Tandon P , Wilczewski CM , Williams CE , Conlon FL .

P40 OD010997 NIH HHS , R21 HD073044 NICHD NIH HHS , R21HD073044 NICHD NIH HHS , R01 HL127640 NHLBI NIH HHS , T32 HL069768 NHLBI NIH HHS , R01 HL135007 NHLBI NIH HHS

             heart development
             epicardium morphogenesis

	Fig. 1. Spatio-temporal analysis of lhx9 isoforms during Xenopus epicardial development. Whole embryo in situ hybridization was performed on Xenopus embryos at stages 36 to 46, using probes specific for lhx9-α (A-D), lhx9-LIM (E,F), lhx9-HD (G-L) and tbx18 (K-N) (see also Figs S2-S5). Views display embryos facing right, left and ventral with anterior to the top. Images show magnified cardiac regions as depicted by red box on tadpole schematic (bottom right). Red arrowheads indicate staining of septum transversum and PE clusters. Asterisks indicate cardiac region. White dashed line outlines heart in ventral views. (E,F) Transverse gelatin section in situ hybridization images of cardiac region demonstrate lhx9-LIM expression exclusively in the septum transversum (stage 36) and proepicardial tissue (stage 41). en, anterior endoderm; h, heart; peo, proepicardial organ; st, septum transversum.
	Fig. 2. Lhx9 is required for proper epicardial layer and PE cluster formation. Epicardial layer formation (as shown with tbx18) analysis in control (A,C) and Lhx9-depleted (B,D) embryos at stage 45. (A,B) Images show lateral view of cardiac region, dorsal to the top and anterior to right. (C,D) Transverse gelatin sections through representative embryo cardiac regions showing tbx18 expression, dorsal to the top. PE cell clustering (red arrowheads) and epicardial cell layer on the ventricular surface is shown. (E) Epicardial layer formation defects were quantified as abnormal when≤50% of the ventricle is covered, as depicted in schematic. (F) Quantification of observed epicardial layer formation defects, as represented in A-D, from three independent experiments, P≤0.0001 by two-tailed Fisher’s exact test. Proepicardial clustering morphology was analyzed using in situ hybridization for tbx18 in control (G,I,K) and Lhx9-depleted (H,J,L) embryos at stage 38 (G,H), stage 41 (I,J) and stage 42 (K,L). Ventral view showing cardiac region, anterior to the top. Tbx18 expression is detected as a cluster of cells in control embryos on the right of the embryo near the atrioventricular sulcus (I,K, red arrowheads), whereas in Lhx9-depleted embryos, the cluster is either not detected and tbx18 expression remains throughout the septum transversum region or is mis-positioned to the caudal side of the heart (abnormal clustering) (J,L, green arrowheads). (M) Quantification of PE clustering defects is depicted in the schematic as being abnormal by bilateral tbx18 expression retention on septum transversum and/or cluster mis-positioning of ≥45° caudal to the AVS compared with controls. (N) Quantification of observed clustering phenotype at stage 41 represented in C and D. Data taken from seven independent experiments, P≤0.0001 by two-tailed Fisher’s exact test. (O-T) In situ hybridization analysis for tcf21 (O,P,U) and wt1 (Q-T) proepicardial expression at stage 41. Images depict lateral view of the cardiac region, dorsal to the top and anterior to the right (M-P) or ventral view, dorsal to top (Q,R). Red arrowheads indicate clustered PE cells. (U) Quantification of observed phenotypes as depicted in M, data taken from six independent experiments, P≤0.0001 by two-tailed Fisher’s exact test. avs, atrioventricular sulcus; pe/peo, proepicardial organ; st, septum transversum; v, ventricle.
	Fig. 3. Integrin-paxillin association is required for PE clustering. In situ hybridization for tbx18 on whole embryos after incubation in stated concentrations of small molecules between stages 38 and 41. Lateral views (A,C,E,G,I) and ventral views (B,D,F,H,J) of the cardiac region from representative embryos are shown; red arrowheads indicate PE cells. (C-F) Images depict maintained tbx18 expression on septum transversum and clustering abnormalities as shown in Fig. 2M when embryos are incubated in 6-B345TTQ. (K) tbx18 expression in embryos taken from two independent experiments; P=0.000248 by Chi-square test. v, ventricle.
