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Cardiovascular Research 1999 44(1):185-196; doi:10.1016/S0008-6363(99)00186-8
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Unique vascular morphology of the fourth aortic arches: possible implications for pathogenesis of type-B aortic arch interruption and anomalous right subclavian artery

Maarten Bergwerffa,*, Marco C. DeRuitera, Susan Hallb, Robert E. Poelmanna and Adriana C. Gittenberger-de Groota

aDepartment of Anatomy and Embryology, Leiden University Medical Centre, P.O. Box 9602, 2300 RC Leiden, The Netherlands
bDepartment of Vascular Biology and Pharmacology, Institute of Child Health, University of London, 30 Guilford Street, London WC1N 1EH, UK

* Corresponding author. Tel.: +31-71-527-6502/6660; fax: +31-71-527-6680 bergwerff{at}mail.medfac.leidenuniv.nl

Received 24 March 1999; accepted 20 May 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objective: Neural crest-derived cells were previously shown to participate in vessel wall formation of the great thoracic arteries, and their contribution was proposed to affect morphology and physiology of these vessels in the chick. The present investigation was undertaken to examine vascular differentiation and morphogenesis of the neural crest-derived aortic arches in mammals. Methods: Using immunohistochemical markers for smooth muscle cell differentiation and a neurofilament marker, we examined morphogenesis of the great arteries in mice, ranging from embryonic day 11.5 to the adult. Results: We observed that in the 4th aortic arch arteries early media formation differed from the other arteries, in that they almost completely lacked (or showed decreased) actin expression in certain areas. This discontinuity in actin expression persisted throughout much of foetal development, in the form of circular segments of cells displaying decreased staining for smooth muscle markers, both at the left and right side of the arterial tree. In adult mice, the 4th arch artery derivatives, segment B of the aortic arch and the proximal right subclavian artery, were observed to differ from adjoining vessels in their smooth muscle and elastic composition. Staining for neurofilaments revealed close association of the developing segments with apparent sensory afferent vascular innervation. Conclusion: The unique areas of the 4th arch artery identified here reflect the basic segmental patterning of the early embryonic pharyngeal arches. These segments correlate with sites that are predisposed to interruption or severe hypoplasia, and may thus reveal part of the aetiology of type-B aortic arch interruptions and arteria lusoria.

KEYWORDS Autonomic nervous system; Congenital defects; Embryology; Morphogenesis; Smooth muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
One of the most characteristic features of both the invertebrate and vertebrate body plan is segmentation of the anterior–posterior body axis. A large number of developmental control genes have been found to be responsible for the formation of segments and for implementing each segment’s identity. Segment-polarity, pair-rule, and gap genes induce segmentation of the body axis, whereas genes from the Antennapedia complex – and their Hox gene homologues in vertebrates – are held responsible for supplying each segment with positional information which results in acquisition of its own identity.

In vertebrate embryos, segmentation of the developing pharyngeal arches is also apparent, and these arches are subject to major remodelling processes in most vertebrates. Among fish, the first and second arch will develop into the mandibular arch and hyoid arch respectively, whereas the more posterior arches retain their segmental nature in the form of gill-bearing arches, each supplied with apparently identical blood circulations. In air-breathing vertebrates, pharyngeal arch 1 and 2 develop to form the mandibular and hyoid complexes, as in fish. All arches are populated with rhombencephalic neural crest cells, and neural crest-derived mesectodermal cells in arches 3, 4 and 6 are retained in the cardiovascular system and in the pharyngeal neural network [1–3]. The pharyngeal arch arteries start as a bilaterally symmetrical and segmented system, which remodels extensively into the asymmetrical tree of the great arteries. A missing link in the early embryonic work is that segments named after their original embryonic positions have not been delineated and followed to their ultimate fate by histological and immunohistochemical means. (For a basic description of the pharyngeal arch arteries and their theoretical transformation into adult structures, see Refs. [4,5].)

