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First published online 14 March 2007
doi: 10.1242/dev.000836

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The negative regulation of Mesp2 by mouse Ripply2 is required to establish the rostro-caudal patterning within a somite

Mitsuru Morimoto^1,*,, Nobuo Sasaki^1,, Masayuki Oginuma², Makoto Kiso¹, Katsuhide Igarashi³, Ken-ichi Aizaki³, Jun Kanno³ and Yumiko Saga^1,2,

¹ Division of Mammalian Development, National Institute of Genetics, Yata 1111, Mishima 411-8540, Japan.
² SOKENDAI, Yata 1111, Mishima 411-8540, Japan.
³ Cellular and Molecular Toxicology Division, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagayaku, Tokyo 158-8501, Japan.

View larger version (64K): [in this window] [in a new window]   Fig. 1. Analysis of the of Ripply2 expression pattern. (A) Comparison of the mRNA expression patterns of Mesp2 and Ripply2 during mouse development. Positive expression is indicated by an arrowhead. (B) Comparison of the spatial expression patterns of Mesp2 and Ripply2 as revealed by section double in situ hybridization. Two representative examples are shown for Mesp2 (green) and Ripply2 (magenta), and merged images of these expression patterns are shown beneath. The green signals in the periphery are artifacts and do not represent Mesp2 expression. In some cases, only a single band could be observed for each gene, and these bands are merged in the image shown in the left-hand bottom panel. Two bands were sometimes visible for Ripply2, the posterior band of which merges with that of Mesp2 (right-hand bottom panel). All samples were prepared from E10.5 embryos. (C) Whole-mount in situ hybridization showing that Ripply2 expression is lost in the E9.5 Mesp2-null embryo.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Mesp2 can directly bind to the enhancer element of the Ripply2 gene and activate its transcription. (A) Comparison of the genomic sequences around the Ripply2 gene in mouse (top line) with those in human, dog and chick using MultiPipMaker sequence alignment software. A conserved region (framed in red) is evident across these species. (B) Sequence alignment of the 171 bp region conserved among the Ripply2 genes, within which a highly conserved E-box is located. HC E-box, highly conserved E-box. (C) The genomic organization of the mouse Ripply2 gene and the corresponding construct used in the transgenic analyses. A 1.5 kb DNA fragment containing this highly conserved 171 bp stretch (shown in A) of the Ripply2 upstream region was ligated to a cassette composed of the hsp promoter and nlacZ (lacZ harboring a nuclear localization signal). E, EcoRI; B, BamHI; N, NcoI. (D) The Ripply2 enhancer drives lacZ reporter gene expression in somitic mesoderm cells at E11.0. The inset shows high magnification of the somitic region. (E) Luciferase reporter assay for Mesp2 activation, with or without E47, using constructs harboring either the 1.5 kb Ripply2 enhancer (left) or six repeats of the conserved 171 bp fragment (right). The addition of E47 had negative effects upon transactivation. The data represent the means±s.d. from four separate experiments. *P<0.01, **P<0.04. (F) EMSA analyses revealing that a DNA fragment containing the conserved E-box (Region B, light-blue shading) from the Ripply2 upstream region can bind Mesp2 in the absence of E47. This binding of Mesp2 thus appears to be different from its binding to the Epha4 enhancer, which is dependant upon E47. Non-specific bands are indicated by the asterisk. (G) The binding specificity of Mesp2 was confirmed by successful competition with cold probe, but not with an E-box mutant probe (shown in B).

View larger version (32K): [in this window] [in a new window]   Fig. 3. The targeting strategy used for the Ripply2 gene and the external morphology of the resulting knockout mouse. (A) The top line shows the genomic organization of the Ripply2 gene, the second line represents the structure of the targeting vector, and the bottom two lines show the predicted structure of the Ripply2 locus following homologous recombination. The first exon of Ripply2 was partially deleted and replaced with a floxed neo cassette (the arrowheads on the line represent loxP sites). A germline chimeric mouse was then generated from recombinant ES cells containing the targeted allele and crossed with a CAG-Cre mouse to remove the neo cassette and establish the Ripply2-knockout mouse line. Ssp, SspI; E, EcoRI; B, BamHI; H, HindIII; N, NcoI; K, KpnI; X, XhoI. (B) The Ripply2-null mouse dies soon after birth and the external morphology at E17.5 is similar to those of segmentation-defective mutants, featuring a short trunk with rudimental tails.

View larger version (102K): [in this window] [in a new window]   Fig. 4. The Ripply2-knockout mouse exhibits segmentation defects. (A-D) Ripply2+/- and Ripply2-/- embryos (n=3 at E10.5) were compared by external morphology (A,B) and by the Hematoxylin and Eosin staining of parasagittal sections of tail regions (C,D). Ripply2-/- embryos display irregularly sized myotomes, and an unclear segmental border. (E-G) Skeletal preparations at E17.5 stained with Alizarin Red-Alcian Blue reveal that the Ripply2-/- fetus harbors fewer pedicles of neural arches and lacks components of the proximal ribs (F; n=4), which is similar to the aberrant phenotype of the Psen1-null fetus (G; n=2).

