{'fraction_non_alphanumeric': 0.03204868154158215, 'fraction_numerical': 0.040432724814063556, 'mean_word_length': 4.680491551459293, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Endothelial TGF-β signaling instructs smooth muscle development in the cardiac outflow tract

The development of the cardiac outflow tract (OFT), which connects the heart to the great arteries, relies on a complex crosstalk between endothelial (ECs) and smooth muscle (SMCs) cells. Defects in OFT development can lead to severe malformations, including aortic aneurysms, which have often been associated with impaired TGF-β signaling. To further investigate the role of TGF-β signaling in OFT formation, we generated zebrafish lacking the type I TGF-β receptor Alk5 and found a strikingly specific dilation of the OFT. alk5 mutants also exhibit increased EC numbers, extracellular matrix (ECM) and SMC disorganization. Surprisingly, endothelial-specific alk5 overexpression in alk5 mutants rescues both endothelial and SMC defects. Furthermore, modulation of the ECM gene fibulin-5, a TGF-β target, partially restores OFT morphology and function. These findings reveal a new requirement for endothelial TGF-β signaling in OFT morphogenesis and suggest an important role for the endothelium in the etiology of aortic malformations.


37
The cardiovascular system is essential to deliver blood to the entire organism. Within the 38 heart, however, the high pressure deriving from ventricular contractions needs to be buffered Despite the evidence for a potential role for ALK5 in the endothelium, most studies in OFT 82 and aortic pathologies, such as aortic aneurysms, have been focused on the role of TGF-β   Here, we generated a zebrafish alk5 mutant and observed a severe dilation of the developing 100 OFT. We show that this phenotype results from early defects in EC proliferation and  Figure S1). 115 We detected alk5b expression in the neural tube starting at 24 hours post fertilization (hpf) 116 and in the gut at 72 hpf (Figure S1A-C'). Notably, in the developing cardiovascular system, 117 alk5b reporter expression appeared to be restricted to the heart ( Figure S1E

121
In order to investigate Alk5 function, we used CRISPR/Cas9 technology to generate mutants 122 for alk5a and alk5b. We obtained a 4 bp and a 5 bp deletion in alk5a and alk5b, respectively, each leading to the predicted generation of truncated proteins, which lack the essential kinase 124 domain ( Figure S1F). Moreover, alk5a and alk5b mutant mRNA levels are decreased in the 125 respective mutant fish while the other paralog does not appear to be upregulated, suggesting 126 mutant mRNA degradation and a lack of transcriptional adaptation (El-Brolosy et al., 2019) 127 ( Figure S1G). Single alk5a and alk5b mutant larvae do not exhibit any gross morphological 128 defects, other than the lack of inflation of the swim bladder in alk5b mutants (Figure S1H-J).

129
Therefore, to achieve a complete blockade of Alk5 signaling, we generated alk5a, alk5b 130 double mutants (alk5a -/-;alk5b -/-, hereafter referred to as alk5 mutants). Loss of Alk5 function 131 does not lead to early developmental defects until 72 hpf, when mutant larvae start exhibiting 132 pericardial edema, evident at 96 hpf ( Figure S1K), suggesting defective cardiac function. By 133 analyzing heart morphology in live Tg(kdrl:eGFP) alk5 mutant embryos, we observed a 134 specific increase in OFT width by 54 hpf (Figure 1C-H). Live imaging on beating hearts 135 showed that the dilation of the mutant OFT grows more severe with time, becoming more 136 than twice as large as wild type by 78 hpf (+162%; Figure 1I-K; Video S1, S2). This 137 expansion of the OFT is accompanied by its inability to pump blood into the connecting 138 vessels, leading to retrograde flow into the ventricle (Video S3).

139
In 78 hpf wild-type zebrafish, the OFT is connected to the aortic arches by a single vessel, the 140 ventral aorta (VA) ( Figure S1L). alk5 mutants fail to form this vessel, leading to two 141 independent connections from the OFT to the left and right aortic arches ( Figure S1M).

151
Taken together, these results identify a previously unknown and specific requirement for Alk5 152 in OFT morphogenesis, structural integrity, and functionality.

154
Alk5 restricts EC proliferation in the cardiac outflow tract and promotes EC migration 155 towards the ventral aorta 156 Given the increased size of the mutant OFT, we asked whether this phenotype was 157 accompanied by an increase in cell number. In wild-type animals, the average number of 158 OFT ECs increases from 21, at 36 hpf, to 45, at 72 hpf (Figure 2A, B). Consistent with the 159 absence of a morphological phenotype, the mutant OFT, at 36 hpf, was composed of an 160 average of 22 ECs, similar to wild type ( Figure 2B). However, the number of ECs in mutant 161 OFT diverges substantially over time, and by 72 hpf twice as many ECs were observed in 162 mutants compared to wild types (85 ECs; Figure 2B). To investigate the underlying cause of 163 this increase, we performed EdU labeling to assess EC proliferation. In the mutant OFTs, we 164 found that, by 36 hpf, ECs were already more likely to undergo cell cycle reentry than ECs in wild-type OFTs ( Figure 2C-E). This abnormal increase in the number of EdU + ECs in mutant 166 OFTs becomes even more pronounced at later stages (48-72 hpf; Figure 2F-H).

167
Along with proliferation, EC migration has been implicated in the patterning and formation of 168 blood vessels, such as in the case of the aortic arches (Rochon et al., 2016). Therefore, we set 169 to investigate if the absence of the VA in alk5 mutants could be attributed to a defect in EC   During early larval stages, the OFT becomes covered by SMCs, which allow it to buffer the 194 high blood pressure caused by ventricular contractions. In order to visualize SMCs, we used 195 the Tg(pdgfrb:eGFP) line, which labels these cells before they differentiate into more mature 196 SMCs and start expressing established markers such as Acta2 (smooth muscle actin; Figure   197 3A-B'). We observed that the OFT endothelium in 75 hpf wild-type larvae is surrounded by  Figure 3D). Furthermore, using TUNEL staining (data 204 not shown), we excluded SMC death by apoptosis.

205
In order to form a compact yet elastic wall, the SMCs are embedded within a specialized 206 extracellular matrix (ECM), which provides essential biomechanical support as well as 207 signaling cues to the SMCs (Raines, 2000). To assess whether and how ECM structure and composition were affected in alk5 mutants, we analyzed the localization of Elastin2 (Eln2; 209 Figure 3E-G), a major ECM component in the OFT (Miao et al., 2007). We observed that in  In order to characterize the EC-specific OFT rescue at a cellular level, we performed EdU

252
Overall, these data suggest that endothelial Alk5 signaling is sufficient to restore OFT 253 morphology and function, including SMC wall formation.

255
Alk5 signaling regulates ECM gene expression 256 We hypothesized that Alk5 is required in the OFT endothelium and that it controls an

Endothelium-smooth muscle interplay in the cardiac outflow tract and aorta
Surprisingly, we found that alk5 overexpression in ECs was sufficient to restore SMC wall 335 formation. Although these results do not resolve whether ECs are the only cell type in which 336 Alk5 is necessary for OFT morphogenesis, they suggest that the SMC phenotype could be a 337 secondary effect from the crucial cross-talk between these two cell types (Gaengel et