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First published online 20 October 2004
doi: 10.1242/dev.01436

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Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation

Jun Hatakeyama¹, Yasumasa Bessho^1,*, Kazuo Katoh², Shigeo Ookawara², Makio Fujioka³, François Guillemot⁴ and Ryoichiro Kageyama^1,

¹ Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
² Department of Anatomy, Jichi Medical School, Tochigi 329-0498, Japan
³ Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan
⁴ National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK

View larger version (112K): [in a new window]   Fig. 1. Hes1 and Hes5 expression in the developing nervous system. In-situ hybridization for Hes1 and Hes5 was performed with mouse embryos at E9.5 or indicated stages. (A-E) Hes1 and Hes5 exhibit mostly complementary expression patterns. Hes1 is expressed in the isthmus (A, arrowhead) and the optic vesicles (C,D, arrows) whereas Hes5 is not (B, arrowhead and E, arrow). (F) In the Hes1-null embryo, Hes5 is expressed in the optic vesicles (arrow). (G-J) Hes1 and Hes5 exhibit complementary expression patterns in the spinal cord. Hes1 and Hes5 expression is expanded in Hes5-null (H, arrows) and Hes1-null spinal cord (J, arrow), respectively. The dorsal is up in G-J. Scale bar: 50 µm in G-J.

View larger version (112K): [in a new window]   Fig. 2. Disorganized structures of the Hes-mutant nervous system. (A-D) Toluidine Blue staining of sections at the level of rhombomere 7 of wild-type (A), Hes1–/– (B), Hes5–/– (C) and Hes1–/–;Hes5–/– (D) at E10.5. Only Hes1–/–;Hes5–/– neural tube is morphologically abnormal. In these panels, dorsal is up. (E-J) SEM analysis was performed with E10.5 mouse embryos. (E,F,I) In the wild type, radial glial cells are aligned radially in the wall of the neural tube. Their endfeet form a smooth inner surface. (G,H,J) In Hes1;Hes5 double mutants, cells are exposed to, and scattered into, the lumen. As a result, the cell arrangement is totally destroyed. In addition, cells become round in shape (J, compare with I). Boxed regions in (E,G) are enlarged in (I,J). (K) Positions of (E-H) are indicated. Scale bars: 100 µm in A-H; 10 µm in I,J.

View larger version (109K): [in a new window]   Fig. 3. Cell death and dorso-ventral patterning of the spinal cord. (A-F) TUNEL assay indicates that cell death (green) is not increased in Hes1–/–Hes5–/– spinal cord (D-F). (G-P) In-situ hybridization analysis of Shh for the floor plate (G,L), Sim1 for V3 interneurons (H,M), Evx1 for V0 interneurons (I,N), Lhx2 for D1A interneurons (J,O), and Wnt1 for the roof plate (K,P) at E10.5. Except for the hypomorphic roof plate, the dorso-ventral patterning is mostly normal in Hes1–/–Hes5–/– spinal cord (L-P). Scale bars: 100 µm.

View larger version (107K): [in a new window]   Fig. 4. Premature loss of radial glia in Hes-mutant spinal cord. Histological analysis was done with the wild-type (A-C,G-I) and Hes1;Hes5 double-mutant (D-F,J-L). The dorsal is up in all panels. At E8.5, the neural plate consists of nestin+ neuroepithelial cells in Hes1;Hes5 double-mutant (D) as in the wild type (A). At E9.5 and E10.5, nestin+RC2+ radial glial cells are present throughout the wall of the spinal cord in the wild type (B,C,H,I). By contrast, in the double mutant, nestin+RC2+ radial glial cells are prematurely lost from the ventral region (E,F,K,L). Scale bars: 100 µm.

View larger version (73K): [in a new window]   Fig. 5. Premature neurogenesis in Hes1;Hes5 double mutants. (A) In the wild type, radial glial cells (Ki67+) are located throughout the wall while neurons (TuJ1+) reside in the outer layer at E10.5. (E) In Hes1;Hes5 double mutants, there are numerous neurons (TuJ1+) while radial glial cells (Ki67+) are significantly decreased. (B-D,F-H) In-situ hybridization analysis shows that expression of Dll1, Mash1 and Math3 is highly upregulated in Hes1;Hes5 double mutants (F-H), compared with the wild type (B-D). Scale bar: 100 µm in A,E.

