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First published online 1 March 2006
doi: 10.1242/dev.02305

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A tyrosine-rich domain within homeodomain transcription factor Nkx2-5 is an essential element in the early cardiac transcriptional regulatory machinery

David A. Elliott^1,*,, Mark J. Solloway^1,*,, Natalie Wise¹, Christine Biben¹, Mauro W. Costa^1,3, Milena B. Furtado¹, Martin Lange^1,, Sally Dunwoodie^1,2 and Richard P. Harvey^1,2,¶

¹ Victor Chang Cardiac Research Institute, Darlinghurst, Sydney 2010, Australia.
² Faculties of Life Science and Medicine, University of New South Wales, Randwick 2031, Australia.
³ Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 20941-000, Brazil.

View larger version (23K): [in a new window]   Fig. 1. Transcriptional activity in the C terminus of Nkx2-5. (A) Domain structure of the Nkx2-5 C terminus fused to GAL4 DNA-binding domain (GAL4). (B-E) Trans-activation of the GAL4-dependent G5T-luciferase reporter in C3H10T1/2 cells by co-transfected GAL-Nkx2-5(232-318) and similar constructs bearing mutations (see text). Relative transcriptional activity compared with vector-only control (bars indicate s.e.m.) is shown on the right of each panel. (F) Western blot showing that GAL-Nkx2-5 proteins are stably expressed in transfected COS cells.

View larger version (52K): [in a new window]   Fig. 2. Cephalopod mollusc cardiac NK2 proteins carry the YRD and show a domain structure resembling their mammalian relatives. (A) Homeodomain and YRD sequences from cephalopod and other cardiac NK2 proteins. Tyrosine 54 within the homeodomain, a defining hallmark of NK2-class homeodomains, is shown in red. Tyrosines and phenylalanines within the YRD are shown in red, with other conserved amino acids in green. Species abbreviations: Ag, Anopheles gambiae (mosquito); Am, Apis mellifera (honeybee); amphi, amphioxus; c, chick; Ce, Caenorhabditis elegans (nematode); Cn, cnidarian Hydra vulgaris (hydra); Dm, Drosophila melanagaster; h, human; m, mouse; Nv, Nematostella vectensis (sea anemone); Ok, Octopus kaurna (octopus); So, Sepia officinalis (cuttlefish); x, Xenopus laevis (frog); z, zebrafish. TN, Tin-domain. (B-D) LpNkx2-5 expression in Loligo paelii (squid) embryos. (B) Schematic diagram of a squid embryo showing location of systemic heart (sh; red) and branchial hearts (bh; yellow). (C) Histological section showing systemic heart and ink sac (is). (D) In situ hybridisation to an adjacent section showing LpNkx2-5 expression in the systemic heart and ink sac (arrow), as well as muscle beneath the fins (f; arrowheads). (E) Phylogenetic relationships and domain structure of NK2 protein from divergent species. G, GIRAW motif;N, Nkx2-5 box; NK2, NK2SD.

View larger version (50K): [in a new window]   Fig. 3. DNA binding and transactivation analysis of the Nkx2-5Y-A mutant protein. (A) EMSA comparing DNA binding of in vitro-translated Nkx2-5 and Nkx2-5Y-A using an Nkx2-5-binding oligonucleotide probe. Anti-Nkx2-5 antibody and specific and non-specific competitor oligonucleotides were used to assess binding specificity. (B,C) In vitro transcriptional analysis using FLAG-tagged Nkx2-5 and Nkx2-5Y-A expression vectors and a multimerised Nkx2-5-binding site synthetic enhancer-luciferase reporter (B) or with and without Gata5 and Tbx20a on the Nppa (–700) and Gja5 (–1190 to +121) promoter-luciferase reporters in C3H 10T1/2 cells (C). Inset in B shows western blot of transfected proteins. Significance assessed using Student's t-test.