Much research has been carried out on pattern formation in the pharyngeal region of the vertebrate head, that is, structures derived from the first and second pharyngeal arches. Sharp boundaries of transient expression of Hox genes of the different Hox clusters were reported to map with the distinct pharyngeal arches [6,7]. A distinct combinatorial Hox code for each arch was initially proposed to be transferred through the neural crest from its origin in the hindbrain towards the branchial region. After migration into the pharyngeal arches, the neural crest may superimpose its Hox code onto surrounding ectodermal, endodermal and mesodermal cell compartments, which finally collaborate in forming, for example, the lower jaw or the hyoid structures after having received specific positional cues. More recent findings, however, suggest a lesser role of the crest’s predetermination, and indicate that additional patterning information resides within the branchial arches themselves. Experimentally induced abnormal expression of Hox genes in the premigratory crest (e.g. by crest rotation) was followed by re-establishment of normal expression patterns in the arches [8–10].

In the pharyngeal arterial system derived from arches 3, 4, and 6, however, segmental morphogenesis has never really been evaluated in this manner. In chicks, the vessel walls of the great arteries were previously shown to be almost entirely derived from the cardiac neural crest [2,11], emanating from the rhombencephalic crest at levels between the otic placode and the third somite [12]. The fact that neural crest cells in the branchial region may be under influence of distinct positional cues in the different pharyngeal arches, and that experimental interference with Hox gene expression in cardiac crest cells may alter vascular identity [13], suggest that vessel segments derived from either arch 3, 4, or 6 may differ in certain characteristics.

The sixth arch-derivate, the ductus arteriosus, has on numerous occasions been postulated as a unique vessel both in its origin [14] as well as its differentiation and definitive morphology [15–19]. However, the alleged third and fourth arch derivates in mammals were never recognised as distinct vascular segments on a morphological basis. Intriguingly though, the 4th arch derivates, (that is, segment B of the aortic arch and the proximal part of the right subclavian artery [4,20], see Fig. 1), are subject to specific interruption/obliteration in a number of recently published gene targeted models [21–23], and throughout the years have presented as a clinical case of type-B aortic interruptions that often combined with anomalous origin of right subclavian arteries (retro-oesophageal right subclavian artery=arteria lusoria) [24–26].


Figure 1
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  		Fig. 1 Diagram of the late foetal mammalian thoracic arterial tree. A, B, and C represent segments of the aortic arch according to the nomenclature proposed by Celoria and Patton [31]. The shaded areas in the aortic arch and proximal right subclavian artery represent the segments that allegedly derive from the fourth arch arteries [4,20]. Ao, aorta; DA, ductus arteriosus; LCA, left carotid artery; LSA, left subclavian artery; RCA, right carotid artery; RSA, right subclavian artery.

 
In this study, we provide immunohistochemical evidence in the mouse for a unique characteristic of those vessel segments that allegedly derive from the fourth pharyngeal arch arteries. This different morphological characteristic is apparent from early embryological stages to the adult. Local sensory vascular innervation also appears to associate with the identified vessel segments. Recognition of these arterial segments as being intrinsically different from adjoining vessel segments may aid in understanding the pathogenesis of neural crest-related type-B aortic interruption and anomalous right subclavian artery.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Animals and tissue preparation
Pregnant Swiss mice were killed by cervical dislocation, and embryos ranging from embryonic day 11.5 (E11.5) to neonates were removed, killed, and fixed in 4% paraformaldehyde (PFA) in phosphate buffer (0.1 mol/l, pH 7.2) (day 0 was designated as the day of detection of the vaginal plug). In addition, the aortic arches with their branches were carefully dissected from two adult female mice and fixed in either 4% PFA or ethanol/acetic acid (98:2%), respectively. Fixed tissues were dehydrated in graded ethanol and xylene and embedded in paraffin. Sections of 5 µm were cut and mounted serially onto egg-white/glycerine-coated glass slides.

