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First published online 25 May 2006
doi: 10.1242/dev.02422

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Identification of Epha4 enhancer required for segmental expression and the regulation by Mesp2

¹ Division of Mammalian Development, National Institute of Genetics, Yata 1111, Mishima 411-8540, Japan.
² Division of Developmental Genetics, RIKEN Research Center for Allergy and Immunology (RCAI) RIKEN Yokohama Institute, 1-7-22 Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan.

View larger version (57K): [in a new window]   Fig. 1. Identification of a somite-specific Epha4 enhancer. (A) The lacZ transgene constructs used to identify the cis-acting somite enhancer within the Epha4 genomic region. The numbers of transgenic mouse embryos that expressed ß-gal in the somites, among the transgene-positive embryos, are indicated on the right. E, EcoRI; H, HindIII; N, NheI; Sa, SacI; Sw, SwaI; P, PmaCI; X, XbaI. (B,C) Lateral view of ß-gal activity driven by the 630 bp (HindIII) Epha4 enhancer fragment in a 10.5 dpc embryo. A magnified image in the somitic region of B is shown in C. (D,E) Comparison by in situ hybridization at 10.5 dpc of the transgene expression (lacZ) in a transgenic embryo (D) with endogenous Epha4 expression in the wild-type embryo (E). In situ signals in the anterior PSM are indicated by arrows in the lower panels showing magnified images. (F,G) ß-Gal activity driven by the 630 bp enhancer in 10.5 dpc wild-type (F) and Mesp2L/L embryos (G).

View larger version (37K): [in a new window]   Fig. 2. Identification of the functional E-box motifs responsible for the somite-specific activation of Epha4. (A) Sequence of the 630 bp Epha4 somite enhancer (core enhancer) region. The four indicated fragments (fragment 1-4) represent deleted sequences from the core enhancer region. The results of transgenic analyses using these deleted constructs are shown in parentheses. (B) Sequence alignment of somite enhancer regions (fragment 2 and fragment 3) of mouse Epha4 with the corresponding regions of human Epha4. The mutations introduced in each E-box and the results of the subsequent transgenic analyses are shown. (C) Schematic representation of artificially constructed enhancers, containing six E-box motif repeats. (D) The artificial enhancers were cloned upstream of a lacZ reporter vector and the results of the subsequent transgenic analyses (representative images are shown) are indicated in parentheses. For the [E2]6-lacZ artificial enhancer, no somite expression was observed among the 11 transgene-positive embryos. Blue letters indicate putative E-box motifs.

View larger version (13K): [in a new window]   Fig. 3. Transactivation of the Epha4 enhancer by the Mesp2/E47 heterodimer. Epha4 somite enhancers (A, 630 bp core sequence; B, six tandem repeats of E-box sequences) were ligated to the pGL3 luciferase vector. Luciferase activity was measured at 36 hours after transfection into NIH3T3 cells. (A) The presence (+) or absence (-) of either Mesp2 or E47 are indicated in each column. (B) Luciferase activity was compared with (black bars) and without (white bars) Mesp2/E47. Mutations in E3 (5'-CATTTG-3'), that give rise to E3m (5'-ACGGGT-3'), results in the loss of reporter activity. The results shown are the mean values from three independent experiments. Standard deviations are indicated by error bars.

View larger version (29K): [in a new window]   Fig. 4. The Mesp2/E47 heterodimer binds to the E3 site of the Epha4 enhancer. (A) The results of EMSA using nuclear extracts from NIH3T3 cells transfected with FLAG-Mesp2 and/or Myc-E47 and incubated with E3 probe. The quantities of nuclear extracts used (µg) are indicated. (B) A competition assay indicating the specificity of the binding of the Mesp2/E47 (2 µg each) complex to the E3 probe. The addition of 100-fold excess of unlabeled E3 probe, but not the E3m mutant probe, abolished the binding. (C) Evidence for the heterodimer formation of FLAG-Mesp2/Myc-E47. The band containing E3 (arrow) was supershifted by the addition of either anti-FLAG or anti-Myc antibodies. The oligonucleotides used were as follows: E3, 5'-TGGGTCACATTTGTCCAAAA-3'; E3m, 5'-TGGGTCAACGGGTTCCAAAA-3' (E-box is shown in the bold; altered nucleotides in the mutant probe are underlined).

