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First published online 5 September 2007
doi: 10.1242/dev.008599

Development 134, 3723-3732 (2007)
Published by The Company of Biologists 2007
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Long form of latent TGF-ß binding protein 1 (Ltbp1L) is essential for cardiac outflow tract septation and remodeling

Vesna Todorovic1,*, David Frendewey2, David E. Gutstein1,3, Yan Chen1, Laina Freyer1,{dagger}, Erin Finnegan1, Fangyu Liu3, Andrew Murphy2, David Valenzuela2, George Yancopoulos2 and Daniel B. Rifkin1,3

1 Cell Biology Department, NYU School of Medicine, New York, NY 10016, USA.
2 Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA.
3 Department of Medicine, NYU School of Medicine, New York, NY 10016, USA.


Figure 1
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  		Fig. 1. Targeted inactivation of the Ltbp1L gene. (A) Schematic representation of the Ltbp1 short (S) and long (L) forms. (B) Targeting of the Ltbp1L locus. The Ltbp1L gene is composed of 34 exons (represented as vertical bars above the gene). Four exons specific for Ltbp1L, and not for Ltbp1S, are presented as red bars. Two null alleles, designated A and B, were generated. Deletion `A' encompasses 457 bp of exon 1 and a portion of intron 1. Deletion `B' is 7.8 kb long and includes most of exon 2 and a portion of intron 2. (C,D) Validation of Ltbp1L gene targeting. (C) Upper panels: northern blot with E14.5 wild-type and knockout (KO) RNA (left) and wild-type and KO RNA isolated from immortalized E17.5 MEFs (right), hybridized to a [32P]-labeled Ltbp1L cDNA. Lower panels: RNA loading control. (D) Western blots with total-protein extracts isolated from wild-type and KO E14.5 hearts incubated with an Ltbp1 antibody (upper panels) and a ß-actin antibody (lower panels). (E) Newborn control and Ltbp1L-/- mice. Ltbp1L-/- neonates are cyanotic and unable to suckle (arrow illustrates the lack of milk in the stomach).

 

Figure 2
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  		Fig. 2. Cardiovascular defects in Ltbp1L-/- mice. Gross dissections of newborn wild-type (A) and Ltbp1L-/- (B-E) mice with left or both atria removed to facilitate visualization of the great vessels. The ductus arteriosus is labeled with an asterisk in A-C. The great vessels are labeled as follows: Ao, aorta; PA, pulmonary artery; dAo, dorsal aorta; PTA, persistent truncus arteriosus. PTA with type B (B,E) and type C (C) IAA in Ltbp1L nulls. (D) PTA and a dextraposed aortic arch. (E) Ectopic position of a coronary artery (arrowhead). (F-I) Hematoxylin and Eosin-stained frontal sections of newborn hearts: (F,H) control; (G,I) mutant heart. PTA (G) is labeled with an asterisk. The membranous ventricular septum (G, arrow) and the atrial septum (I, arrowhead) are absent in the mutants.

 

Figure 3
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  		Fig. 3. Ltbp1L expression in the septating heart. Whole-mount (A) and lateral sections (B,C) of an E9.5 Ltbp1L heterozygous heart, stained with X-gal. Myocardial cells underlying outflow tract (OFT; B) and atrio-ventricular (AV; C) endocardial cushions express Ltbp1L (black arrows). Red arrows in A and B mark the thyroid primordium. (D-F) Lateral sections of E10.5 (D) and E11.5 (E,F) heterozygous hearts, stained with X-gal and Eosin. Myocardium (My) underlying OFT and AV cushions, mesenchymal cells (M) within the cushions and some E11.5 endocardial cells (E) express Ltbp1L. (F) Endoderm of pharyngeal pouches expresses Ltbp1L (arrows). The third, fourth and sixth pharyngeal arch arteries (PAAs) are labeled as 3, 4 and 6, respectively. (G-I) Transverse sections of an E12.5 heterozygous heart at the level of the ductus arteriosus (G), the fourth PAA (H) and the third PAA (I) stained with X-gal and Eosin. Arrows indicate Ltbp1L-expressing smooth muscle cells (SMCs) surrounding dorsal aortae and PAAs.