The investigations were performed conforming to the Netherlands Legislation on Animal Welfare and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Immunohistochemistry
After deparaffination, sections were autoclaved in citric acid buffer (0.01 mol/l, pH 6.0) at 120°C for 20 min, cooled down to room temperature and briefly rinsed in phosphate-buffered saline (PBS, pH 7.3) followed by treatment with 0.3% H2O2 in PBS (10 min) to quench endogenous peroxidase activity.

Standard immunohistochemistry was performed by incubating (overnight, room temperature) with the primary monoclonal antibodies 1A4 (anti-{alpha}-smooth muscle actin, Sigma, St. Louis, MO, USA) 1:3000 diluted in PBS with 0.05% Tween-20 and 0.5% bovine serum albumin; anti-{gamma}-actin (a generous gift from Dr J. Lessard, Cincinnati, OH, USA) diluted 1:100; hHCD (anti-heavy caldesmon, Sigma) diluted 1:400; VN-11-5 (anti-vinculin, Sigma) diluted 1:100; and RMO-270 (anti-neurofilament-M protein (NF-M) [27], kindly provided by Dr J.Q. Trojanowski) diluted 1:400. Sections were then thoroughly rinsed and incubated with horseradish peroxidase-conjugated rabbit-anti-mouse antibodies (1:250, 90 min, DAKO A/S, Glostrup, Denmark) and rinsed again before further treatment (see below).

A polyclonal antibody against S-100 protein (1:50, Zymed, San Francisco, CA, USA; used to reveal vascular dendritic cells [28]) was used without prior autoclaving of the sections. Primary incubation with S-100 (overnight, room temperature) was followed by rinsing in PBS and subsequent administration of an excess of goat-anti-rabbit immunoglobulins (GAR/Ig, 1:50; Nordic, Tilburg, The Netherlands). After rinsing, a rabbit peroxidase–anti peroxidase complex (R/PAP, 1:500; Nordic) was applied. PFA-fixed adult murine spinal chord served as a positive control for the S-100 antibody.

After thorough rinsing, bound monoclonal and polyclonal antibody complexes were finally visualised by treatment with 0.04% diaminobenzidine tetrahydrochloride (DAB)/0.06{per thousand} H2O2 in 0.05 mol/l TRIS–maleic acid (pH 7.6) for 10 min at room temperature. Sections were briefly counterstained with Mayer’s haematoxylin, dehydrated, and embedded in Entellan (Merck, Darmstadt, Germany). A standard resorcin/fuchsine staining [29] was applied to visualise elastic fibres.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 E11.5: media layers in early pharyngeal arch 4 display discontinuous {alpha}-actin
At E11.5, the major arteries in the thorax have acquired smooth muscle {alpha}-actin expression. The dorsal aorta presented with three to five heavily stained layers of primary SMCs, and the small pulmonary arteries showed a thin line of peri-endothelial actin expression. Likewise, the pharyngeal arterial apparatus, consisting of the just septated aortic sac and arch arteries 3, 4, and 6, had acquired SM-actin expression in a primary tunica media of approximately two to three cell layers thick (not shown).

It was evident, however, that the actin-expressing primary media was not continuous along the entire system. Both 6th arch arteries showed solid and continuous actin expression between their origin in the pulmonary trunk and the dorsal aortae (Fig. 2a). This was seen in the left 6th arch, or prospective ductus arteriosus (DA), as well as in the right 6th arch artery, which had a smaller lumen compared with the left arch illustrating its prospective degeneration.