View larger version (41K): [in a new window]   Fig. 4. Targeted alleles of Nkx2-5. (A) Targeting strategies used to create Nkx2-5 `knock-in' alleles (see text). (B,C) Wild-type Nkx2-5 locus with targeting vectors and targeted alleles shown before and after Cre deletion of drug selection cassettes (H, pgkHygro; N, pgkNeo). Coding region exons shaded black. Arrows show loxP sites. HD indicates the small homeobox deletion (amino acids 141-184). 5'P and 3'P, and p1-p4, respectively, indicate positions of Southern probes and PCR primers for validation of recombinations and deletions. Restriction endonucleases: Nc, NcoI; Bc, BclI; Bg, BglII; M, MluI; N, NotI; X, XbaI; Xh, XhoI. (D) Southern blot analysis of ES cell DNA of indicated genotype digested with BclI (left) or NcoI (right) using probe 3'P. The 23 kb band is the wild-type allele, while 9 and 6 kb bands represent targeted alleles. (E) PCR validation of Cre-deleted ES cell clones using primers specific for the pgkHygro cassette, as indicated in B and C, and those specific for the GFP and pgkNeo cassettes.

View larger version (35K): [in a new window]   Fig. 5. The Nkx2-5Y-A allele impairs cardiac differentiation in EBs. (A) FACS profiles of dissociated wild-type and Nkx2-5GFP/+ EBs showing side scatter (SSC-H) versus GFP fluorescence (FL1-H). Gated cells were collected for expression profiling. (B) Comparison of GFP fluorescence versus cell counts in EBs of different genotype. (C) Comparative RT-PCR analysis of gene expression in FACS-purified GFP+ cells from EBs of genotypes indicated. (D) Real-time RT-PCR analysis of gene expression from FACS-purified GFP+ cells as in C. Results reported as percent expression relative to Nkx2-5GFP/+(N) cells (two independent experiments performed in duplicate; error bars indicate ±s.e.m.).

View larger version (63K): [in a new window]   Fig. 6. High-level Nkx2-5GFP/lacZ or Nkx2-5GFP/Y-A ES cell contribution to chimaeric embryos perturbs heart development. (A,B) Nkx2-5GFP/Y-Awild-type chimaeras at 7.5 and 8.5 dpc stained for lacZ. (C) Chimaera showing abnormal cardiac development at 9.0 dpc with pericardial oedema (arrows). (D-I) Comparison of heart morphology (upper panels) and histology (lower panels) in 9.0 dpc chimaeric embryos composed of indicated ES cell genotypes. Nkx2-5lacZ/+wild-type chimaeras (D,G) were morphologically normal. Nkx2-5GFP/lacZwild-type (E,H) and Nkx2-5GFP/Y-Awild-type (F,I) chimaeras showed arrested heart development and absence of ventricular trabeculation (arrows). (J,K) High-level chimaera at E9.5 carrying Nkx2-5Y-A:IRESlacZ/+ ES cells (stained for lacZ) and section, showing relatively normal cardiac morphology. Abbreviations: CA, common atrium; LV, left ventricle; OT, outflow tract; PV, primitive ventricle; RA, right atrium.

View larger version (116K): [in a new window]   Fig. 7. Heterozygous Nkx2-5Y-A ES cell chimaeras show cardiac defects. (A) Control chimaeric heart at 14.5 dpc produced from an Nkx2-5lacZ/+wild-type ES cells. (B-D) Three examples of Nkx2-5GFP/Y-Awild-type chimaeras at 14.5 dpc, the section in D stained for lacZ. Arrows in B and C show finger-like projections from the myocardium. (E,F,I,J) Section in situ hybridisation for Myl2 transcript showing low Myl2 transcript levels across the chimaeric heart (F) and in finger-like projections (J), and epicardial blistering (I). (G,H) Enlargements of boxed area in B showing finger-like projections that contain mutant Y-A cells (lacZ staining in H). (K-P) Histological examination of control Nkx2-5lacZ/+wild-type chimaera (K,O) and mutant Nkx2-5Y-A:IRESlacZ/+wild-type chimaeric hearts (L-N,P) highlighting right ventricular non-compaction (black arrows, L), fenestration of the ventricular septum (red arrow, L), ventricular septal defect (broken arrow in M), tricuspid valve showing Ebstein's anomaly (arrowhead, M; brackets, P) and transmural lesion (arrow, N). Abbreviations: IVS, interventricular septum; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle.