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First published online 25 May 2006
doi: 10.1242/dev.02403

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Persistent and high levels of Hes1 expression regulate boundary formation in the developing central nervous system

Joung Hee Baek, Jun Hatakeyama^*, Susumu Sakamoto, Toshiyuki Ohtsuka and Ryoichiro Kageyama

Institute for Virus Research, Kyoto University, Shogoin-Kawahara, Sakyo-ku, Kyoto 606-8507, Japan.

View larger version (73K): [in a new window]   Fig. 1. High levels of Hes1 expression in boundaries. (A) Whole-mount in situ hybridization at E10.5. Hes1 mRNA is expressed at high levels in the ZLI (left arrow) and the isthmus (right arrow). Left and right lines indicate sections shown in (H-O) and (B,C), respectively. (B) Immunohistochemistry for Hes1. Hes1 protein is expressed at high levels in the isthmus (boxed) and the floor plate (asterisk). (C) A higher magnification of a boxed region in B. Inset in C shows Hes1 protein expression in the isthmus at E9.5. (D-G) Serial parasagittal sections of the isthmic region at E11.5 (boxed in A). Wnt1-expressing cells (D, arrow) also express Hes1 (E, arrow) at high levels in the isthmus. These cells do not efficiently take up BrdU (F,G, arrows), compared with the surrounding region. (H-K) Hes1 protein is expressed at high levels in the ZLI (H, asterisk). Cells expressing Hes1 (I, bracket) also express Shh (J,K, brackets) in the ZLI. Inset in J shows a lower magnification of in situ hybridization for Shh. (L-O) Hes1 protein (L) and Hes1 mRNA (M) are highly expressed in the ZLI (asterisks). Cells in the ZLI display lower BrdU uptake (N,O, asterisks). (P) Flat-mount in situ hybridization of Hes1 at E9.5. The midbrain-hindbrain region was cut along the dorsal midline. Hes1 mRNA is expressed in the interrhombomeric boundaries. Arrowheads indicate the isthmus. Top is rostral. (Q-V) Frontal section of the hindbrain at E9.5. Cells at interrhombomeric boundaries express Hes1 protein at high levels. They are mostly negative for phosphorylated histone 3 (pH3) (U,V, arrows), indicating that interrhombomeric boundary cells do not actively proliferate. The boxed region in Q is enlarged in the insets in Q-S. PI, propidium iodide. Scale bars: 500 µm in O; 200 µm in V; 100 µm in C,G; 50 µm in K.

View larger version (51K): [in a new window]   Fig. 2. Hes1 expression in the spinal cord. (A-C) Double immunostaining of Hes1 and Ki67 at E10.5. Hes1-expressing cells in non-boundary regions are mostly positive for Ki67 (C, bracket). In contrast, Hes1-expressing cells in the roof plate and the floor plate are negative for Ki67 (C, arrowheads). (D-G) BrdU uptake in the spinal cord at E11.5. BrdU-positive cells are present in non-boundary regions but very rare in the roof plate (E, upper box, and F) or the floor plate (E, lower box, and G). Boxed regions in E are enlarged in F and G. (H-J) Hes1 protein expression in the roof plate at E10.5. Inset in H shows Wnt1 expression in the roof plate. (K-M) Hes1 protein expression in non-boundary regions at E10.5. (N-P) Hes1 protein expression in the ventral spinal cord including the floor plate at E10.5. Inset in N shows Shh expression in the floor plate. In non-boundary regions just dorsal to the floor plate, Hes1 protein is expressed at variable levels (P, bracket). (Q) Hes1 protein expression (top). (Bottom) Relative intensity of Hes1 signal was measured (see Materials and methods): error bars represent s.e.m. (a total of 108 roof plate cells, 109 floor plate cells and 1,106 non-boundary cells in six different sections were examined). Scale bars: 100 µm (C-E), 50 µm (F, G and P).