Figure 2
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  		Fig. 2 Light micrographs of the pharyngeal arch arteries of an E11.5 mouse embryo illustrating primary media formation as revealed by the 1A4 ({alpha}-SM actin) antibody. (a) The sixth arch arteries (VI) showed continuous SM-actin expression of two to three cell layers thick along their entire length. DAo, dorsal aorta; Oe, oesophagus; T, trachea. Scale bar=100 µm. (b) SM-actin expression in the fourth arch arteries (IV) of the same embryo. AoS, aortic sac; DAo, dorsal aorta. Scale bar=100 µm. (c) Higher magnification of the distal portion of the left fourth arch artery (IV), illustrating that actin expression is not continuous in this area. Sites (arrows) in the vicinity of the vagal nerve (VN) clearly lack SM-actin expression, in contrast to arch 6. Scale bar=50 µm. (d) Adjacent section of Fig. 1c, stained for RMO-270, an anti-neurofilament antibody, to illustrate the exact location of the vagal nerve (VN) beside the fourth arch artery. Scale bar=100 µm. (e) Actin expression in the third arch arteries (III) was continuous along the entire length, like that of the sixth arch arteries (Fig. 1a), with exception of a small area (arrow) near the vagal nerve (VN) and junction with the dorsal aorta. P, pharynx. Scale bar=100 µm.

 
The 4th arch arteries showed a clear discontinuity in actin expression. Along the most proximal parts downstream of the left cardiac outflow tract, actin was expressed in a continuous pattern of two to three cell layers. In the distal half of the 4th arch arteries, by contrast, actin-positive peri-endothelial cells were only sparsely present and did not form a continuous layer (Fig. 2b,c). This was most marked in the region near the vagal nerve (Fig. 2d), but also extended toward the junction with the dorsal aortae. Actin-negative areas showed condensation of mesenchymal cell layers, despite the absence of actin expression. The discontinuity in actin expression was found in both 4th arch arteries. The dorsal aortae had no regions of unstained cells along their entire length.

The 3rd arch arteries had a well developed actin-positive media along most of their length from the aortic sac towards the dorsal aortae. However, at the junction with the dorsal aortae, small areas of a few non-stained cells were observed in the arch arteries’ media (Fig. 2e). Although much less prominent in the 3rd arch arteries, as in the fourth arch arteries, the regions in which these cells were observed in close proximity to the vagal nerves, which pass just laterally from the arteries. Later stages did not reveal irregularities along the common carotid arteries which allegedly derive from the 3rd arch arteries.

3.2 E12.5–E14: development of a nonmuscular ring of cells in 4th arch artery derivates
At E13, the pharyngeal arterial apparatus had further remodelled. The right sixth arch artery was obliterated and left behind a residual non-lumenised strand of actin-positive cells. Concomitantly, regression of the right sided dorsal aorta occurred between the subclavian artery and the junction with the left dorsal aorta. The (left) aortic arch had developed a media of five to six peri-endothelial cell layers that showed {alpha}-actin expression. At one site in the aorta, however, between the vagal and recurrent laryngeal nerves, the vascular media showed groups of non- or weakly stained cells, separated from the endothelium by a single layer of actin-positive cells. On the right side of the arterial tree a similar phenomenon was observed near the junction of the prospective brachiocephalic, subclavian and common carotid arteries (Fig. 3a–c).


Figure 3
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  		Fig. 3 Development of a nonmuscular ring of cells in fourth arch artery derivates. (a) SM-actin expression in the left aortic arch (Ao) of an E13 mouse embryo. Oe, oesophagus; RV, right ventricle; T, trachea. Scale bar=150 µm. (b) Higher magnification of Fig. 3a. Groups of cells (arrows) that clearly lack expression of SM-actin partially interrupt the media of the arch, which has otherwise attained a solid media of approximately five to six peri-endothelial layers of primary SMCs. VN, vagal nerve. Scale bar=75 µm. (c) On the right side in the same embryo, a similar phenomenon of groups of non-staining cells (arrows) was observed at the origin of the prospective subclavian artery. Ao, aortic arch; T, trachea. Scale bar=75 µm. (d) Graphic reconstruction of the arterial tree of an E13.5 embryo reveals the ring-like formation of both left and right segments that did not express SM-actin. The sites at which this phenomenon occurs correspond with the alleged 4th arch derivates as described by Congdon [20]. Ao, aorta; DA, ductus arteriosus; LCA, left carotid artery; LSA, left subclavian artery; RCA, right carotid artery; RSA, right subclavian artery. (e,f) Adjacent sections through the left 4th arch of an E12.5 embryo stained for actin (e) and RMO-270 (f). From E12.5 onwards, the ring of non-actin expressing cells between the vagal (VN) and recurrent (RN) nerves was seen to be clearly interspersed with a maze of thin nerve fibres (arrowheads). Scale bar=75 µm. (g,h) Light micrographs of two adjacent sections, showing the aortic arch (Ao) of an E14 mouse foetus. The arrows indicate the area with reduced {gamma}-actin (g) expression, especially in the outer media. Vinculin expression (h) appears more homogeneous throughout the aortic wall and does not clearly delineate an aberrant segment. VN, vagal nerve. Scale bar=75 µm.

