Transcriptional profiles in the chicken ductus arteriosus during hatching

Abstract

The ductus arteriosus, an essential embryonic blood vessel between the pulmonary artery and the descending aorta, constricts after birth or hatching and eventually closes to terminate embryonic circulation. Chicken embryos have two long ductus arteriosi, which anatomically differ from mammal ductus arteriosus. Each long ductus arteriosus is divided into two parts: the pulmonary artery-sided and descending aorta-sided ductus arteriosi. Although the pulmonary artery-sided and descending aorta-sided ductus arteriosi have distinct functional characteristics, such as oxygen responsiveness, the difference in their transcriptional profiles has not been investigated. We performed a DNA microarray analysis (GSE 120116 at NCBI GEO) with pooled tissues from the chicken pulmonary artery-sided ductus arteriosus, descending aorta-sided ductus arteriosus, and aorta at the internal pipping stage. Although several known ductus arteriosus-dominant genes such as tfap2b were highly expressed in the pulmonary artery-sided ductus arteriosus, we newly found genes that were dominantly expressed in the chicken pulmonary artery-sided ductus arteriosus. Interestingly, cluster analysis showed that the expression pattern of the pulmonary artery-sided ductus arteriosus was closer to that of the descending aorta-sided ductus arteriosus than that of the aorta, whereas the morphology of the descending aorta-sided ductus arteriosus was closer to that of the aorta than that of the pulmonary artery-sided ductus arteriosus. Subsequent pathway analysis with DAVID bioinformatics resources revealed that the pulmonary artery-sided ductus arteriosus showed enhanced expression of the genes involved in melanogenesis and tyrosine metabolism compared with the descending aorta-sided ductus arteriosus, suggesting that tyrosinase and the related genes play an important role in the proper differentiation of neural crest-derived cells during vascular remodeling in the ductus arteriosus. In conclusion, the transcription profiles of the chicken ductus arteriosus provide new insights for investigating the mechanism of ductus arteriosus closure.

Fig 1. Distinct morphological changes in the proximal portions of the chick DA during development.

Representative histological sections of the proximal and distal DA from ED16 to AB. Serial sections were stained with HE (a) and EVG staining (b). (a) The nucleus in the middle lamella of the distal DA were aligned in parallel to the internal elastic laminar and layered throughout the hatching, whereas the layers were disorganized in the proximal DA from the IP stage. Scale bars are 50 μm. (b) The proximal DA and the distal DA had a muscular and an elastic type of artery, respectively. Moreover, in the proximal DA, the poor elastic fiber formation was observed in the middle lamella from the IP stage. Scale bars are 50 μm. (c) The vessel wall thickness of the chick DA at developmental stages. The vessel wall of the proximal DA was thin compared to the distal DA at the ED16 and ED19 stage. Moreover, the vessel wall of the proximal DA drastically thickened from the ED19 to the IP stage. * and ** indicate p < 0.05 and p<0.01, †† indicates p<0.01 compared to the same tissues of the ED16 stage, # and ## indicate p < 0.05 and p<0.01 compared to the same tissues of the ED19 stage, ‡ and ‡‡ indicate p < 0.05 and p<0.01 compared to the same tissues of the IP stage, § indicates p<0.05 compared to the same tissues of the EP stage, n = 4–5. (d) The vessel luminal area of the chick DA at developmental stages. * indicates p < 0.05. n = 4–5.

Fig 2. Quantitative PCR analysis for proximal DA-enriched or aorta-enriched genes.

The relative mRNA levels of col8a1 (a), tnc (b), mamdc2 (c), hpse2 (d) or fam19a2 (e) to gapdh were quantified by RT-PCR. * and ** indicate p < 0.05 and p<0.01, † and †† indicate p < 0.05 and p<0.01 compared to the same tissues of the ED16 stage, # and ## indicate p < 0.05 and p<0.01 compared to the same tissues of the ED19 stage, ‡ and ‡‡ indicate p < 0.05 and p<0.01 compared to the same tissues of the IP stage, §§ indicates p<0.01 compared to the same tissues of the EP stage, n = 5–6.

Fig 3. Quantitative PCR analysis for melanogenesis/tyrosine metabolism-related genes.

The relative mRNA levels of each gene, dct (a), ednrb2 (b), tyr (c), tyrp1 (d) or wnt11 (e), to gapdh were quantified by RT-PCR. We performed this experiment in quintuplicate and noted results as ND only if we could not detect any signals from two or more samples among the five available samples. * and ** indicate p < 0.05 and p<0.01, † and †† indicate p < 0.05 and p<0.01 compared to the same tissues of the ED16 stage, # and ## indicate p < 0.05 and p<0.01 compared to the same tissues of the ED19 stage, ‡ and ‡‡ indicate p < 0.05 and p<0.01 compared to the same tissues of the IP stage, §§ indicates p<0.01 compared to the same tissues of the EP stage, n = 5. ND: not detected.

Fig 4. Hierarchical clustering of normalized DNA array data.

The graphical representation showed that the profiles of gene expression were grouped into two distinct clusters: a DA-related cluster and an aorta-related cluster (distance = 1 − Pearson’s correlation coefficient, Ward’s method).