Backgrounds

In mammals, pharyngeal arch arteries are initially formed as symmetric pairs of arteries that connect aortic sac with dorsal aortae. They undergo a remodeling process and finally form thoracic arteries including thoracic aorta, pulmonary artery and ductus arteriosus. The abnormal remodeling process of pharyngeal arch arteries in human results in congenital vascular anomalies such as hypoplastic or interrupted aortic arch.

Cranial neural crest cells are known to be committed to the remodeling process of pharyngeal arch arteries. During neural tube closure, they migrate in distinct streams and contribute to head mesenchyme together with mesodermal cells. Cardiac neural crest cells, constituting a subpopulation of cranial neural crest cells, migrate to the 3rd, 4th and 6th pharyngeal arch arteries, aortic sac, and the conotruncal region of heart, and contribute to the medial layer of great arteries and aortopulmonary septum.

Endothelin-1 (Edn1) was originally identified as a vascular endothelium-derived vasoconstrictor. Edn1 binds to its G-protein coupled receptor, Endothelin receptor type A (Ednra), and contributes to the development of pharyngeal arches. Defects in the Edn1/Ednra pathway result in the malformation of pharyngeal-arch-derived craniofacial structures and thoracic arteries in mice.

Homeotic transformation of the lower jaw into the upper jaw structure with downregulation of the homeobox genes Dlx5/Dlx6 in Edn1-null embryos indicate the involvement of the Edn1/Ednra pathway in the dorsoventral axis patterning of pharyngeal arch system as a positive regulator of Dlx5/Dlx6 expression.

In contrast to the Edn1/Ednra-dependent pathway involved in craniofacial patterning, the pathway involved in pharyngeal arch artery remodeling to form thoracic arteries is largely unknown. The aim of this study is to clarify the mechanism by which the Edn1/Ednra signaling regulates pharyngeal arch artery remodeling.

Methods

Ednra(+/lacZ), Ednra(+/EGFP) (lacZ or EGFP knock-in) mice and Edn1(-/-) mice have been described previously. By crossing these mice, the EdnralacZ/EGFP (Ednra-null) mice were obtained. Ednra(Ednrb/Ednrb) (Ednrb knock-in) mice have been also reported, in which not Ednra but Ednrb is expressed under the control of the Ednra gene promoter. Dlx5/Dlx6 double knock-out mice have been reported previously. Wnt1-Cre mice, in which Cre recombinase is expressed in early neural crest cells, were crossed with R26R reporter mice containing a loxP-flanked lacZ cassette in the Rosa26 locus, to generate Wnt1-Cre;R26R mice, in which β-galactosidase expression is observed along the neural tube and in most neural crest-derived cells. We further generated Wnt1-Cre;R26R;Ednra(EGFP/EGFP)(Ednra-null) mice by crossing above-mentioned mice.

Using these mice, I performed phenotypic evaluation by ink injection, wholemount or section immunostaining for several markers, and β-galactosidase stainig.

Results

1. Edn1/Ednra signaling regulates the remodeling of thoracic arteries.

First, the phenotype of thoracic arteries in E18.5 embryos was examined by ink injection. In wild-type embryos, like in humans, three vessels branch from aortic arch; brachiocephalic, left common carotid, and left subclavian arteries. The brachiocephalic artery then bifurcates to form right subclavian and right common carotid arteries. By contrast, in Edn1- or Ednra-null embryos, abnormal vessels from bilateral common carotid arteries and abnormal bifurcation of the brachiocephalic artery were observed with high incidence, as previously described. Some Edn1-null embryos demonstrated more severe phenotype like type-B aortic arch interruption or hypoplastic aortic arch. These aortic arch anomalies were not observed in Ednra-null embryos of our ICR background, in contrast to a previous report on C57BL/6 background.

