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First published online January 27, 2005
doi: 10.1242/10.1242/dev.01597

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Differential contributions of Mesp1 and Mesp2 to the epithelialization and rostro-caudal patterning of somites

¹ Cellular & Molecular Toxicology Division, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagayaku, Tokyo 158-8501, Japan
² Division of Mammalian Development, National Institute of Genetics, Yata 1111, Mishima 411-8540, Japan

View larger version (102K): [in a new window]   Fig. 1. Mesp1 and Mesp2 are co-expressed in the anterior PSM but have differing roles in somitogenesis. (A) Overlapping expression of Mesp1 and Mesp2 is revealed by in-situ hybridization using the left and right halves of the same embryo. The lines show most recently formed somite boundaries. (B-C) A Mesp1-null embryo (B) shows the same normal somite formation as a wild-type embryo (C). Arrowheads indicate somite boundaries. (D) In Mesp2-null embryos, no somite formation is observed but Mesp1 is expressed at comparable levels to wild type, although its expression is anteriorly extended and blurred. (E-H) Mesp1-null embryos show normal rostro-caudal patterning of somites. (E,F) Expression of a caudal half marker, Uncx4.1. (G,H) Expression of a rostral half marker, EphA4. The lines indicate presumptive or formed somite boundaries and the dotted line indicates approximate position of somite half boundary.

View larger version (21K): [in a new window]   Fig. 2. Schematic representation of chimera analysis method. Either Mesp2-null or Mesp1/Mesp2 double-null embryos, genetically labeled with Rosa locus, were aggregated with wild-type embryos at the 8-cell stage, and the resulting chimeras were subjected to analysis at 11.0 dpc.

View larger version (101K): [in a new window]   Fig. 3. Mesp2-null cells tend to be excluded from the epithelial region of the somites. (A) The control chimeric embryo undergoes normal somite formation and shows random distribution of labeled cells. The right panel is a high-power view of a somite. (B) In the Mesp2-null chimeric embryo, incompletely segmented somites are formed. Mesp2-null cells tend to be localized at the rostral and central region of these incomplete segments. Red arrows: wild-type cell clusters; blue arrows: Mesp2-null cell clusters. (C,D) Other sections indicating multiple small epithelial cell clusters (arrows). Note that Mesp2-null cells only partially contribute to the epithelial clusters (blue arrows). (E,F) A small number of Mesp2-null cells are distributed in the dermomyotome and are mostly localized at the caudal end. Scale bars: 100 µm.

View larger version (65K): [in a new window]   Fig. 4. Mesp2 function is cell autonomously required for rostral properties. (A-D) Expression of lacZ and Uncx4.1 transcripts at the site of initial somite formation in control (A,B) and Mesp2-null (C,D) chimeric embryos. In the control, lacZ-expressing cells are randomly distributed and Uncx4.1 expression is normal. In the Mesp2-null chimera, lacZ-expressing Mesp2-null cells at the rostral part of the incomplete segments (arrows in C) ectopically express Uncx4.1 (arrows in D). Lines indicate somite boundaries. (E,F) In the dermomyotome, Mesp2-null cells are mostly localized at the caudal end, and the Uncx4.1 expression pattern is normal. (G,H) In the sclerotome, the distribution of Mesp2-null cells results in expansion of Uncx4.1 expression (arrows). (I) The control chimeric fetus shows normal vertebrae. (J) The Mesp2-null chimeric fetus exhibits partial fusion of the neural arches. (K) The control chimeric fetus shows normal ribs. (L) The Mesp2-null chimeric fetus shows proximal rib fusion. Scale bars: 100 µm. C, caudal compartment; R, rostral compartment.

