Dynamic expression and essential functions of Hes7 in somite segmentation - PubMed (original) (raw)

Dynamic expression and essential functions of Hes7 in somite segmentation

Y Bessho et al. Genes Dev. 2001.

Abstract

The basic helix-loop-helix (bHLH) gene Hes7, a putative Notch effector, encodes a transcriptional repressor. Here, we found that Hes7 expression oscillates in 2-h cycles in the presomitic mesoderm (PSM). In Hes7-null mice, somites are not properly segmented and their anterior-posterior polarity is disrupted. As a result, the somite derivatives such as vertebrae and ribs are severely disorganized. Although expression of Notch and its ligands is not affected significantly, the oscillator and Notch modulator lunatic fringe is expressed continuously throughout the mutant PSM. These results indicate that Hes7 controls the cyclic expression of lunatic fringe and is essential for coordinated somite segmentation.

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Figures

Figure 1

Figure 1

Dynamic expression of Hes7 in the PSM. (A) Variable expression patterns for Hes7 of E9.5 embryos. (B_–_D) Comparison of the expression of Hes7 (left) and Lfng (right) in the bisected caudal portions of E9.5 embryos. The expression patterns of the two genes were variable, but similar to each other in all three stages (stage I, n = 4; stage II, n = 4; stage III, n = 2). Each pair is dissected from the same embryo. The established somites were stained with the Uncx4.1 probe for spatial alignment. (E_–_G) Hes7 is differentially expressed after explant culture. The right halves were cultured for 60 min (E), 90 min (F), or 120 min (G), and compared with the left halves, which were fixed immediately. (G) Somite boundaries and a newly formed boundary are indicated by arrows and arrowhead, respectively. Somite −I is indicated by asterisks.

Figure 2

Figure 2

Generation of Hes7_-null mice. (A) Targeting strategy. The top line shows the structure of the wild-type Hes7 gene and the middle line shows the structure of the targeting vector. Most of the coding region of Hes7 was replaced by IRES–_LacZ and PGK–neo (inverted orientation). The bottom line indicates the resultant disrupted locus. Diphtheria toxin gene (DT) was used for negative selection. (E) _Eco_RV; (S) _Sac_I; (K) _Kpn_I. The positions of 5′-external and 3′-external probes are indicated on top. (B) Southern blot analysis. The 5′-external probe detected 10-kb wild-type and 7-kb mutant bands of _Sac_I-digested genomic DNA. (C) Kinked tails of adult heterozygous mutants. (D) Whole-mount in situ hybridization for Hes7 of wild-type (left) and homozygous-mutant (right) embryos at E9.5. In the mutant embryo, Hes7 expression is completely missing (arrows). (E) The appearance of heterozygous- (left) and homozygous-mutant (right) neonates. A homozygous mutant has a short trunk and a short tail (arrow).

Figure 3

Figure 3

Segmentation defects in _Hes7_-null mice. (A_–_F) Neonates of heterozygous (A,C,E) and homozygous (B,D,F) mutants were stained for bone (red) and cartilage (blue). (A,B) Dorsal view of the whole neonates; (C,D) lateral view of vertebrae and; (E,F) dissected ribs and sternums. (G,H) HE staining of sagittal sections of Hes7(+/−) (G) and Hes7(−/−) (H) neonates. In Hes7(−/−) embryos, vertebral bodies and neural arches (arrows) are fused, and the patterning of vertebral bodies and intervertebral disks is defective. (ID) Intervertebral disk; (NA) neural arch; (SC) spinal cord; (VB) vertebral body. (I,J) Parasagittal sections of E9.5 embryos. Compared with Hes7(+/−) (I), somites are irregularly arranged in Hes7(−/−) embryos (J). Incomplete and almost complete fusion of epithelial somites are indicated by arrow and arrowhead, respectively. Bar, 1 mm (G,H); 100 μm (I,J).

Figure 4

Figure 4

Defects of somitogenesis in _Hes7_-null embryos. (A) Whole-mount lacZ staining. In a Hes7(+/−) embryo, weak staining is observed in the PSM and several caudal somites. In contrast, in a Hes7(−/−) embryo, much stronger staining is observed in the PSM and irregular somites. A higher magnification is shown in the lower panels. (B_–_H) Expression of Paraxis (B), pMesogenin (C), MesP2 (D), Uncx4.1 (E,F), Ephrin-B2 (G), and EphA4 (H) was examined by in situ hybridization. (E,F) The disorder of Uncx4.1 expression is more severe in the caudal parts (brackets) than in the rostral parts (arrows). In E, rostral is to the left. (G) Ephrin-B2 expression disappears in somites but remains in the PSM of _Hes7_-null embryos. (H) In the wild type, EphA4 is expressed in the anterior half of somite I (arrowhead) and the PSM (arrow). In _Hes7_-null embryos, the expression in somite I disappears.

Figure 5

Figure 5

Whole-mount in situ hybridization for Notch signaling molecules. (A) In the _Hes7_-null embryo, the expression is not significantly affected in the PSM, but the anterior border becomes diffuse. (B) In the wild type, Notch2 is expressed in somites I (arrow) and −I (arrowhead). In the _Hes7_-null embryo, only a broader expression is observed in the anterior PSM. (C) In the wild type, Dll1 is expressed in the PSM and the posterior half of each somite. In the _Hes7_-null embryo, it is expressed in the PSM, but the anterior border is diffuse and the somite expression is undetectable. (D) Dll3 expression in the PSM is not significantly affected but the anterior border is diffuse in _Hes7_-null embryos. (E) In the wild type, Hes1 is expressed in the PSM (brackets) and the posterior half of somites (arrowheads). In the _Hes7_-null embryo, the expression is observed in somite −I (bracket) but undetectable in other regions (n = 5). (F) In the wild type, Hey2 is expressed in the PSM (arrow) and the posterior half of somites (arrowheads). In the _Hes7_-null embryo, the expression is almost undetectable (n = 4). (G) In the wild type, Lfng displays various expression patterns at E8.5 (stage I, 9; stage II, 16; stage III, 12 out of 37 embryos) and E9.5 (stage I, 6; stage II, 9; stage III, 10 out of 25 embryos). (H) In contrast, all Hes7_-null embryos (E8.5, n = 8; E9.5, n = 9) exhibit the same expression pattern—_Lfng is expressed continuously throughout the mutant PSM.

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