Pharyngeal pouches provide a niche microenvironment for arch artery progenitor specification

The paired pharyngeal arch arteries (PAAs) are transient blood vessels connecting the heart with the dorsal aorta during embryogenesis. Although PAA malformations often occur along with pharyngeal pouch defects, the functional interaction between these adjacent tissues remains largely unclear. Here we report that the ablation of pouches in zebrafish embryos impairs PAA progenitor specification and leads to the absence of PAA structures. Through time-lapse imaging studies, we reveal that the segmentation of pharyngeal pouches coincides spatiotemporally with the emergence of PAA progenitor clusters. These pouches physically associate with pharyngeal mesoderm in discrete regions and provide a niche microenvironment for PAA progenitor commitment by expressing BMP proteins. Specifically, tissue specific knockdown experiments demonstrate that pouch-derived BMP2a and BMP5 are the primary niche cues responsible for activating the BMP/Smad pathway in pharyngeal mesoderm, thereby promoting progenitor specification. In addition, BMP2a and BMP5 play a primary inductive function in the expression of the cloche gene npas4l in PAA progenitors. Mutation of the cloche locus represses the specification of PAA progenitors and generates ectopic muscle precursors in the pharyngeal mesoderm. Therefore, our results support a critical role of pharyngeal pouches in establishing a progenitor niche for PAA morphogenesis via BMP2a/5 expression.


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The paired pharyngeal arch arteries (PAAs) are transient blood vessels connecting the heart 24 with the dorsal aorta during embryogenesis. Although PAA malformations often occur along 25 with pharyngeal pouch defects, the functional interaction between these adjacent tissues 26 remains largely unclear. Here we report that the ablation of pouches in zebrafish embryos 27 impairs PAA progenitor specification and leads to the absence of PAA structures. Through Introduction 45 During vertebrate development, the pharyngeal arch arteries (PAAs), also known as 46 aortic arch arteries, are transient embryonic blood vessels that connect the heart to the dorsal 47 aorta and establish the circulatory system (Hiruma et al., 2002). These arteries form in a 48 cranial-to-caudal sequence, followed by the regression of the first and second PAAs, whereas 49 the PAAs 3, 4 and 6 undergo asymmetric remodeling and contribute to the carotid arteries and 50 great vessels of the heart, including the aorta and pulmonary arteries (Congdon, 1922;  To test this hypothesis, we evaluated the expression pattern of nkx2.5, the specific marker The endogenous nkx2.5 transcripts in wild-type embryos were also sequentially observed, but 133 eventually decreased when the PAA progenitors differentiated into angioblasts (S1C Fig). 134 Importantly, the transcripts of both ZsYellow and nkx2.5 were segregated in different domains 135 (S1C Fig). To further investigate this observation, we combined immunofluorescence and 136 fluorescence in situ hybridization experiments, and found that most of the nkx2.5 + progenitors 137 were restricted to the PAA clusters within ZsYellow + pharyngeal mesoderm, and the etv2 + and 138 scl + PAA angioblasts located in the ventral root of each sprouts (Fig 1A and 1B). Taken 139 together, these results show that the pharyngeal mesoderm within pharyngeal arches 3-6 is 140 composed of distinct subpopulations with or without nkx2.5 expression.

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The above findings raised an interesting question as to whether these subpopulations in internal control ( Fig 1C). As expected, the derivatives of the photoconverted cells were found 150 in the sprouts of PAAs 3-5 at 36 hpf and in the endothelium of the aortic arches 3-6 as well as 151 the ventral aorta at 60 hpf (Fig 1D and 1E). Interestingly, less red fluorescence and more 152 green fluorescence were observed in the cells of caudal PAAs 5-6 and the posterior portion of 153 ventral aorta (Fig 1E), suggesting that the progenitors of these structures are specified at later 154 8 stages and might undergo more proliferation during vasculogenesis after photoconversion. 155 Nevertheless, these results indicate that the endothelial cells of PAAs and ventral aorta 156 originate from the Kaede + pharyngeal mesoderm.

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Next, we specifically photoconverted the Kaede proteins in PAA cluster 5 at 36 hpf (Fig   158   1F). The photoconversion process resulted in red derivatives in PAA 5 at 54 hpf, but not in 159 other PAAs (Fig 1G). Interestingly, a few cells with red fluorescence were observed in the 160 junction of PAA 5 and ventral aorta (Fig 1G), suggesting the occurrence of endothelial cell 161 rearrangements during blood vessel fusion (Herwig et al., 2011). In contrast, the 162 photoconversion of Kaede + cells located between PAA cluster 4 and 5 led to red derivatives 163 housed specifically in ventral aorta (Fig 1H and 1I). Based on these observations, we 164 concluded that the ZsYellow + pharyngeal mesoderm cells within PAAs 3-6 are specified into 165 two vascular progenitor subpopulations: nkx2.5 + cells that give rise to PAAs and nkx2.5cells 166 that generate the connective ventral aorta. formed and reached the sprouting ZsYellow + cluster that would eventually give rise to PAA3 177 (Fig 2A). At later stages, the fourth, the fifth and the sixth pouches successively made contact 178 with the developing ZsYellow + clusters for PAAs 4-6 (Fig 2A), indicating a close interaction 179 between the endodermal pouches and the progenitor clusters within pharyngeal mesoderm.    strong GFP protein expression in the ZsYellow + clusters and the other nearby tissues (Fig 3C).

