Intercalated cushion cells within the cardiac outflow tract are derived from the myocardial troponin T type 2 (Tnnt2) Cre lineage

Abstract

Background: The origin of the intercalated cushions that develop into the anterior cusp of the pulmonary valve (PV) and the noncoronary cusp of the aortic valve (AV) is not well understood.

Results: Cre transgenes in combination with the Rosa TdTomato-EGFP reporter were used to generate three-dimensional lineage mapping of AV and PV cusps during intercalated cushion development. Tie2-Cre;EGFP was used to mark endothelial-derived mesenchymal cells, Wnt1-Cre;EGFP for cardiac neural crest and cardiac Troponin T (Tnnt2)Cre;EGFP, for myocardial lineage. The highest percentage of intercalated cushion cells at embryonic day (E) 12.5 was Tnnt2-Cre; EGFP positive; 68.0% for the PV and 50.0% AV. Neither Tnnt2 mRNA nor Tnnt2-Cre protein was expressed in the intercalated cushions; and the Tnnt2-Cre lineage intercalated cushion cells were also positive for the mesenchymal markers Sox9 and versican. Tnnt2-Cre lineage was present within the forming intercalated cushions from E11.5 and was present in the intercalated cushion derived PV and AV cusps and localized to the fibrosa layer at postnatal day 0.

Conclusions: Intercalated cushions of the developing outflow tract are populated with Tnnt2-Cre derived cells, a Cre reporter previously used for tracing and excision of myocardial cells and not previously associated with mesenchymal cells. Developmental Dynamics 247:1005-1017, 2018. © 2018 Wiley Periodicals, Inc.

Keywords: aortic valve; cardiac valves; endocardial cushion; mesenchymal; pulmonary valve; versican.

Figure 1. The intercalated cushions of the aortic and pulmonary valves are morphologically distinct, and form in an extracellular matrix of versican

Cardiac outflow tract (OFT) sections of an E12.5 heart shown from proximal to distal denoted on schematic in A. Panels B-D, stained for intact versican (Vcan, green). Panels A′–D′ immunolocalized for cleaved Vcan, (DPEAAE, green). Proximal to distal progression of AV-IC, (B–D; white outlines). Yellow bar-thinning of myocardial wall (B, C); white outline-AV-IC and PV-IC (D); aorticopulmonary septum, condensed cells, (B–D, B′–D′; asterisk); yellow lines-unfused margins of the major OFT cushions (B); white arrows-Vcan rich mesenchyme; yellow arrowhead-Vcan in myocardial sleeve (C); white arrowhead-subendothelial localization of cleaved Vcan, DPEAAE (C′, D′, green). Three-dimensional reconstructions (3D) of the entire cardiac outflow tract at E12.5 (E–G). D-Distal; P-proximal; LA-left atrium; RV-right ventricle; LV-left ventricle; Ao-developing aortic outlet; P-developing pulmonary outlet; PV-IC-pulmonary valve intercalated cushion; AV-IC-aortic valve intercalated cushion; AVC-atrioventricular cushions; AV-RC-aortic valve right coronary cusp; AV-LC-aortic valve left coronary cusp, PV-R-pulmonary valve right cusp; PV-L-pulmonary valve left cusp; I-inferior septal cushion; S-superior septal cushion. 3D orientation: A-anterior; P-posterior; R-right; L-left; V-ventral; D-dorsal. Cyan (E–F) denotes inferior septal cushion and P-R and A-RC cusps; yellow denotes superior septal cushion and P-L and A-LC cusps; magenta-intercalated cushions; *green-condensed mesenchymal cells. Bar in B=150μm applies to C-G. Data is representative of n=8 3D reconstructions and 10 additional E12.5 OFT samples evaluated in 2D.