View larger version (114K): [in a new window]   Fig. 6. Formation of the intercellular junctional complex at the apical side of the neuroepithelium. (A,D) Toluidine Blue staining of the wild-type and Hes1;Hes5 double-mutant neuroepithelium at E8.5. (B,C,E,F) TEM analysis of the wild-type and Hes1;Hes5 double-mutant neuroepithelium at E8.5. The junctional complex (arrowheads) is formed at the apical side of the neuroepithelium in both wild-type (B,C) and Hes1;Hes5 double-mutant embryos (E,F). Kissing points of the tight junction are indicated by arrows (C,F). (G-L) At E8.5, the adherens junction molecule N-cadherin and the tight junction molecules claudin-15 and -18 are expressed at the apical side of the neuroepithelium in both wild-type (G-I) and Hes1;Hes5 double-mutant embryos (J-L). Scale bars: 100 µm in A,D,G-L; 500 nm in B,E; 50 nm in C,F.

View larger version (85K): [in a new window]   Fig. 7. Premature loss of the apical intercellular junctions in Hes1;Hes5 double-mutant spinal cord. (A-D) Sections of the wild type (A,C) and Hes1;Hes5 double mutant (B,D) at E9.5. Cells in the right and left walls of the double mutant are intermingled (D). (E,F) TEM analysis revealed the junctional complex at the apical side of the wild type (E, arrows). By contrast, the junctional complex is already lost in the Hes1;Hes5 double mutant (F, arrowheads). The boxed regions in C,D are enlarged. (G-I) Claudin-10 is expressed at the apical side of radial glia (nestin+) in the wild type. The en face view of the ventricular surface shows a ring-like distribution of claudin-10 (H). (J,K) TEM analysis shows the tight junction (kissing points are indicated by arrows in K) and adherens junction at the apical side in the wild type (J, brackets). (L-N) Claudin-18 is expressed at the apical side of radial glia (Ki67+) while neurons are located in the outer region (TuJ1+). (O-Q) In the double mutant, claudin-18 expression is lost in the ventral region, where radial glial cells are depleted (Q, arrow). This region is occupied by TuJ1+ neurons (P, arrow). Scale bars: 50 µm in A,B; 25 µm in C,D; 500 nm in E; 1 µm in F; 10 µm in G-I; 300 nm in J; 70 nm in K; 100 µm in L-Q.

View larger version (139K): [in a new window]   Fig. 8. Premature loss of the basal lamina in the Hes1;Hes5 double mutant. (A) In the wild type, the spinal cord and dorsal root ganglia (DRG, arrows) are clearly separated. (B) The basal lamina is formed at the outer barrier of the wild-type spinal cord (open arrows). (C) Neurons (TuJ1+) in the spinal cord and DRG (arrows) are clearly separated in the wild type. (D) In the double mutant, neurons (TuJ1+) in the spinal cord and DRG are intermingled (arrow). (E) In the double mutant, the outer boundary of the spinal cord is not clear. Some cells are erupted from the spinal cord (arrowheads). (F) The basal lamina is not formed at the boundary of the double-mutant spinal cord (arrows). (G,H) Neurons (TuJ1+) are erupted from the spinal cord (arrowheads), and some of them are intermingled with the DRG. (I-K) Laminin-1 is expressed at the basal side of radial glia (nestin+) in the spinal cord. (L) Laminin-1 mRNA is expressed in the ventricular zone. (M-O) In the double mutant, laminin-1 expression is lost from the ventral region where radial glia (nestin+) are lacking but is maintained at the dorsal boundary where radial glia still remain. Arrowheads indicate the end points of laminin-1 expression. (P) Expression of laminin-1 mRNA is lost in the ventral region of the double mutant. Scale bars: 100 µm in A,C,E,G,I-P; 100 µm in D,H; 10 µm in B,F.

View larger version (80K): [in a new window]   Fig. 9. Ablation of radial glia by diphtheria toxin (DT) disrupts the inner and outer barriers of the developing brain. (A-L) Each vector was introduced into the telencephalic cells by electroporation at E13, and the sections were examined at E15. When pCL-GFP was introduced (A-C), both cells in the ventricular zone (nestin+) and the outer layers were found to express GFP. No dying cells (TUNEL+) were detected (C). When pNes-DT-A was introduced (E-G), many cells underwent apoptosis in the ventricular zone (TUNEL+, G). When pNes-GFP was introduced, only nestin+ ventricular cells (I,J), but not NeuN+ neurons (K), expressed GFP. The schematic structures of the vectors are shown in D,H,L. (M-Z) Each vector was introduced into the telencephalic cells by electroporation at E13, and the sections were examined at E18. When pCL-GFP was introduced (M-S), GFP was expressed in both radial glia and neurons (O) and did not affect radial glia (nestin+, P), the apical junction (ZO-1+, Q, arrowheads), the basal lamina (Laminin 1+, N, arrowheads) or neurons (NeuN+, R). Cell arrangement was not disturbed (DAPI, S). The indicated region in M is enlarged in O-S. When pNes-DT-A was introduced (T-Z), GFP+ cells were almost undetectable (T,T'). Only a small number of GFP+ neurons is detectable, which shows the electroporated region (T,T'). pNes-DT-A specifically killed nestin+ radial glia, which disappeared from the electroporated region (V,V', arrowheads). This region lost ZO-1 expression at the apical side (W, arrowheads, compare with the normal expression indicated by arrows). The electroporated region also lost laminin 1 at the basal side (X, between the two arrowheads). In this region, the cortical lamination is destroyed with rosette-like structures, and some neurons protrude into the outer region and the lumen (Y,Y',Z, arrowheads). The indicated region in (T) and (U) is enlarged in (T') and (V',W,Y',Z), respectively. Scale bars: 100 µm in A-C,E-G,I-K,N S,T',V',W,Y',Z; 200 µm M,T-V,X,Y.