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These results demonstrate that BMP/Smad signal is highly activated in the presumptive PAA 240 progenitors. 241 We then ablated the pharyngeal pouches in Tg(nkx2.5:ZsYellow) and Tg(BRE:EGFP) 242 embryos, respectively. Interestingly, the pouch-depletion led to an evident decrease in BMP 243 signal activity in both ZsYellow + clusters and other pharyngeal tissues (Fig 3D and 3E).

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These findings imply that pharyngeal pouches function as a niche for activating BMP signals   growing sprouts at 60 hpf (Fig 5A and 5B). Therefore, when BMP inhibition is removed, the 311 pharyngeal mesoderm cells may recover their endothelial potential. specificity of these MOs. We observed that the expression of PAA angioblast marker etv2 was 323 not obviously changed in embryos injected with bmp4 MO (Fig 6A and 6B). It was surprising 324 that the expression of etv2 was almost abolished in bmp2b morphants (Fig 6A and 6B). We a steady reduction in etv2 + cluster numbers, and this reduction became more pronounced 332 when these two genes were knocked down at the same time (Fig 6A and 6B). Furthermore, 333 the phosphorylation of Smad1/5/8 and the expression of nkx2.5 were evidently decreased in 334 the pharyngeal region (Fig 6C and 6D). Together, these data suggest that bmp2a together with 335 bmp5 plays an important role in PAA progenitor specification.

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To examine the direct function of pouch-expressed BMP ligands, we performed    Fig 6E). By contrast, injection of 375 bmp5 MO into to bmp2a -/embryos induced more obvious defects in PAA progenitor 376 specification in a much higher proportion of animals ( Fig 6E). Finally, at 38 hpf, a significant 377 reduction in etv2 + angioblast clusters was observed in bmp2a -/mutants injected with bmp5 378 MO when compared to control animals and bmp2a -/or bmp5 -/embryos (Fig 6F and 6G).

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Collectively, these data suggest that, among the BMPs emerged from the pouch   region was analyzed by in situ hybridization. We found that npas4l was not expressed in the 395 pharynx at 20 hpf ( Fig 7A). But then npas4l transcripts was detected in the presumptive PAA 396 progenitor cluster 3 at 24 hpf, approximately 2 hours later than the initial expression of 397 nkx2.5 in the same PAA cluster (Fig 7A). Over the next 14 hours, npas4l transcripts were 398 gradually appeared in a craniocaudal sequence in the PAA clusters (Fig 7 B). Moreover, the 399 expression of npas4l in the PAA clusters was further confirmed by the colocation of npas4l 400 and nkx2.5 transcripts (Fig 7C).

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Previous study has shown that the expression of nkx2.5 is reduced following the Indeed, compared to control animals, embryos injected with nkx2.5 MO showed much higher 412 levels of nkx2.5 expression (Fig 7D). By contrast, the expression levels of npas4l was not 413 obviously changed in the pharynx upon nkx2.5 MO injection (Fig 7E). These results may 414 imply that although the inhibition of nkx2.5 function led to excess PAA progenitors at the 415 expense of angioblasts, the total number of cells with endothelial potential was unchanged.

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Thus, npas4l is expressed in both PAA progenitors and angioblasts. 417 We next examined whether npas4l plays a role in PAA development. In situ hybridization 418 20 analyses revealed that, in comparison to wild-type and heterozygous siblings, cloche 419 homozygous (cloche -/-) mutants exhibited normal nkx2.5 expression in pharyngeal clusters 420 3-5 at 32 hpf (Fig 7F). To our surprise, the expression of etv2, the PAA angioblast marker, 421 was completely missing in the cloche -/mutants at 38 hpf (Fig 7G), suggesting a successful progenitors. Therefore, before npas4l expression, the pharyngeal mesoderm might maintain 434 its multiple differentiation potential into at least the endothelial and muscular lineages.

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Together, these data suggest that npas4l plays a pivotal role in the specification of PAA 436 progenitors from pharyngeal mesoderm.

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To learn whether pharyngeal pouches are required for npas4l expression, pharynx at 38 hpf ( Fig 7I). Moreover, both DMH1 treatment and injection with MOs 441 targeting bmp2a and bmp5 induced a dramatic reduction in npas4l transcripts (Fig 7I). We 442 also found a steady decrease of the number of npas4l + PAA clusters in bmp2a -/or bmp5 -/-443 single mutants and bmp2a -/embryos injected with bmp5 MO (Fig 7J and 7K). Taken together, 444 these findings support the idea that the pharyngeal pouches provide a niche 445 microenvironment for the commitment of multipotent pharyngeal mesoderm toward PAA 446 progenitors through expressing BMP2a and BMP5 (Fig. 7L).

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The amplified fragments were identified with Sanger DNA sequencing for genotyping. F2