View larger version (115K): [in a new window]   Fig. 3. Persistent Hes1 expression in the roof plate and the floor plate. (A-X) In situ hybridization for Wnt1 and Shh, Hes1 immunostaining and PI staining were performed with embryos at E9.5, E11.5 and E13.5. Cells in the roof plate and the floor plate persistently express Hes1 protein at high levels during this period. Scale bar: 50 µm.

View larger version (103K): [in a new window]   Fig. 4. Inverse correlation between Hes1 and Mash1 expression levels in the spinal cord. (A-D) Double immunostaining for Hes1 and Mash1 at E10.5. Boxed region in C is enlarged in D. (E-G) A higher magnification of a boxed region in D. Cells expressing Hes1 at a high level (green) do not express Mash1 whereas a cell expressing Mash1 at a high level (red) does not express Hes1. Cells expressing Hes1 at an intermediate level express Mash1 at an intermediate level. (H) The results in E-G are shown schematically. Green and red cells are Hes1 positive only and Mash1 positive only, respectively, whereas striped cells are Hes1;Mash1 double-positive. Scale bars: 100 µm (C), 50 µm (D).

View larger version (52K): [in a new window]   Fig. 5. Boundary defects in the developing nervous system. (A-H) SEM analysis of wild-type (A,B,E,F) and Hes1;Hes5 double-null (C,D,G,H) embryos. Parts of A and C are enlarged in B and D, respectively. At E10.5, the ZLI (arrows) and the isthmus (arrowhead) are clearly formed in the wild type (A,B). By contrast, in Hes1;Hes5 double-null embryo (C,D), the ZLI is lacking (asterisks) and the isthmus is only partially formed (arrowheads). The interrhombomeric boundaries are clearly formed in the wild type at E9.5 (E, arrowheads) and E10.5 (F, arrowheads). They are not significantly affected at E9.5 (G, bracket) but become ambiguous at E10.5 (H, bracket) in Hes1;Hes5 double-null embryos. (I,J) In situ hybridization for Shh at E10.5. Shh is expressed in the wild-type ZLI (I, arrowhead) but not in Hes1;Hes5 double-null ZLI (J, asterisk). (K-N) In situ hybridization for Lfng at E9.5 (K,L) and Pax6 at E10.5 (M,N). In the wild-type ZLI (K,M), Lfng and Pax6 are not expressed (arrowheads). In contrast, in Hes1;Hes5 double-null embryos (L,N), the Lfng- and Pax6-negative regions are lacking (asterisks). (O-Cc) In situ hybridization for Wnt1 (O-Q), Fgf8 (R-T), En1 (U-W), Otx2 (X-Z) and Gbx2 (Aa-Cc) at E9.5 and E8.5 (insets of X-Cc). Wnt1, Fgf8 and En1 are normally expressed in the isthmus of Hes3;Hes5 double-null embryos (O,R,U, arrowheads). This expression is downregulated in Hes1;Hes5 double-null embryos (P,S,V, arrowheads) and more severely impaired in Hes1;Hes3;Hes5 triple-null embryos (Q,T,W, asterisks). In Hes3;Hes5 double-null embryos, Otx2 is expressed rostrally whereas Gbx2 is expressed caudally to the isthmus (X,Aa, arrowheads). The Otx2 and Gbx2 expression domains are less clearly separated in Hes1;Hes5 double-null embryos at E9.5 (Y,Bb, asterisks), and their borders become even more ambiguous in Hes1;Hes3;Hes5 triple-null embryos at E8.5 and E9.5 (Z,Cc, asterisks).

