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Hypomorphic Mesp allele distinguishes establishment of rostrocaudal polarity and segment border formation in somitogenesis

¹ Cellular and Molecular Toxicology Division, National Institute of Health Sciences, 1-18-1 Kamiyohga, Setagaya-ku, Tokyo 158-8501, Japan
² Division of Early Embryogenesis,
³ Division of Mammalian Development, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan

View larger version (41K): [in a new window]   Fig. 1. Comparison among members of the Mesp-related gene family (A) and strategy of gene replacement of mouse Mesp2 with zebrafish mespb (B,C). (A) Comparison of amino acid sequences in the bHLH motif. The percentage homology to Mesp2 within and outside the bHLH motif are shown. Differences are indicated in green (Mespb) and purple (Mesp1). (B) Knock-in strategy. The top line shows the genomic organization of the Mesp2 gene; the second shows the structure of the targeting vector; the third is the predicted structure of the Mesp2 locus following homologous recombination. Mesp2 exons (pink boxes) were completely deleted and replaced with the zebrafish mespb cording region flanked with floxed neo cassette and poly(A) signal (the arrowheads on the line represent loxP sites). Chimeric mice generated from recombinant ES cells containing targeted allele, Mesp2neo–mespb, were mated with CAG-Cre mice to excise the floxed neo cassette, resulting in the generation of the Mesp2mespb allele. The probe used for Southern blot analysis is indicated. Restriction enzymes: B, BamHI; E, EcoRI; Hi, HincII; H, HindIII; K, KpnI; P, PstI; S, SacI; Sm, SmaI; X, XbaI. Arrows indicate PCR primers. (C) Genomic Southern blot analysis of SacI-digested DNA from embryos with various Mesp2 alleles. Arrowheads show the 6.0 kb fragment of the wild-type allele, the 2.3 kb targeted Mesp2neo–mespb, and the 4.7 kb Cre-excised Mesp2mespb allele. Genotypes of progeny are indicated at the top of each lane. All represent genotypes of the Mesp2 allele.

View larger version (58K): [in a new window]   Fig. 2. Characterization of mespb-knockin mice. (A) Intercrossing of heterozygous mespb/+ mice gives rise to viable mespb/mespb mice with kinked tails. In these mespb/mespb (C) and neo-mespb/neo-mespb (D) embryos, the mespb genes introduced are expressed in the expected region, which are similar to that of Mesp2 (B). Segmented somites are observed in both mespb/mespb (C) and neo-mespb/neo-mespb embryos, but not in Mesp2–/– embryos (E). However, the skeletal morphology (F-I) showed various defects in vertebrae formation in both mespb/mespb (G) and neo-mespb/neo-mespb (H) embryos. Embryo samples were prepared at 11.5 dpc. Skeletal specimens were prepared at 18.5 dpc. Anterior is to the left. Genotypes for various mice are schematically represented on the left. Mesp2, endogenous allele; neo, pgk-neo cassette replaced with Mesp2 for gene targeting (ref); mespb, zebrafish mespb gene; gray arrowhead, lox sequence; pA, polyadenylation signal.

View larger version (74K): [in a new window]   Fig. 3. mespb gene dosage effect revealed by the skeletal morphology at 18.5 dpc (A-F) and gene expressions at 11.5 dpc (G-R) reflecting RC polarity and subsequent resegmentation. (A-F) The lumber regions of the vertebral columns stained with Alcian Blue-Alizarin Red. The wild-type embryo (A) exhibits clear separation of the lamina (l), pedicle (p) and transverse process (t). (B-E) Varying degrees of fusion of the pedicles and laminas are observed in the mespb or neo-menpb fetuses. (F) A Mesp2-null fetus with a completely fused pedicle and lamina. (G-R) The segmental pattern is prefigured by the expression pattern of Uncx4.1 (G-L) in segmented somites and Dll1 (M-R) in the anterior PSM. Expression of these genes, normally localized in the caudal half of each somite and CPM, is expanded rostrally in mespb, or neo-mespb embryos.

View larger version (66K): [in a new window]   Fig. 4. Expression of Mesp2 or mespb is autoregulated. Mesp2/+, mespb/+ or neo-mespb/+ mice were crossed with Mesp2-lacZ/+ mice and the expression pattern of Mesp2 or mespb was visualized. Mesp2 (A), mespb (B-C) and Mesp2-lacZ (D-G) transcripts are shown by whole-mount in situ hybridization. The lack of autoregulation that results in the loss of Mesp2-lacZ restriction to the rostral compartment is revealed by the caudally extended expression pattern of ß-gal activities (I-K), which are different from the striped expression pattern of Mesp2+/L (H; Mesp2 heterozygous embryo).

View larger version (96K): [in a new window]   Fig. 5. Differential regulation between segment border formation and its maintenance. The border formation in nascent somites of embryos of various mespb genotypes was compared in serial horizontal sections. In all embryos (A-E, A'-E') except for the Mesp2-null embryo (F,F'), the initial segmental borders are generated. However, the borders are not maintained in the neo-mespb/– embryo (E,E'). Various levels of border fusion were observed in mespb/– and neo-mespb/neo-mespb embryos. The initial segmental borders are indicated by black arrowheads. Partial fusion between segmented epithelial somites is indicated by green arrowheads. All specimens were prepared at 11.5 d.p.c., but the AP level of these samples are not always same. (A'-F') Higher magnification of the boxed areas in A-F.

View larger version (79K): [in a new window]   Fig. 6. Gene expression implicated in the segmental border formation. The expressions of Lfng (A-F) EphA4 (G-L) and PAPC (M-R) are compared in various mespb embryos at 11.5 dpc. Lfng is expressed in a highly dynamic manner. Therefore, 8-10 embryos of each genotype were analyzed by whole-mount in situ hybridization and we chose those with similar expression patterns for the comparison. The anterior thinner band of Lfng (black arrowhead) is rostrally extended in the Mesp2-null embryo. In contrast, the sharpness of the band was recovered in all mespb embryos. The rostral band of EphA4 (green arrowhead) and PAPC (purple arrowhead) expression, which disappeared in the Mesp2-null embryo, is also present in mespb embryos.

View larger version (14K): [in a new window]   Fig. 7. Possible models of events leading to the somite border formation and the establishment of RC polarity. (A) Mesp2 or Mespb might regulate these two events using different genetic pathways. Mesp2 is known to suppress Dll1 via the Notch signaling pathway. (B) However, the pathway might be important for the normal expression of EphA4, Lfng and PAPC, which is required for the border formation. (C) Finally the suppression of Dll1 required for the establishment of RC polarity might play a role in the border formation. In all cases, Notch signaling is required for the autoregulation of Mesp2, and RC polarity is required for the maintenance of the segmental border. Only the anterior most bands of Lfng and PAPC are Mesp-dependent.