View larger version (75K): [in a new window]   Fig. 5. Mox1 and Mox1-cre expression and the lineage analysis. Whole-mount in situ hybridization analysis of Mox1 expression in 9.5 dpc embryos (A,B) and Mox1-cre in 8.25 (C), 8.5 (D) and 11.5 (E) dpc embryos. (F-I) Whole-mount expression patterns and sagittal sections of ß-gal-stained R26R/Mox1-cre double heterozygous embryos at 10.5 dpc. (H,I) The sections were counterstained with Eosin. Arrowheads indicate somite borders forming between S0 and S-1.

View larger version (100K): [in a new window]   Fig. 6. Ectopic Mesp2 expression in somites leads to the activation of Epha4. Expression of Mesp2 in 10.5 dpc wild-type (A) and CAG-CAT-Mesp2; Mox1-Cre double heterozygous embryos (B-D). In addition to the normal Mesp2 expression in the anterior PSM, ectopic expression of Mesp2 was observed throughout the entire somitic region (B-D). The expression pattern of Epha4 at 11.5 dpc in wild-type (E,G) and double heterozygous embryos (F,H) is also shown. An expression pattern for Epha4 that was similar to Mesp2 was observed in the double heterozygote (F,H). Histological analyses of 10.5 dpc wild-type (M) and double heterozygous (I-L,N) embryos. Ectopic Mesp2 protein (I,K) and Epha4 expression (J,L) were evident in serial sections of double heterozygotes. Magnified images of square parts of I and J are shown in K and L, respectively. Another consecutive section was stained with phalloidin (N) and a similar region of the wild-type embryo is shown in M. In double heterozygotes, abnormal epithelial cells were observed within the somite (N). A paraffin section stained with nuclear Fast Red revealed gaps in epithelialized somites in the double heterozygote at 10.5 dpc (O). Black arrows in I and J indicate the endogenous expression of Mesp2 (I) or Epha4 (J). Red arrows in K,L,N indicate separated cell clusters. White arrow in N indicates an abnormal epithelialized feature. Black arrows in O indicate local gaps. Scale bar: 100 µm.

View larger version (86K): [in a new window]   Fig. 7. Ectopic expression of Mesp2 leads to skeletal malformations. Skeletal preparations of 18.5 dpc wild-type (A-D) and CAG-CAT-Mesp2; Mox1-Cre fetuses (E-H) stained with Alcian Blue and Alizarin Red. Lateral (A,E) and dorsal (B,F) views of whole skeletons are shown. Higher magnifications of the lumber region from wild-type (C,D) and double heterozygous fetuses (G,H) are also shown. The lack of pedicles of the neural arches could be observed in the double heterozygotes (G,H). The rib structure was also severely affected in the double heterozygotes (G,H). pd, pedicle; lm, lamina.

View larger version (88K): [in a new window]   Fig. 8. Early defects in chondrogenesis and gene expressions affected in the CAG-CAT-Mesp2;Mox1-Cre double heterozygotes. Skeletal morphology was revealed by Alcian Blue staining in the wild-type (A) and double heterozygous (B) embryos at 13.5 dpc. (C-H) Uncx4.1. expression was reduced in double heterozygotes (D,F) at both 10.5 (C,D) and 11.5 dpc (E,F) compared with wild-type embryos (C,E). The section of 11.5 dpc embryonic tail revealed Uncx4.1-negative cells (arrowheads) in the caudal compartment of somites in the double heterozygote (H). Comparison of expression patterns of Pax3 (I,J), Sox9 (K,L) and Tbx18 (M-P) between wild-type (I,K,M,O) and double heterozygous (J,L,N,P) embryos. Outlines in K and L show Sox9 expression in the rib primordia.