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First published online 13 August 2003
doi: 10.1242/dev.00672

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Enhancer timing of Hox gene expression: deletion of the endogenous Hoxc8 early enhancer

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA

View larger version (25K): [in a new window]   Fig. 1. Hoxc8 early enhancer sequence and targeted disruption/deletion of Hoxc8 early enhancer locus. (A) The 200 bp region of the EE. Sequences underlined represent putative transcription factor binding sites. Cis-acting elements A-E are identified by mutational analysis of reporter genes (Shashikant et al., 1995; Shashikant and Ruddle, 1996). (B) Schematic representation of the EE (oval), targeting vector, disrupted EE locus (EEneo) and deleted EE locus (EElox). Exons of Hoxc8 (black boxes) and Hoxc9 (gray boxes), and restriction enzyme sites (A, AvrII; B, BamHI; D, DraI; H, HindIII, X, XhoI) are shown. A PGKneo cassette (neo) flanked by loxP sites (arrowheads) and two copies of the HSV thymidine kinase gene (tk) are used for positive and negative selection, respectively. Arrows indicate different primer sets used for PCR screening. The position of the 5' probe used for Southern hybridization is also indicated. Broken lines represent the homologous recombination regions. (C) PCR analysis of genomic DNA isolated from ES cells using different primer pairs. 5ee1 and 5neo2, 3neo1 and 3sb3r and 5neo1 and 3sb3r amplify 2.5 kb, 2 kb and 3 kb product for EEneo allele. 5neo1 and 3sb3r amplify 2.2 kb product for wild-type allele. 5neo1 and 3neo2 amplify 300 bp product for EElox allele, 500 bp product for wild-type allele and 1.5 kb product for EEneo allele. N, negative control without DNA. (D) Southern hybridization of ES cell genomic DNA using an 5' external probe (box labeled as probe in B). XhoI digestion gives a 5 kb fragment for EEneo allele, and a 10 kb fragment for wild-type allele. BamHI and HindIII digestion gives a 3 kb fragment for EEneo allele, and a 3.7 kb fragment for wild-type allele. AvrII and DraI digestion gives a 5.5 kb fragment for EElox allele, and a 3.2 kb fragment for wild-type allele. (E) PCR analysis of mouse tail DNA using primer set 5neo1 and 3neo2. Amplification of wild-type alleles produces a 472 bp fragment, while amplification of mutated alleles gives a 1472 bp fragment (EEneo) or 306 bp fragment (EElox).

View larger version (61K): [in a new window]   Fig. 2. Hoxc8 expression in the wild-type and EEneo mouse embryos at different developmental stages by whole-mount in situ hybridization. (A) 8.0 dpc embryos. Expression of Hoxc8 is detected in the allantois (a) and in the posterior region of the wild-type (wt) embryo. No Hoxc8 transcript is detected in EEneo embryo. (B) 8.5 dpc embryos with 10-12 somites. EEneo embryo shows delay of Hoxc8 expression in both neural tube (red arrow) and paraxial mesoderm (green arrow) when compared with the wild-type embryo. (C) Embryos (9.0 dpc) with 15-17 somites. In the wild-type embryo, the anterior boundaries of Hoxc8 expression in the neural tube and somite are established in the level of the 10th and 14th somites (s10 and s14), respectively. In EEneo embryo, expression of Hoxc8 is still restricted to the paraxial mesoderm. Note that although the anterior limit of Hoxc8 expression domain in the neural tube is established correctly in EEneo embryo, the expression level is very low and can only be seen under the microscope. (D) 10.0 dpc embryos with 25-27 somites. Hoxc8 expression domain within the somites is from s15 to s23 in the wild-type embryo. In EEneo embryo, although the anterior boundaries of Hoxc8 expression domains are established in both neural tube and somite, the posterior boundary is anteriorized to s21. (E) The same somite expression pattern is observed in 11.0 dpc EEneo embryo. (F) At 11.5 dpc, the expression domains of Hoxc8 within both neural tube and somite are comparable in the wild-type and EEneo embryos. Note that the expression level at s22 and s23 is stronger in EEneo embryo.