 

Figure 4
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  		Fig. 4. The fate of CNCCs in Ltbp1L mutants. (A,B) Whole-mount in situ hybridizations of E9.5 wild-type (A) and knockout (KO; B) embryos, using a Crabp1 ribo-probe. Broken lines outline streams of cardiac neural crest cells (CNCCs) migrating into the outflow tract (OFT). (C,D) Fate mapping of CNCCs in Ltbp1L mutants. E11.5 wild-type (C) and Ltbp1L-/- (D) transverse sections at the level at which the fourth pair of pharyngeal arch arteries (PAAs) branches out of the aortic sac. Green cells represent CNCCs. Arrowheads points to the aortico-pulmonary (AP) septum (C), or where it is missing (D). (E,F) Transverse sections of E11.5 heterozygous (E) and KO (F) embryos, stained with X-gal and Eosin. Blue cells express Ltbp1L. Black arrowheads indicate the AP septum (E) or its absence (F). Red arrowheads indicate Ltbp1L-expressing smooth muscle cells (SMCs) surrounding dorsal aortae. Ms, pharyngeal mesenchyme. (G) Quantification of mesenchymal cells and proliferating mesenchymal cells in E11.5 control and KO OFTs. Average of three sets of samples is shown. (H-K) Whole-mount in situ hybridization of E11.5 control (H,J) and KO (I,K) hearts using plexin A2 (H,I) and FoxC1 (J,K) ribo-probes. Plexin A2 and FoxC1 visualize post-migratory CNCCs in the control (arrows in H and J) but not Ltbp1L-/- OFT.

 

Figure 5
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  		Fig. 5. Abnormal remodeling of PAAs in Ltbp1L mutants and normal differentiation of CNCCs into SMCs. (A,B) Visualization of pharyngeal arch arteries (PAAs) by India ink injection in E10.5 control (A) and knockout (KO; B) hearts. The third, fourth and sixth PAAs are properly formed in Ltbp1L-/- embryos. (C,D) Semi-transverse sections of E11.5 control and mutant embryos, stained with {alpha}SMA antibody and Hematoxylin. A ring of smooth muscle cells (SMCs; brown) surrounds all three pairs of PAAs both in control (C) and in KO (D) embryos. (E,F) Normal differentiation of CNCCs within the OFT of control and Ltbp1L-/- into SMCs. SMCs are visualized by staining with an antibody to {alpha}SMA. (G-J) Left fourth PAA is shown. Transverse sections of E12.5 control (G,I) and Ltbp1L-/- (H,J) embryos stained with active caspase 3 (G,H) or {alpha}SMA (I,J) antibody. Notice the clusters of apoptotic cells in the mutant left fourth PAA (arrowheads in H).

 

Figure 6
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  		Fig. 6. Decreased Tgf-ß signaling in the septating OFT of Ltbp1L mutants. (A) Quantification of Tgf-ß1, Tgf-ß2, Tgf-ß3, Ltbp3, Ltbp4 and c-Myc expression by Q-RT-PCR, using cDNA isolated from three sets of E11.5 control (blue) and knockout (KO, burgundy) hearts. (B) Distribution of pSmad2 in E11.5 control (upper) and KO (lower) OFTs. Framed parts of the outflow tracts (OFTs) are magnified to show cardiac neural crest cell (CNCC)-specific attenuation of Smad2 phosphorylation. (C) Ctgf expression in E11.5 control and KO hearts estimated by in situ hybridization on sections taken at levels (a, b, c) indicated in the diagram and (D) semi-quantitative RT-PCR on cDNA isolated from E11.5 hearts.

 

Figure 7
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  		Fig. 7. Summary and a model for Ltbp1L requirement in the septating OFT. (A) Cardiac neural crest cells (CNCCs; orange) initially (E9.5-10.5) express genes necessary for proper specification, delamination and migration. As CNCCs relocate to the outflow tract (OFT) at E11.5, their gene expression program changes (blue CNCCs). This reprogramming correlates with morphological changes, such as aortico-pulmonary (AP) septum formation, remodeling of pharyngeal arch arteries (PAAs) and differentiation into smooth muscle cells (SMCs). At E12.5, CNCCs continue their gene expression program (blue cells) that enables them to elongate the AP septum, to continue remodeling PAAs and to differentiate into SMCs. Ltbp1L-/- CNCCs are normal and have a migratory trajectory undistinguishable from control littermates. The gene expression pattern characteristic for the migrating CNCCs also appears normal in Ltbp1L-/- embryos (orange CNCCs). However, when CNCCs invade the ECM in the OFT of Ltbp1L nulls, their gene expression program and function alters (E11.5, green CNCCs rather than blue) because they fail to form the AP septum. At E12.5, other morphological changes are evident - remodeling of defective PAAs and AP septum absence. However, CNCCs in Ltbp1L nulls successfully differentiate into SMCs. (B) Functional Tgf-ß signaling is required for the maintenance of the appropriate gene expression program and function of post-migratory CNCCs. The ECM of the OFT contains both Ltbp1L-LLC and Ltbp3-LLC. Post-migratory CNCCs specifically recognize Ltbp1L-LLC and activate the latent Tgf-ß. When the extracellular matrix (ECM) of the OFT is deprived of Ltbp1L, post-migratory CNCCs do not sufficiently propagate Tgf-ß signals and, therefore, do not express genes required for their proper function (green CNCCs), resulting in persistent truncus arteriosus (PTA). Ltbp3 cannot substitute for Ltbp1L function, because post-migratory CNCCs do not recognize and act on Ltbp3-LLC.

 

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© The Company of Biologists Ltd 2007