Next, the thoracic arteries of Ednra(Ednrb/Ednrb) (Ednrb-knock-in) embryos were examined. Ednrb-knock-in embryos demonstrated vascular anomalies similar to those of Ednra-null embryos, indicating that Ednrb gene expression cannot rescue the thoracic artery anomalies of Ednra-null mice. Thus, Edn1/Ednra signaling regulates the morphogenesis of thoracic great vessels, like in craniofacial development.

2.Regulation of vascular morphogenesis by the Edn-1/Ednra signaling is independent of Dlx5/Dlx6-mediated regional identification of pharyngeal arches.

In craniofacial development, the Edn1/Ednra signaling regulates the expression of the homeobox genes Dlx5 and Dlx6. Deletion of Edn1 or Ednra results in downregulation of Dlx5/Dlx6 in mandibular arch and induces homeotic transformation of the lower jaw into the upper jaw structure. To investigate whether this Dlx5/Dlx6-mediated pathway is involved in vascular morphogenesis, the phenotype of thoracic great vessels in E18.5 Dlx5/Dlx6 double knock-out embryos was examined. Almost all Dlx5/Dlx6 double knock-out embryos demonstrated the normal pattern of great vessels, although they presented the formerly-reported craniofacial anomalies, the homeotic transformation of the lower jaw into the upper jaw structure. These data indicate that, unlike craniofacial development, vascular morphogenesis does not depend on the Dlx5/Dlx6-dependent ventral identification of pharyngeal arches, and requires an alternative Dlx5/Dlx6-independent molecular pathway downstream of the Edn1/Ednra signaling.

3.The abnormal vessels from common carotid arteries derive from the abnormally persistent first and second pharyngeal arch arteries.

I focused on the abnormal vessels from the common carotid arteries in Ednra-null mice and sought for their origin. First, I performed the CD31 (vascular endothelial marker) immunostaining of the pharyngeal arch arteries at the forming stage (E9.5-10.5). The first and second pharyngeal arch arteries equally formed both in Ednra-null and heterozygous embryos at E9.5, but the regression of these arteries was less eminent in Ednra-null embryos at E10.5. Next, the pharyngeal arch arteries at the remodeling stage (E10.5-E12.5) were visualized by ink injection. In heterozygous embryos five pairs of symmetrical pharyngeal arch arteries underwent a remodeling process to form the final pattern of thoracic great vessels at E12.5. In this process, the first and second pharyngeal arch arteries regressed to a large extent. By contrast, the first and second pharyngeal arch arteries persisted beyond E12.5 in Ednra-null embryos. These abnormally persistent first and second pharyngeal arch arteries were likely to be the origin of the abnormal vessels from common carotid arteries, which are known to derive from the third pharyngeal arch arteries.

4.The Edn1/Ednra signaling regulates the properly-directed differentiation of neural crest cells into smooth muscle cells in pharyngeal arch arteries.

PDGFRβ is known as a marker of developing smooth muscle cells and pericytes. In E10.5 Ednra-heterozygous embryos, PDGFRβ-positive cells surrounded the third, fourth and sixth pharyngeal arch arteries, but not the first and second pharyngeal arch arteries. On the other hand, PDGFRβ expression was detected also around the first and second pharyngeal arch arteries in Ednra-null embryos. In E11.5 Ednra-null embryos, αSMA, a marker of differentiated smooth muscle cells, started to be expressed in the first and second pharyngeal arch arteries, in addition to PDGFRβ. These data indicate that smooth muscle cells are aberrantly developed along the first and second pharyngeal arch arteries in Ednra-null embryos.