Figure 2. Wnt1-Cre, cardiac neural crest contributes a relatively small volume to the intercalated cushions of the pulmonary and aortic valves

Cardiac outflow tract (OFT) sections of an E12.5 heart from proximal to distal denoted in A inset. Panels A–F, stained for versican (Vcan, blue), with Wnt1-Cre cell contribution (green); white arrowhead-condensed region of Wnt1-Cre (C–F). Panels G-L, Amira™ images with colormap intensity scale (blue = lesser; red=greater) for OFT septal cushions Wnt1-Cre; white-Wnt1-Cre contribution to intercalated cushions (IC); pink- Wnt1-Cre contribution to the pulmonary subendothelium (P-SE). Amira™ orthoslices with Wnt1-Cre overlay appear in G–I. Amira™ three-dimensional (3D) reconstructions of Wnt1-Cre shown in J–L. Transparent white-total volumes of IC; yellow asterisk-regions of the septal OFT cushions devoid of Wnt1-Cre; red arrowhead- Wnt1-Cre cells subjacent to endocardium; yellow lines (K) sections from 3D reconstruction shown in G, H and I respectively. RV-right ventricle; Ao-developing aortic outlet; P-developing pulmonary outlet; AV-RC-aortic valve right coronary cusp; AV-LC-aortic valve left coronary cusp, PV-R-pulmonary valve right cusp; PV-L-pulmonary valve left cusp; *-aorticopulmonary septum; P-SE (pink)-pulmonary subendothelial Wnt1-Cre. 3D orientation: A-anterior; P-posterior; R-right; L-left; V-ventral; D-dorsal. Bar in A=150μm applies to B–E and G–L; F=50μm. Data is representative of n=6 3D reconstructions of Wnt1-Cre and an additional n=12 hearts evaluated in 2D with technical replicates for each heart.

Figure 3. Tie2-Cre, EMT-lineage cells comprise a small volume of the intercalated cushions at E12.5

Cardiac outflow tract (OFT) sections of an E12.5 heart from proximal to distal denoted in A inset. Panels A, C–F, stained for Vcan (blue), and Tie2-Cre contribution (green). Yellow asterisk-regions of the major OFT Tie2-Cre positive cell in Vcan rich ECM (C, D, blue); white asterisk-aorticopulmonary septum; blue arrow-unfused proximal superior cushion. Panels G–L Amira™ images with Tie2-Cre lineage mesenchymal cells: bright yellow- IC; orange- RC, R; cream- LC, L; light gray-proximal inferior; dark gray- proximal superior region of septal OFT cushions; transparent yellow-total volumes of IC. Amira™ 2D orthoslices with Tie2-Cre mesenchymal cell overlay appear in G, I. 3D reconstructions of Tie2-Cre shown in H, J–L. Yellow lines (K) sections from 3D reconstruction shown in G and I respectively. RV-right ventricle; Ao-developing aortic outlet; P-developing pulmonary outlet; AV-RC-aortic valve right coronary cusp; AV-LC-aortic valve left coronary cusp, PV-R-pulmonary valve right cusp; PV-L-pulmonary valve left cusp; 3D orientation: A-anterior; P-posterior; R-right; L-left; V-ventral; D-dorsal. Bar in A=100μm; C=150μm applies to G–L; D=50μm applies to E, F. Data is representative of n=13 samples of Tie2-Cre evaluated in 3D with an additional n=4 evaluated in 2D sections with technical replicates for each heart.

Figure 4. Tnnt2-Cre, myocardial lineage comprises the majority of cells within the intercalated cushion of the pulmonary and aortic valves

Panels A–D, versican (Vcan, A, C, D; blue), cleaved Vcan (DPEAAE, B; blue) and Tnnt2-Cre;EGFP positive lineage (green). Panels E–H Amira™ images with Tnn2-Cre cells; pink-intercalated cushions (ICs); red-AV-RC, and PV-R; light red-AV-LC, and PV-L and transparent pink-total volumes of ICs. Amira™ orthoslices with Tnnt2-Cre mesenchymal cell overlay appear in E, F. Amira™ three dimensional (3D) reconstructions of Tnnt2-Cre shown in G, H. AV-RC-aortic valve right coronary cusp; AV-LC-aortic valve left coronary cusp, PV-R-pulmonary valve right cusp; PV-L-pulmonary valve left cusp. 3D orientation: A-anterior; P-posterior; R-right; L-left; V-ventral; D-dorsal. Panels A-K,′ data is representative of n=4 samples in 3D and an additional 7 samples evaluated in 2D with technical replicates for each heart. Immunolocalization of Mef2C (I, green) Nkx2.5 (J, green) and Cdh2 (K, K′, green). Graph in L depicts cell condensation in the PV-R, PV-IV and PV-L. *P < .0015; **P < .001; n=7 hearts each. Panels M–P, Sox9 (green); MF20 immunolocalization of myosin heavy chain (Q) and Cre protein immunolocalization (R, yellow arrows). Tnnt2 mRNA in situ localization (S, T, black arrows). Tnnt2-Cre;EGFP (blue, M, N, P, Q, R) and Propidium iodide (PI, red, C, D, I–K, M–R). Bar in A=150μm applies to B, E–H, M, S; C=50μm applies to D; I–K′, Q, R; Bar in N=20μm applies to O, P, T.