View larger version (62K): [in a new window]   Fig. 6. Ectopic neurogenesis in Hes-mutant boundaries. (A-I) Whole-mount in situ hybridization at E9.5. Mash1, Ngn2 and Math1 are not expressed in the ZLI or the isthmus of Hes3;Hes5 double-null embryos (A,D,G, arrowheads). In Hes1;Hes5 double-null embryos, Mash1 and Ngn2 are ectopically expressed in the ventral regions of the ZLI (B,E, asterisks) and the isthmus whereas Math1 expression is extended rostrally into the midbrain without interruption (H, arrow). In Hes1;Hes3;Hes5 triple-null embryos, ectopic expression of Mash1 and Ngn2 in the ZLI (C,F, asterisks) and the isthmus (C,F, arrowheads) is further enhanced. Ectopic Math1 expression in the midbrain is also enhanced (I, arrow). Note that embryos in H,I have an open brain with open isthmus (arrowheads). (J-L) Dll1 is not expressed in the ZLI or the isthmus of Hes3;Hes5 double-null embryos at E9.5 (J, arrowheads). Dll1 is ectopically expressed in the ZLI and the isthmus of Hes1;Hes5 double-null (K, asterisks) and Hes1;Hes3;Hes5 triple-null embryos (L, asterisks). (M-O) Flat-mount in situ hybridization of Ngn2 in the hindbrain. The hindbrain was cut along the ventral midline. Top is rostral. Ngn2 is not expressed in the interrhombomeric boundaries of Hes3;Hes5 double-null embryos (M, arrowheads). The interrhombomeric Ngn2-negative regions become obscure in Hes1;Hes5 double-null embryos (N, arrowheads) and are lacking in Hes1;Hes3;Hes5 triple-null embryos (O, bracket). (P-X) In situ hybridization of the spinal cord at E9.5. Math1 is ectopically expressed in the roof plate of Hes1;Hes3;Hes5 triple-null embryos (R, asterisk) but not of Hes3;Hes5 and Hes1;Hes5 double-null embryos (P,Q, arrowheads). Wnt1 expression is almost lost in the roof plate of Hes1;Hes3;Hes5 triple-null embryos (U). Ngn2 is ectopically expressed in the floor plate of Hes1;Hes3;Hes5 triple-null embryos (X, asterisk). (Y-Aa') Immunostaining (Y-Aa) and in situ hybridization (Y'-Aa') for Shh at E9.5. Shh is normally expressed in the floor plate (arrowheads) and the notochord (N) in Hes3;Hes5 double-null embryos (Y,Y'). Shh expression in the floor plate is downregulated in Hes1;Hes5 double-null embryos (Z,Z', arrowheads) and mostly lost in Hes1;Hes3;Hes5 triple-null embryos (Aa,Aa', asterisks) whereas that in the notochord (N) is not affected. Scale bars: 100 µm.

View larger version (51K): [in a new window]   Fig. 7. Reduction of cell proliferation and neurogenesis by persistent Hes1 expression. (A) The schematic structures of recombinant retroviruses. (B-G) Immunostaining for GFP. E11.5 telencephalic neural progenitors infected with CLIG or CLIG-Hes1 were examined at different time points. (H) Clonal sizes (with s.e.m.) of the virus-infected cells. Clonal sizes of CLIG-Hes1-infected cells are smaller than those of CLIG-infected cells. Experiments were repeated at least six times at each time point. (I) Ratios (with s.e.m.) of TUNEL-positive cells in the virus-infected cells at 72 hours after infection (at least 2,000 cells in three independent experiments were examined). Although there is a tendency for cell death to increase in CLIG-Hes1 infection compared to CLIG infection, the effect is not sufficient for the reduction of growth rates in CLIG-Hes1 infection. (J) Ratios (with s.e.m.) of proliferating cells in the virus-infected cells at 72 hours after infection (at least 500 cells in three independent experiments were examined). Ki67-positive cell ratios are similar between CLIG- and CLIG-Hes1-infected cells, whereas a ratio of cells positive for the G1 phase-specific cyclin D1 is higher in CLIG-Hes1 infection, suggesting that Hes1 overexpression prolongs G1 phase. (K-Z) Immunocytochemical staining of cells infected with CLIG (K-N,S-V) and CLIG-Hes1 (O-R,W-Z). After 5 days in culture, some CLIG-infected cells have differentiated into TuJ1-positive neurons (K-N) or GFAP-positive astrocytes (S-V) whereas virtually none of the CLIG-Hes1-infected cells (closed arrowheads) are neurons (O-R) or astrocytes (W-Z). Some neurons (TuJ1-positive cells) and astrocytes (GFAP-positive cells) are indicated by open arrowheads (R,V).

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