Failure of cardiac looping, malformation of AV canal, and loss of AV conduction delay in slipjig mutants. (A) Bright-field micrographs of 48-hpf wild-type (WT) and sli mutant (s644^−/−) embryos. The white arrow points to pericardial edema. (B,C) Nomarski micrographs of wild-type and sli mutant hearts at 48 hpf. (B) Green circle delineates the wild-type ventricle, and blue circle indicates the wild-type atrium. (C) Yellow circle delineates the sli mutant heart with no discernible AV boundary. (D,E) Fluorescence micrographs of 48-hpf Tg(cmlc2:gfp) wild-type and sli mutant hearts. sli mutant hearts fail to loop and to form an AV canal. (F–H,J) Confocal micrographs of 40-hpf Tg(cmlc2:ras-GFP)^s883; Tg(flk1:ras-cherry)^s896 wild-type (F) and sli mutant (G) hearts and AV canal region (H,J). Myocardium and endocardium are in green and red, respectively. sli mutant AV myocardial and endocardial cells fail to undergo characteristic cell shape changes. (I,K) Schematic representation of wild-type and sli mutant AV region. (L,M) Forty-hour-post-fertilzation Tg(cmlc2:gCaMP)^s878 wild-type and sli mutant heart optical maps of calcium-dependent fluorescence represented by isochronal lines every 60 msec. Numbers indicate temporal sequence of calcium activation in the heart. sli mutant hearts fail to develop an AV conduction delay by 40 hpf. The white arrowheads point to the AV canal. (Atr) Atrium; (Ven) ventricle.

Molecular analyses of AV-specific cardiac genes reveal AV canal defects in sli mutant hearts. Schematized representations are shown to the right of whole-mount RNA in situ hybridization data. Red lines indicate myocardium, green lines indicate endocardium, and blue lines indicate gene expression. In 48-hpf wild-type hearts, bmp4 (A,B), tbx2b (E,F), and versican (I,J) are expressed in the AV myocardium and notch1b (M,N) is expressed in the AV endocardium. In 48-hpf sli mutant hearts, bmp4 (C,D) and versican (K,L) expression is expanded throughout the ventricular myocardium, tbx2b (G,H) expression is absent within the AV canal, and notch1b (O,P) expression is diffusely expanded throughout the endocardium. The red arrowhead points to the AV canal. The red box surrounds sli mutant hearts. (A) Atrium; (V) ventricle.

sli encodes Foxn4. (A) Genetic map of the sli region. Numbers below SSLP markers indicate recombination events. Two genes were identified within the critical region, which spans two BACs. (B) Knockdown of foxn4 by the injection of 2 ng of an ATG MO into Tg(cmlc2:GFP) embryos results in failure of the hearts to form an AV canal constriction (arrowhead) and absence of cardiac looping. (C) Calcium-dependent fluorescence optical map of 48-hpf Tg(cmlc2:gCaMP)^s878 foxn4 MO knockdown heart reveals loss of AV conduction delay at 48 hpf. Isochronal lines depict the distance traveled every 60 msec. Numbers indicate temporal sequence of calcium activation in the heart. (D–G) foxn4 is expressed in the AV canal at 24, 36, 48, and 72 hpf. The black arrows point to the AV canal. The white dashed lines outline the heart. (D) Dorsal view. (E–G) Ventral view. (Atr) atrium; (Ven) ventricle.

Foxn4 and Tbx5 directly regulate expression of tbx2b, a gene required for AV canal formation. (A,B) EMSA reveals specific binding of Foxn4 and Tbx5 to their respective binding elements within the tbx2b enhancer. Lane 1 (−/−) contains reticulocyte lysate without recombinant Foxn4 or Tbx5 protein. Recombinant Foxn4 (A) and Tbx5 (B) proteins were combined with radiolabeled double-stranded oligonucleotides representing the canonical Foxn4-binding site (Foxn4 control) and Tbx5-binding site (Tbx5 control) as well as the tbx2b enhancer Foxn4-binding element (tbx2bpro, Foxn4) and Tbx5-binding element (tbx2bpro, Tbx5). Foxn4 and Tbx5 interactions to respective binding sites were further tested through competition assays using excess unlabeled canonical wild-type/mutant (WTc, MTc) and tbx2b enhancer wild-type/mutant (tbx2b WT, tbx2b MT), foxn4, and tbx5 oligonucleotides. (C–F) tbx2b MO knockdown hearts exhibit absence of AV canal and cardiac looping at 48 hpf. Fluorescence micrographs of 48-hpf control (C) and tbx2b MO-injected (D) Tg(flk1:gfp)⁸⁴³ embryos. The arrowheads point to the AV boundary. (E) Confocal micrograph of 48-hpf Tg(cmlc2:ras-GFP)^s883; Tg(flk1:ras-cherry)^s896 tbx2b MO knockdown heart. (Inset) AV boundary region. (F) Calcium-dependent fluorescence optical map of 48-hpf Tg(cmlc2:gCaMP)^s878 tbx2b MO knockdown heart reveals loss of AV conduction delay at 48 hpf. Isochronal lines depict the distance traveled every 60 msec. Numbers indicate temporal sequence of calcium activation in the heart. (G,J) tbx2b expression in foxn4 mRNA rescued sli mutant heart. The black arrow points to the AV canal. (H,I,K,L) Cardiac-specific overexpression of tbx2b results in lack of cardiac looping and failure to form the AV canal. Fluorescence micrographs of 80-hpf Tg(cmlc2:dsRed)^s879 wild-type (H,I) and Tg(cmlc2:Tbx2b-GFP)^s900 (K,L) hearts. The white arrow points to pericardial edema.