Figure 5. Tnnt2-Cre;EGFP positive cells are present at initiation of intercalated cushion formation

Cardiac OFT sections from proximal to distal of E12 (A–F) and E11 (G–L) to examine intercalated cushion formation. Sections denote Tnnt2-Cre;EGFP positive cells (blue), Vcan (green) and α smooth muscle actin (αSMA) localization. Arrows (B, C) denote Tnnt2-Cre;EGFP cells in region where the intercalated cushions of the aortic valve forms at E12; arrows in D–F indication Tnnt2-Cre;EGFP cell contribution to the forming pulmonary valve intercalated cushion at E12. Arrows (H, I) indicate Tnnt2-Cre;EGFP cells in regions where the aortic valve intercalated cushion forms; arrow in L early formation of pulmonary valve intercalated cushion Tnnt2-Cre positive cells. Sections AVC-atrioventricular cushion; AV-IC-aortic valve intercalated cushion; PV-IC-pulmonary valve intercalated cushion. Bar in A=150μm applies to B, C; D=75μm applies to E, F; G=75μm applies to H–L.

Figure 6. Postnatal day 0 anterior pulmonary valve and non coronary aortic valve cusps, derived from intercalated cushions contain Tnnt2-Cre;EGFP positive cells

Frontal sections from a postnatal day 0 heart (P0) from ventral to dorsal show the pulmonary valve (A–D) and aortic valve (E–H). Sections denote Tnnt2-Cre;EGFP positive cells (blue), Vcan (green) and α smooth muscle actin (αSMA) localization. Solid arrows (A, B) indicate Tnnt2-Cre;EGFP cells in the Anterior cusp of the pulmonary valve. Open arrow (A, B, D) indicate regions of Vcan localization devoid of Tnnt2-Cre lineage. Solid arrows (G, H) indicate Tnnt2-Cre; EGFP cells in the intercalated cushion derived noncoronary cusp; open arrows (F, G) Vcan rich regions of aortic valve cusps devoid of Tnnt2-Cre; EGFP cells. PV-pulmonary valve; R-right cusp; An-anterior cusp; L-left cusp; AV-aortic valve; RCC-right coronary cusp; LCC-left coronary cusp; NCC-noncoronary cusp. Bar in A=100μm applies to B–H.

Figure 7. Schematic depicting the lineage of the intercalated cushions that contribute to the pulmonary and aortic valves

Distal region schematic of the lineage contribution to the pulmonary and aortic valve mesenchyme at E12.5 (A). Green-intact Vcan; light green-cleaved DPEAAE Vcan; IC (intercalated cushion); red-OFT myocardium; dark red circles-Tnnt2-Cre lineage; yellow stellate-Tie2 lineage mesenchymal cells; yellow line-Tie2-Cre positive endothelium; white stellate-aorticopulmonary Wnt1-Cre; white small stellate; condensed Wnt1-Cre; black arrows-movement of Tnnt2-Cre lineage from myocardium to intercalated cushions. Percentage of lineage contribution, of total IC cells (B). Each symbol represents the average of three technical replicates of each Cre-lineage traced heart; (Tie2-Cre PV-IC, n=5; Wnt1-Cre PV-IC, n=5; Tnnt2-Cre PV-IC, n=5; Tie2-Cre AV-IC n=3; Wnt1-Cre, n=3, Tnnt2-Cre, n=3). C–G: 3D Amira™ reconstruction of E12.5 cardiac OFT, dark red-Tnnt2-Cre lineage; solid yellow-Tie2-Cre; transparent yellow-superior cushion derived mesenchyme; transparent cyan-inferior cushion contribution based on a total of n>3 complete outflow tract reconstructions *Wnt1-Cre (white) positive transient aorticopulmonary septum; 3D pulmonary intercalated cushion (PV-IC) lineage contribution shown in D and F. Aortic intercalated cushion (AV-IC) lineage contribution viewed in E and G. 3D orientation: V-ventral; D-dorsal, R-right; L-left; A-anterior; P-posterior. RC-Right coronary cusp; LC-left coronary; S-superior OFT cushion.