Next, Wnt1-Cre;R26R mice were analyzed, to examine whether these aberrant smooth muscle cells in the first and second pharyngeal arch arteries derive from neural crest cells. As early as at E9.5, the forming stage of pharyngeal arch arteries, β-galactosidase-positive neural crest cells were surrounding the first and second pharyngeal arch arteries in Wnt1-Cre;R26R embryos, suggesting that neural crest cells are the primary source of smooth muscle cells in pharyngeal arch arteries. At E11.5, the distribution of β-galactosidase-positive neural crest cells was not different between Ednra-null and heterozygous embryos, but only in Ednra-null embryos, β-galactosidase-positive cells of the first and second pharyngeal arch arteries were positive for PDGFRβ. In E18.5 Ednra-null embryos, whole-mount β-galactosidase staining confirmed that neural crest-derived cells existed around the abnormal vessels from common carotid arteries. α-SMA and β-galactosidase were coexpressed at the cells around the abnormal vessels from common carotid arteries. These results suggest that, in Ednra-null mice, neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arch arteries, which result in the abnormal persistence of these arteries and the formation of the abnormal vessels from common carotid arteries. This means that the Edn1/Ednra signaling is involved in appropriate deployment of neural crest-derived smooth muscle cells in pharyngeal arch arteries, which contributes to the normal patterning of thoracic great vessels.

Conclusion

The remodeling of pharyngeal arch arteries does not depend on Dlx5/Dlx6-mediated ventral identification of pharyngeal arches, and the Edn1/Ednra signaling regulates the properly-directed differentiation of neural crest cells into smooth muscle cells in pharyngeal arch arteries.

本研究は、エンドセリンシグナルによる胸部血管発生調節のメカニズムを明らかにするため、Endothelin-1 (Edn1), Endothelin type A receptor (Ednra)のノックアウトマウスやDlx5/Dlx6ダブルノックアウトマウスの胸部血管表現型を解析するとともに、発生過程における咽頭弓動脈の血管平滑筋分化マーカーの発現を調べたものであり、下記の結果を得ている。

1.Edn1およびEdnraノックアウトマウスにおいて、総頸動脈からの異常血管などの血管異常を認めた。一方、Endothelin type B receptor (Ednrb)遺伝子をEdnra座にノックインしたマウスにおいてこれらの異常がレスキューされなかったことから、エンドセリンによる胸部血管発生調節は主にEdnraを介することが示された。

2.エンドセリンシグナルによる頭蓋顔面発生調節には、ホメオボックス遺伝子Dlx5/Dlx6を介することが知られているが、今回Dlx5/Dlx6ダブルノックアウトマウスの胸部血管を解析したところEdn1/Ednraノックアウトマウスに見られた血管異常を認めなかった。このことから、エンドセリンシグナルによる胸部血管発生調節は、Dlx5/Dlx6による咽頭弓領域決定を介さず、他の機構で行われることが示された。

3.咽頭弓動脈リモデリング期の血管形態を継時的に観察することにより、Ednraノックアウトマウスにおいてみられた総頸動脈からの異常分枝は、第1・2咽頭弓動脈が異常に残存することによって生じることが示された。

咽頭弓動脈リモデリング期 (E10.5-E11.5)において、Ednraノックアウトマウスでは、正常マウスと比較し血管平滑筋分化マーカーであるPDGFRβおよびαSMAの発現が亢進していた。また神経堤細胞を標識するWnt1-Creマウスを用いた検討により、これらの平滑筋マーカーを発現している細胞は神経堤細胞であることが示された。また出生前における総頸動脈からの異常分枝血管においても、その壁に神経堤細胞由来の血管平滑筋細胞が存在した。これらの結果から、Ednraノックアウトマウスにおいて、第1・2咽頭弓動脈において神経堤細胞から血管平滑筋細胞への分化が異常に亢進しており、これによって同血管が残存し血管形態異常を生じると考えられた。

以上、本論文はエンドセリンシグナルにより咽頭弓動脈における神経堤細胞から血管平滑筋への分化を調節し、この調節はこれまで知られていた下流遺伝子であるDlx5/Dlx6による咽頭弓の領域決定を介さないことを明らかにした。本研究は、胸部血管の形態形成や、その異常によるヒト先天性心血管異常の病因の解明に貢献すると考えられ、学位の授与に値するものと考えられる。