View larger version (112K): [in a new window]   Fig. 10. Lack of the optic vesicles and the ganglionic eminences in Hes1;Hes5 double-mutant brain. (A,B) SEM analysis shows the optic vesicle (arrow) and the ganglionic eminence (arrowhead) in the wild type (A) while such structures are not formed in the Hes1;Hes5 double-mutant brain (B). In some regions, cell arrangement is disrupted (B, arrows). (C-G) Immunohistochemistry for TuJ1. Neurons (TuJ1+, green) are not generated in the wild type optic vesicle at E9.5 (C) and E10.5 (E,F). By contrast, in Hes1;Hes5 double-mutants, neurons are already differentiated at E9.5 (D) and E10.5 (G) in the region that should normally become optic vesicles. The nuclei are counterstained with PI (red). (H,I) HE staining of the wild type (H) and Hes1;Hes5 double mutant (I). In the double mutant, the eye is not formed (I, arrow). (J) Rx is expressed similarly in both the wild type and Hes1;Hes5 double-mutant. (K-N) Pax2 and Chx10 are expressed in the wild-type eye (K,M) but not in the double-mutant (L,N). Scale bars: 500 µm in A,B; 100 µm in C-E,G; 200 µm in H,I.

View larger version (76K): [in a new window]   Fig. 11. Premature loss of neuroepithelial and radial glial cells in Hes1;Hes3;Hes5 triple mutants. (A-D) At E8.0, like Hes1 (A), Hes3 is expressed in the nervous system (B) whereas Hes5 and Dll1 are not (C,D). (E-H) At E8.5, while Hes1 is still diffusely expressed (E), Hes3 expression domain is restricted to the medial to dorsal region (F). Hes5 and Dll1 expression is observed at this stage (G,H). (I,J) Hes3 is widely expressed in the developing nervous system at E8.0 (I) and E8.5 (J). The head region is indicated by arrowheads. (K-M) Hes3 expression is not significantly affected in the Hes1;Hes5 double mutant, compared with the wild type. (N-R) At E8.5, very few neurons (TuJ1+) are generated in both the wild type and Hes1;Hes5 double mutant (N,O) whereas in Hes1;Hes3;Hes5 triple-mutant, many neurons are prematurely differentiated (P). There are still many neuroepithelial cells (nestin+Ki67+) in the Hes1;Hes5 double mutant (Q,R). (S) At E9.5, more neurons are prematurely differentiated in the ventral region of Hes1;Hes3;Hes5 triple-mutant spinal cord. (T-V) At E10.0, the structure of the spinal cord is severely disorganized in Hes1;Hes3;Hes5 triple mutants (T). Strikingly, virtually all cells become neurons (U), and radial glia are missing (V). Scale bars: 100 µm in A-H,N-V.

View larger version (49K): [in a new window]   Fig. 12. Characters and functions of neural stem cells. (A) Embryonic neural stem cells change their characters in the following order: Hes-independent neuroepithelial cells, transitory Hes-dependent neuroepithelial cells, and Hes-dependent radial glial cells. Based on the expression patterns, transitory Hes-dependent neuroepithelial cells seem to depend on Hes1 and Hes3, while Hes-dependent radial glial cells mostly depend on Hes1 and Hes5. (B) Roles of radial glia in the inner and outer barrier formation. (a) Radial glia form the tight and adherens junctions at the apical side and contribute to the basal lamina formation at the basal side. (b) Radial glia establish the framework of the neural tube by forming the inner and outer barriers. In the absence of radial glia (Hes1–/–;Hes5–/–), neurons escape from the wall of the neural tube, thereby destroying the structural integrity.

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