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First published online 11 February 2009
doi: 10.1242/dev.034025

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DNA binding-dependent and -independent functions of the Hand2 transcription factor during mouse embryogenesis

¹ Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA.
² Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Generation of Hand2EDE mutant mice. (A) Schematic of the Hand2 protein. Amino acid sequences of the basic domain (++) of wild-type and EDE mutant protein are shown. The EDE mutant protein is also referred to as Hand2EDE. (B) Targeting strategy. Homologous recombination resulted in replacement of the region of exon 1 encoding the basic region and part of the intron with the exon 1 containing mutations and a neor cassette flanked by FRT sites (red diamond). After FLPe-mediated excision, one FRT site remained in the intron. Mutations representing the amino acid changes within the basic domain in exon 1 are shown. The positions of the 5' long arm (4.6 kb), the 3' short arm (1.7 kb), and the 5' and 3' probes used for Southern blotting are shown. (C) Southern blot analysis. Genomic DNA from ES cell clones was isolated and analyzed by Southern blotting with the 5' probe after NdeI digest and the 3' probe after NcoI digest. Hybridization of NdeI-digested ES cell DNA with the 5' probe yielded an 11.8-kb fragment for the wild-type allele and a 13.5-kb fragment for the targeted allele because of the insertion of the neor cassette. Hybridization of NcoI-digested ES cell DNA with a 3' probe yielded a 4.9-kb DNA fragment for the wild-type allele and a 4-kb fragment for the targeted allele due to an additional NcoI site in the neor cassette. The positions of wild-type and mutant bands are shown. Genotypes are shown at the top. (D,E) Analysis of Hand2 transcripts by RT-PCR and sequencing. RNA was isolated from wild-type, Hand2EDE/+ and Hand2EDE/EDE embryos at E9.5 and analyzed by RT-PCR to detect Hand2 transcripts. Transcripts for HPRT were detected as a control for RNA loading and integrity. The Hand2 PCR products were then gel-isolated and subjected to sequencing. The sequencing traces from wild-type, Hand2EDE/+ and Hand2EDE/EDE embryos are shown in E and the amino acid sequences are indicated. Because the Hand2 locus was sequenced with a reverse primer, the antisense sequences are shown and amino acid sequences of Hand2 are shown from right to left. (F) The Hand2 mRNA level was analyzed by quantitative real-time PCR. RNA isolated from wild-type and Hand2EDE/EDE embryos at E9.5 was analyzed by real-time PCR to detect levels of Hand2 mRNA. Relative expression normalized to GAPDH in wild-type and Hand2EDE/EDE embryos is shown. (G) Hand2 protein expression was detected by western blotting. Protein isolated from pooled E9.5 hearts from wild-type and Hand2EDE/EDE embryos was blotted against anti-Hand2 antibody. GAPDH protein was detected as a loading control. (H) Summary of homozygous Hand2EDE/EDE mutants obtained from heterozygous intercrosses.

View larger version (80K): [in this window] [in a new window]   Fig. 2. Hand2EDE/EDE embryos from E9.5 to E12.5. (A) Wild-type, Hand2EDE/EDE and Hand2KO/KO embryos at E9.5 are shown. (a-c) Right view; (d-f) higher magnification shows hearts from the right view; (g-i) ventral view; (j-l) higher magnification shows hearts from the ventral view. Hand2EDE/EDE embryos were morphologically indistinguishable from wild-type embryos and showed significant formation of the RV at E9.5. By contrast, Hand2KO/KO embryos showed growth retardation and a hypoplastic RV. Lv, left ventricle; rv, right ventricle; v, single ventricular chamber. (B) Embryos at E10.5, E11.5 and E12.5. Hand2EDE/EDE embryos appear relatively normal but show multifocal hemorrhages at E11.5. The surviving Hand2EDE/EDE embryo at E12.5 showed no obvious heart defects. Delayed growth of the limb buds of Hand2EDE/EDE embryos was apparent at E10.5 and became more severe at E11.5 and E12.5. (a,b,e-h) Right view of the embryos; (c,d) ventral view. Limb buds are highlighted by dashed lines. flb, forelimb bud; hlb, hindlimb bud.

