C) so that all laminae (IV-I) are incompletely formed on E8 tecta (A). In the Grg4-misexpressing region, expansion of the ventricular layer by postmitotic neuronal precursors is observed (D,E,G). Scale bars: 100 µm for A-E; 50 µm for F,G.

 


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  		Fig. 7. Disruption of retinotectal projection by Grg4. (A,B) Terminal arborizations in the control (A) and Grg4-misexpressing region (B). HRP was injected in the contralateral eye at E15, the tectum was fixed at E17, and fibers were visualized with anti-HRP antibody on the slice. (C) Grg4 misexpression was assessed by immunohistochemical staining with anti-gag antibody on the same slice as B. In the control tectum, retinal fibers arborize superficial to lamina g (A), but in the Grg4-misexpressing tectum, retinal axons pass deeper beyond lamina g (arrowheads in B). The rectangle on C indicates approximate area of B. (D,E,E') Immunostaining with anti-neurofilament (D,E) and DAPI staining (E') on the sagittal section of the control (D) and Grg4-misexpresing region (E) on E12 tecta.

 




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