View larger version (91K): [in a new window]   Fig. 5. Mesp1/Mesp2 double-null cells fail to contribute to epithelial somites or to the dermomyotome. (A-D) Tail regions from X-gal-stained whole-mount specimens of control (A,C) and double-null (B,D) chimeric embryos. (A,B) Lateral view. (C,D) Dorsal view. The blue double-heterozygous cells are randomly distributed in the control embryo, whereas the Mesp1/Mesp2 double-null cells are excluded from the lateral region of the somites (arrowheads in D). (E,F) Parasagittal sections of tails from chimeric embryos. (E) The labeled cells are randomly located in the control chimera. (F) The two types of cells are randomly mixed in the PSM, whereas the dermomyotome-like epithelium consisted exclusively of wild-type cells and the sclerotome-like compartment contained mostly Mesp1/Mesp2 double-null cells. Note that normal epithelial somites are not formed in this chimera. (G,H) Transverse sections show elimination of Mesp1/Mesp2 double-null cells from the dermomyotome. (I,J) The dermomyotome-like epithelium in the Mesp1/Mesp2 double-null chimeric embryo gives rise to dermomyotome, myotome (arrowhead in J) and the dorsal part of the sclerotome. Red arches indicate the inner surface of dermomyotome. (K,L) AlexaFluor 488-labeled phalloidin staining shows normal epithelialization of somites in the control chimera (K) and restriction of epithelialization in the dermomyotome-like compartment in the Mesp1/Mesp2 double-null chimera (L). dm, dermomyotome; dml, dermomyotome-like epithelium; dsc, dorsal part of the sclerotome; my, myotome; nt, neural tube; sc, sclerotome; scl, sclerotome-like compartment; tg, tail gut.

View larger version (93K): [in a new window]   Fig. 6. (A,B) Mesp1/Mesp2 double-null cells express Paraxis. Adjacent parasagittal sections of the Mesp1/Mesp2 double-null chimeric embryo were stained for either Paraxis (A) or lacZ (B). Note that the expression domains of the two genes overlap in the medial sclerotomal region (brackets). (C,D) The rostro-caudal pattern in the dermomyotome is formed in a partially segregating wild-type cell population. Adjacent sections of the Mesp1/Mesp2 double-null chimeric embryos were stained for Dll1 (C) or lacZ (D) mRNA. Red outlines demarcate the dorsal dermomyotome-like compartments. Note that suppression of Dll1 expression occurs in a region mostly occupied by wild-type cells (arrows). Scale bar: 100 µm.

View larger version (104K): [in a new window]   Fig. 7. Rostro-caudal patterning of the sclerotome is disrupted in Mesp1/Mesp2 double-null chimeric embryos. (A) The control chimeric embryos exhibit normal stripe patterns of Uncx4.1 expression throughout the somite region. (B) The Mesp1/Mesp2 double-null chimeric embryos exhibit continuous Uncx4.1 expression in the ventral sclerotomal region. Note that caudal localization of Uncx4.1 expression is normal in the dermomyotome and dorsal sclerotome. The insets show a higher magnification of lumbar somites. (C,D) Adjacent sections showing that lacZ-expressing Mesp1/Mesp2 double-null cells express Uncx4.1. (E-H) The Mesp1/Mesp2 double-null chimeric fetus exhibits caudalization of the vertebrae and of the proximal ribs. (E) The control chimeric fetus shows normal metameric arrangement of the neural arches. (F) The Mesp1/Mesp2 double-null chimeric fetus shows severe fusion of the pedicles and the laminae of neural arches. (G) The control chimeric fetus has normal arrangement of ribs. (H) The double-null chimeric fetus shows severe fusion of the proximal elements of the ribs. Scale bars: 100 µm.

View larger version (30K): [in a new window]   Fig. 8. A schematic summarization of the Mesp1/Mesp2 chimera experiments. Mesp1/Mesp2 double-null cells can contribute to neither epithelial somite nor dermomyotome formation, whereas Mesp2-null cells can partially contribute to both somites and dermomyotome. Red outlines indicate epithelialized tissues (epithelial somites, dermomyotomes and abnormal small clusters).