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Figure 2 hapln1a is regionally expressed in the heart tube and secreted asymmetrically into the cardiac jelly. (A) Schematic representation of Tomo-seq pipeline. GFP-expressing hearts are manually excised from embryos at 26hpf, and frozen in tissue freezing medium. Heart tubes are sectioned along the atrioventricular axis. RNA extracted from individual slices is labelled with a slice-specific molecular barcode during reverse transcription before generating sequencing libraries. (B) Example Tomo-seq traces from a single 26hpf heart tube, with individual slices from venous pole to arterial pole along the x-axis and normalized read number along the y-axis. Read numbers for atrial marker myh6 (green) and ventricular marker myh7 (blue) allows identification of chamber position within the dataset. hapln1a expression (magenta) is up-regulated in atrial sections. (C–F) mRNA in situ hybridization analysis of hapln1a expression in the heart between 19hpf and 50hpf. At cardiac disc stage, hapln1a is up-regulated in the posterior (arrow C), which is maintained as the heart forms the cardiac cone prior to tube formation (arrow D), with lower expression in the anterior cone. Once the heart cone has extended to form the tube, the previously posterior hapln1a expression is positioned on the left side of the tube (bracket, E), and expressed at higher levels in the atrium (A) than the ventricle (V). By 50hpf hapln1a expression in the heart is restricted to low levels in the atrioventricular canal (AVC, asterisk, F). Schematics above in situ panels indicate heart morphology at each stage, and hapln1a expression domain within the heart (blue) V, Ventricle; A, Atrium. Scale bar = 100 μm. (G–I) Fluorescent in situ hybridization analysis of hapln1a (magenta) in Tg(myl7:lifeActGFP) transgenic embryos shows hapln1a is expressed in myocardial cells at 26hpf. (J–L) Fluorescent immunostaining of Hapln1a (magenta) in Tg(myl7:lifeActGFP) transgenic embryos demonstrating the protein is secreted into the extracellular space predominantly on the left side of the heart tube (magenta) at 26hpf. (G and J) Dorsal views. (H and I, K and L) Transverse views. (M–Q) Fluorescent immunostaining of Hapln1a (magenta) in Tg(myl7:lifeActGFP) transgenic embryos at 50hpf revealing Hapln1a is maintained in the cardiac ECM as looping progresses. (M–O) Ventral views. (P and Q) Transverse views. L, left; R, right, scale bar = 50 μm