View larger version (52K): [in this window] [in a new window]   Fig. 3. Heart development in Hand2EDE/EDE mutant embryos. (A) Hearts from wild-type, Hand2EDE/EDE and Hand2KO/KO mutant embryos at different time points were sectioned and stained with Hematoxylin and Eosin. Note that Hand2EDE/EDE hearts show significant formation of the RV compared with the lack of RV in the Hand2KO/KO embryos at E9.5. a, atrium; lv, left ventricle; rv, right ventricle; v, single ventricular chamber. (B) Expression of Hand2 and its target genes in the heart as detected by real-time PCR. RNA isolated from pooled E9.5 hearts from wild-type and Hand2EDE/EDE embryos was analyzed by quantitative real-time PCR. Relative expression normalized to GAPDH in wild-type and Hand2EDE/EDE embryos is shown.

View larger version (77K): [in this window] [in a new window]   Fig. 4. Limb development in Hand2EDE/EDE embryos. (A) At E11.5, Hand2EDE/EDE embryos have underdeveloped forelimb and hindlimb buds. Embryos were stained in 0.2% CoCl2 for visualization of the limb buds. (Left) Right view of the whole embryos; (middle and right) dorsal views of the forelimb and hindlimb buds with distal on the top and anterior to the left. (B) Transverse section of wild-type and mutant limb buds at E11.5 with distal on the top and anterior to the left. (C) Fgf8 expression in wild-type and Hand2EDE/EDE embryos detected by whole-mount in situ hybridization at E10.5 and E11.5. (Top) Views of the whole embryos; (middle and bottom) dorsal views of the forelimb and hindlimb buds with distal on the top and anterior to the left. (D) Sox9 expression in wild-type and Hand2EDE/EDE embryos detected by whole-mount in situ hybridization at E10.5. Bottom panel shows dorsal view of the forelimb buds. (E) Shh expression in wild-type and Hand2EDE/EDE embryos at E10.5 analyzed by whole-mount in situ hybridization. Arrows point to the Shh expression domain at the posterior margin of the forelimb and hindlimb buds of wild-type embryos. Expression of Shh was absent in the limb buds of Hand2EDE/EDE embryos. Upper images show the entire embryo, and lower images show higher magnification of the limb buds. (F) Expression of Alx4, Gli3 and gremlin (Grem) in wild-type and Hand2EDE/EDE embryos at the stages indicated. Dorsal views of the forelimb buds were shown with distal on the top and anterior to the left.

View larger version (48K): [in this window] [in a new window]   Fig. 5. Branchial arch development in Hand2EDE/EDE embryos. (A) Transverse sections of branchial arches are shown from E9.5 to E11.5. Hand2EDE/EDE embryos have normal growth of branchial arches until E10.5. At E11.5, the mandibular components of the first brachial arch (man) of the mutant embryo were not fused (compare with those of the wild type). ba, branchial arch; man, mandibular components of the first branchial arch; max, maxillary components of the first branchial arch. (B) Expression of Msx1 transcripts at E10.5 was analyzed by whole-mount in situ hybridization. There is no significant difference in expression level of Msx1 between wild-type and Hand2EDE/EDE embryos. Arrows indicate branchial arches. Top panel, lateral view; bottom panel, ventral view. ba, branchial arches; flb, forelimb bud.

View larger version (119K): [in this window] [in a new window]   Fig. 6. Craniofacial analysis of wild-type, Hand2BA/BA and Hand2EDE/BA mice at P1. (A,D,G,J) Wild-type mouse; (B,E,H,K) Hand2BA/BA mutant mouse; (C,F,I,L) Hand2EDE/BA mutant mouse. (A-C) Lateral view; (D-F) ventral view of the isolated jaws; (G-L) ventral view. (A-C) The mandibles (arrows) are smaller and are deformed in Hand2EDE/BA and Hand2BA/BA mice. (D-F) The mandibles in both mutant mice are shorter and are deformed. The angle between the left and right mandible is wider than that in the wild-type mouse. (G-I) In the wild-type mouse, the secondary palate is formed by the fusion of bilateral palatine processes (dotted vertical line). In Hand2BA/BA and Hand2EDE/BA mice, the palatine processes are not formed (dotted vertical lines), so the presphenoid (ps) bone becomes visible. (J-L) Tympanic rings (ty) are shortened and are deformed in the Hand2BA/BA and Hand2EDE/BA mice. pa, palatine; ps, presphenoid; ty, tympanic ring.

View larger version (12K): [in this window] [in a new window]   Fig. 7. A model of Hand2 functions during development. Hand2 regulates gene expression by two different mechanisms: DNA-binding dependent and DNA-binding independent. During limb development, Hand2 functions through DNA-binding-dependent mechanisms. During early branchial arch development, Hand2 mainly acts through a DNA-binding-independent mechanism, but DNA binding is required for mandible development later. Both the DNA-binding-dependent and -independent functions of Hand2 are necessary for heart morphogenesis.

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