	Fig. 4. Lhx9 regulates integrin α4-paxillin signaling in PE clusters. (A-D) Whole embryo in situ hybridization for itga4, stages 36 to 46. Lateral views of anterior portion (left panels) with magnified image of cardiac region (right panels). Red arrowheads indicate PE cluster; asterisks indicates heart. (E,F) In situ hybridization for itga4 in control (E) and Lhx9-depleted embryos (F) at stage 41. Representative cardiac region shown, anterior to right, dorsal to top; red arrowhead marks PE cluster. (O) Quantification of observed itga4 expression (reduced expression denoting ≤50% stain intensity compared with controls) and clustering defects shown in E,F (see Fig. 2M for phenotype assessment), embryos taken from six independent experiments, P≤0.0001 by Chi-square test. (G-J′) Transverse cardiac region agarose sections from control (G-H′) and Lhx9-depleted (I-J′) stage 41 embryos post-in situ hybridization for tcf21 (G,I), nuclei expression with DAPI (H,J, blue) and Itgβ1 immunohistochemistry (H′,J′, magenta). White outlines (H,H′,J,J′) indicate PE cluster as depicted by tcf21 and Itgβ1. (K-N′) Transverse cardiac region agarose sections from control (K-L′) and Lhx9-depleted (M-N′) stage 45 Xla.Tg(Cardiac-actin:GFP)Mohun embryos depicting representative immunohistochemical analysis for DAPI (blue), GFP (green) to label cardiomyocytes, Itgβ1 (magenta) to label epicardial cells and phosphorylated Y118-paxillin (red). Magnified images (L,L′,N,N′) from white boxes in K,M. White arrowheads indicate PE cluster. (P) Pixel intensity (integrated density) levels for phosphorylated Y118-paxillin from five control and 10 Lhx9-depleted embryos; P=0.0134 by two-tailed Student’s t-test. v, ventricle.
	Fig. 5. Disrupted Lhx9-integrin signaling alters epicardial ECM environment. (A-E) Transverse cardiac agarose sections from control (A,B) and Lhx9- depleted (C,D) Xla.Tg(Cardiac-actin:GFP)Mohun embryos at stage 45. Nuclei stained with DAPI (blue), GFP labels cardiomyocytes (green) and immunohistochemical expression for Itgβ1 (B,D, magenta) to label epicardial cells and fibronectin (B′,D′, red). Threshold binary images (ImageJ) in B′ and D′ show Itgβ1-positive epicardial cells used to quantify pixel intensity in B′ and D′. (E) Pixel intensity (integrated density) levels for fibronectin from five control and 10 Lhx9-depleted embryos, P=0.064 by two-tailed Student’s t-test. (F-K) Transverse cardiac agarose sections from control (F,H,I) and Lhx9-depleted (G,J,K) embryos at stage 45. Nuclei stained with DAPI (blue) and immunohistochemical stain for tropomyosin (cardiomyocytes, F,G, green), laminin (F,G, magenta) and β-dystroglycan (H′-K′, magenta). Tcf21 expression (H-K) demonstrates PE cell attachment to the heart. Red boxes in H,J are magnified in I,K; white boxes in H′,J′ are magnified in I′,K′. White arrowheads label PE (F′,G′,I′,K′) and migrating epicardial cells (F′,G′). Yellow arrowheads label expression in endocardial tissue (B′,D′,I′,K′). v, ventricle. Representative images from seven (laminin) and six (β-dystroglycan) embryos per condition, from two independent experiments.
	Fig. 6. Model depicting role of Lhx9 in epicardial development in Xenopus. (A) During early tadpole stages, the epicardial lineage is determined and marked by transcription factors tcf21, tbx18, wt1 and lhx9 (blue) as a bilateral population of cells on the septum transversum caudal to the heart (red). (B) Lhx9 functions to drive clustering of cells to form the proepicardial cluster on the right side of the embryo (blue), whereby itga4 expression is activated. At this stage, signaling factors, most likely BMP (yellow arrows), from the heart atrioventricular sulcus (AVS) direct epicardial migration. (C,D) Lhx9-integrin-mediated signaling, including focal adhesion (FA) formation and phosphorylation of paxillin (pPaxillin), allows the PEO bridge (blue) to attach to the heart (red) at the AVS. (E,F) Once the PEO has attached to the heart, deposition of essential ECM components such as fibronectin and laminin are required for the epicardial layer (epic) to adhere and spread over the heart surface.
	Figure S1. Lhx9 genomic loci and isoform organization Schematics of X. laevis Lhx9 genomic locus (top) and mRNA (bottom), showing localization of LIM protein binding domains (red) in exon 2 and 3, and the DNA-binding homeodomain in exons 4 and 5 (blue). Not to scale. Note that lhx9α isoform harbors a truncated HD due to alternative splicing to exon 5a. Translation-blocking (purple) and splice-blocking (orange) Morpholinos are depicted on exon 1 of genomic locus. Green bars on mRNA schematics depict localization of in situ hybridization probes.