View larger version (66K): [in a new window]   Fig. 3. Hoxc8 expression in the wild-type and EElox mouse embryos at different developmental stages visualized by whole-mount in situ hybridization. (A) Embryos at 8.0 dpc. Expression of Hoxc8 is detected in the allantois (a) and in the posterior region of the wild-type and EElox embryos. Note that the expression domain in EElox embryo does not spread forward to more anterior regions when compared with the wild-type embryo. (B) Embryos with 10-12 somites. The expression patterns of Hoxc8 in both neural tube (red arrow) and paraxial mesoderm (green arrow) are identical in the wild-type and EElox embryo. (C) 9.0 dpc embryos with approximate 16-18 somites. In both wild-type and EElox embryos, the anterior boundaries of Hoxc8 expression in the neural tube and somite are established at s10 and s14, respectively. (D) 10.0 dpc embryos with 24-26 somites. The anterior boundaries of Hoxc8 expression are identical in both neural tube and somite in the wild-type and EElox embryos. Nevertheless, the posterior boundary of the somite expression domain is anteriorized to s21 in EElox embryo. (E) The same somite expression pattern is observed in 11 dpc EElox embryo. (F) At 11.5 dpc, the expression domains of Hoxc8 within both neural tube and somite are comparable in the wild-type and EElox embryos.

View larger version (103K): [in a new window]   Fig. 4. Morphological alteration of the axial skeletons in newborn EEneo mice. (A,B) Dorsal view of the thoraco-lumbar regions. (A) Wild-type animal showing 13 thoracic vertebrae and 6 lumbar vertebrae. (B) EEneo mouse showing a fully developed extra pair of ribs on L1. (C,D) Ventral view of the thorax. (C) In the wild-type mouse, there are seven pairs of ribs attached to the sternum. The sixth and seventh ribs attach to the same point of the sternum without sternebra separation (arrow). (D) EEneo mouse with nine pairs of ribs attached to the sternum, extra sternebra developed between the sixth and the seventh ribs (arrow). (E-H) Lateral view of lower thoracic/upper lumbar regions. (E) Arrowhead indicates that the position of the transitional vertebra is in T10 in the wild-type mouse. (F) The transitional vertebra (arrowhead) shifted from T10 to T13 in EEneo mouse. (G,H) Anteriorization of the 12th rib in EEneo mouse is revealed by the total length and the length of the cartilage. (G) Total length of the 12th rib in the wild-type animal is about two thirds of the length of the 11th rib and the cartilage part of the 12th rib is half the length of the bony part. (H) The length of the 12th rib is almost equal to the length of the 11th rib and the length of the cartilage segment is equal to the bony part of the 12th rib in EEneo mouse. (I,J) Lateral view of the cervicothoracic transition. (I) Wild-type mouse showing the anterior tuberculum (AT) is on C6 (asterisk) and the first rib is attached to T1. (J) EEneo mouse showing the AT is on C5 (asterisk) instead of C6 and an ectopic rib is developed on C7 and fused to the first rib (arrow).

View larger version (46K): [in a new window]   Fig. 5. (A-C) Comparison of the expression pattern of Hoxc6 (A), Hoxc9 (B) and Hoxb8 (C) in 9.5 dpc wild-type and EEneo embryos by whole-mount in situ hybridization. (A,C) The expression patterns of Hoxc6 and Hoxb8 are identical in the wild-type and EEneo embryos. (B) The anterior boundary of Hoxc9 expression in the somites is at the level of s20, one somite posterior when compared with the anterior level of Hoxc9 in the wild-type embryo.

View larger version (36K): [in a new window]   Fig. 6. Sequence similarity between the EE and Hoxd11 RVIII. (A) Nucleotide sequence comparison of 200 bp Hoxc8 EE and 276 bp Hoxd11 RVIII region. The yellow shaded boxes represent 100% identical sequences of CDX and HOX binding motifs within the two enhancers. Red and blue underlines indicate the putative protein binding motifs within the EE and Hoxd11 RVIII, respectively. Stars indicate conserved nucleotides. (B) Schematic comparison of the EE and Hoxd11 in relationship to the order of the putative transcription factor binding motifs. Blue boxes indicate identical sequences of CDX and HOX-binding motifs within the two enhancers. Note that the spacing between these two binding motifs in the EE and Hoxd11 RVIII differs by only one base pair out of a total of 76. The yellow and green boxes represent the first and second Forkhead/Sry within the two enhancers, respectively. These two Forkhead/SRY pairs within each enhancer also show high sequence similarities and differ by only a few base pairs.

View larger version (22K): [in a new window]   Fig. 7. Comparisons of the skeletal phenotypes between Hoxc8 null, EEneo and EElox mutations. Cervical, thoracic and lumbar vertebrae are shown as blue, yellow and green blocks, respectively. The transformed vertebrae are indicated by red dots. Posterior and anterior transformation are represented by pink and blue arrows. Table represents penetrance (%) of vertebral column defects in Hoxc8 null, EEneo and EElox animals. Data of null 1 and null 2 were obtained from Le Mouellic et al. (Le Mouellic et al., 1992) and van den Akker et al. (van den Akker et al., 2001), respectively.