View larger version (80K): [in this window] [in a new window]   Fig. 5. Altered gene expression in the Ripply2-null embryos. Whole-mount in situ hybridizations were employed to characterize somitogenesis in the Ripply2-/- embryo. The expression of caudal genes such as Uncx4.1 (A,B) and Dll1 (C,D) was found to be reduced, whereas rostral genes such as Tbx18 (E,F) and Epha4 (G,H) show an expanded pattern in Ripply2-/- embryos at E11.5. (I-N) Comparisons of the expression patterns of Mesp2 mRNA, detected by exon (I,J) and intron (K,L) probes, and protein levels (M,N), at E10.5. An additional Mesp2 expression band appears rostrally in the Ripply2-/- embryos (J,L). Mesp2 protein expression, visualized by Mesp2-venus, was compared between the Ripply2+/- (M, n=2) and Ripply2-/- (N, n=3) genetic backgrounds. The confocal images were visualized by fluorescence, detected using anti-GFP antibodies. (O,P) Comparison of the Lfng expression patterns at different cyclic phases (indicated by I to III) at E10.5. The oscillatory expression of Lfng (asterisks) in the posterior PSM was unaffected, but the rostral-most expression bands (brackets) are slightly expanded in the Ripply2-/- embryos (P), as compared with the Ripply2+/- embryos (O). (Q,R) The prolonged expression of Lfng in the anterior PSM. The PSM of E10.5 Ripply2+/- (Q) and Ripply2-/- (R) embryos was separated into two halves, with one being fixed immediately and the other fixed after explant culturing for 20 minutes. Both were then analyzed for Lfng mRNA. The expression of Lfng in the anterior PSM is maintained for longer in the Ripply2-/- embryos.

View larger version (83K): [in this window] [in a new window]   Fig. 6. Notch signaling is reduced in the anterior PSM in the Ripply2-/- embryo. (A-F) Notch1 mRNA (A, n=2; B, n=2), Notch1 protein (C, n=2; D, n=2) and Hes5 mRNA (E, n=2; F, n=4) expression patterns were compared between wild-type (A,C,E) and Ripply2-/- (B,D,F) embryos at E11.0. (G-I) Double immunostaining with anti-Mesp2 (green) and anti-active Notch1 (magenta; the white lines indicate activities in the anterior PSM) antibodies using sections of wild-type (G) and Ripply2-/- (H,I) E11.0 embryos. In the Ripply2-/- background, Mesp2 expression is upregulated but Notch activity is reduced.

View larger version (114K): [in this window] [in a new window]   Fig. 7. Genetic analyses using double-knockouts of Ripply2 and either Lfng or Mesp2. The skeletal morphologies and Uncx4.1 expression patterns were compared among wild-type (A), Lfng-null (B), Ripply2/Lfng double-null (C), Mesp2-null (D) and Ripply2/Mesp2 double-null (E) E17.5 fetuses or E9.5 embryos. The skeletal defects in the Ripply2-/- fetus were found to be further enhanced by the additional loss of Lfng, and the pedicles of the neural arches were almost completely absent in this compound-null fetus (C). By contrast, the Ripply2/Mesp2 double-null fetus (E) shows a similar morphology to that of the Mesp2 single-null fetus (D). The Uncx4.1 expression pattern was independently examined at E10.5 (A, n=2; B, n=2; C, n=1) and E9.5 (A, n=4; B, n=2; C, n=2; D, n=4; E, n=2). Only representative images of E9.5 embryos are shown.

View larger version (28K): [in this window] [in a new window]   Fig. 8. Genetic cascades in the anterior PSM regulating somitogenesis. (A) Schematic of the positive (red line) and negative (blue line) regulation surrounding Mesp2. The transcription of Mesp2 is enhanced by both Notch signaling and Tbx6. At the same time, Mesp2 suppresses Notch signaling by activating Lfng and suppressing Dll1 expression. Mesp2 proteins are also rapidly degraded via a proteasome-dependent pathway. We herein propose a new negative regulatory system for Mesp2 via Ripply2. (B) Schematic illustrating how the rostrocaudal polarity is established or disrupted in the anterior PSM of the wild type and Ripply2-/- mutants. In the anterior PSM, Mesp2 is localized in the rostral compartment of S-1 and suppresses Notch signaling through the suppression of Dll1. By contrast, in the caudal compartment of S0, both Dll1 expression and Notch signaling are retained because of the lack of Mesp2. In the Ripply2-/- embryo, Mesp2 expression persists for a longer period in both the rostral and caudal compartments, although the suppression on Notch signaling is incomplete. This results in the expansion of the rostral properties within the somites.