	Figure S2. Spatio-temporal analysis of lhx9α during Xenopus embryogenesis. In situ hybridization right, left, ventral and dorsal views of wild-type embryos showing lhx9a expression of the anterior region, from stage 34 to stage 46. 1; neural tube, 2; retina, 3; kidney, 4; septum transversum, 5; proepicardial cluster, 6; jaw cartilage.
	Figure S3. Spatio-temporal analysis of tbx18 and itga4 during Xenopus embryogenesis. (A-H) In situ hybridization of tbx18 showing right, left and ventral views of wild-type embryos from stage 34 to 46, anterior region of embryo. (I-P) In situ hybridization of itga4 showing right and left views of anterior region from stage 34-46. 1; cranial mesoderm, 2; somites, 3; branchial arches, 4; septum transversum, 5; proepicardial cluster, 6; epicardium.
	Figure S4. Spatio-temporal analysis of lhx9 during Xenopus embryogenesis. In situ hybridization of whole embryo anterior region using probe specific for the LIM domains of lhx9. Right, left, ventral and dorsal views of wild-type embryos from stage 34 to stage 46. 1; neural tube, 2; septum transversum, 3; retina, 4; kidney, 5; nasal placode, 6; proepicardial cluster, 7; otic placode, 8; jaw cartilage.
	Figure S5. Spatio-temporal analysis of lhx9HD during Xenopus embryogenesis. In situ hybridization right, left, ventral and dorsal views of wild-type embryos showing lhx9HD expression of the anterior region over time, from stage 34 to stage 46. 1; septum transversum, 2; retina, 3; neural tube, 4; nasal placode, 5; otic placode.
	Figure S6. Spatio-temporal analysis of lhx2 during Xenopus embryogenesis. In situ hybridization right, left, ventral and dorsal views of wild-type embryos showing lhx2 expression of the anterior region over time, from stage 34 to stage 46. 1; lung bud, 2; neural tube, 3; retina, 4; mandibular arch, 5; branchial arches, 6; pineal gland, 7; otic placode, 8; lower jaw.
	Figure S7. Validation of Lhx9 depletion assays. (A) Validation of MO-specific inhibition of Lhx9 translation by GFP western blot on stage 11 embryos, injected with 1ng Lhx9-5’UTR-GFP RNA and MO at various concentrations (20-40 ng). Shp2 is used as a protein loading control. MOT targets the translational start site from both the short and long genomic versions of X. laevis Lhx9. (B) RT-PCR analysis of cardiac cDNA from stage 42 embryos injected with both MO1 (30ng each) targeting the short and long genomic versions of X. laevis Lhx9. Negative control lanes (-) are without superscript II enzyme, GAPDH PCR as loading control.
	Figure S8. RT-PCR validation of epicardial marker expression in Lhx9-depleted hearts. RT-PCR was performed on equivalent amounts of RNA from control or Lhx9-MO1-depleted hearts from stage 41-42. Lhx9 is significantly depleted by two PCR amplification methods, as well as decreased itg4. Tbx18 and tcf21 appeared indistinguishable. Gapdh used as loading control.
	Figure S9. Lhx9 splice-blocking MO depletion strategy gives comparable PE clustering defects to translation-blocking MO. Clustering defects at stage 41 as assessed by (A) tbx18 and (B) tcf21 whole embryo in situ hybridization expression were present in Lhx9-MO1-depleted embryos (Fishers exact test p = <0.0001). (C) Defects observed in itga4 expression and localization was significant by Chi-square test (p = <0.0001) in Lhx9-MO1-depleted embryos. From three independent experiments.
	Figure S10. Lhx9 depletion has no obvious effects on vcam1 expression Lhx9-depletion at stage 41(B) did not significantly alter the expression of vcam1 in the heart compared to controls (A) by in situ hybridization, 6 embryos per condition. v; ventricle. Development • Supplementary information
	Figure S11. Lhx9α expression correlates with epicardial maker Integrin β1 Transverse agarose sections demonstrate the co-localization of lhx9α in situ hybridization (A, C) with the epicardial cell marker Itgβ1 (B, D, magenta) and DAPI (blue) at stage 40. Magnified images (C, D) shown in boxes (A, B). h, heart; peo, proepicardial organ.
	tbx18 (T-box 18) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 38, lateral view, anterior right, dorsal up.

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