 
Three-dimensional analysis of both sites in the 4th arch arteries of a slightly older embryo (E13.5), by means of graphic reconstruction, illustrated that the actin-negative cells at this stage form a ring entirely surrounding the vessel segments concerned (Fig. 3d). This ring of unstained or very weakly stained cells was separated from the endothelium by one or two cell layers of actin-positive cells. At the ablumenal side of the media, the ring of cells was separated from the surrounding loose mesenchyme by a thin discontinuous layer of actin-positive primary SMCs. The posterior (caudal) part of the aortic ring of non-stained cells lay within that part of the vascular wall which is shared by the aortic arch and the ductus arteriosus. At this interface, the ring was thickened considerably, having the appearance of a signet ring.

Staining with the anti-neurofilament marker RMO-270 clearly revealed the presence of thin nerve branches in the vessel wall, colocalising with the area of cells that lack SMC-markers, between the vagal and recurrent laryngeal nerves. At E12.5, nerve fibres in the media were visible surrounding the aorta in a ring-like shape within the above-mentioned area free of SMC markers (Fig. 3e,f).

Additional staining for {gamma}-actin at E14 showed a pattern similar to {alpha}-actin (Fig. 3g). Vinculin staining appeared to be present, but at decreased levels in the ring areas (Fig. 3h).

3.3 E15: neonate
From E15 onwards it appeared as though the cells that previously had not stained for {alpha}- and {gamma}-actins had somewhat shifted in position towards the periphery of the media (Fig. 4a). The presence of nerve fibres within the ring of non-staining cells was clearly established by neurofilament-M staining, both in the aorta and in the proximal right subclavian artery (Fig. 4b–d). Neurofilament staining was never observed elsewhere in the media of the embryonic thoracic arteries. At E16.5, segment B of the aorta still contained actin-negative cells, preferentially located in the outer media (Fig. 4e). Their relative abundance appeared to have diminished in comparison with earlier stages. At E16.5, staining with an anti-caldesmon antibody, a relatively late SMC-differentiation marker [30], showed that actin-negative cells did not express caldesmon, whereas most cells expressing SM-specific proteins now also contained caldesmon (Fig. 4f). In addition, lower levels of expression of caldesmon were visible in the entire B-segment of the aorta, compared with the adjacent vessel segments. Thus, weak caldesmon staining marked a more extensive area than the distinct ring of actin-indigent cells.


Figure 4
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  		Fig. 4 (a) {alpha}-SM-actin, and (b) neurofilament-M distribution in adjacent sections through the aortic arch of an E15 mouse embryo. The aortic arch (Ao) at this stage in development still reveals a partial interruption in its actin expression at the site where it was also reported at earlier stages. BA, brachiocephalic artery; VN, vagal nerve; Oe, oesophagus; RN, recurrent nerve; T, trachea. Scale bar=150 µm. (c) Higher magnification of boxed area in (b), showing close association of thin nerve fibres with cells in the aortic media that lack SM-actin expression (see a). Scale bar=50 µm. (d) Section at a more anterior level in the same embryo through the left carotid artery (CA) and the top wall of the aortic arch (Ao). NF-M staining demonstrates the upper edge of the ring-formed nerve fibre network within the aortic wall. Scale bar=100 µm. (e,f) At a slightly later stage in development (E16.5), the nonmuscular aortic ring as revealed by {alpha}-SM-actin staining (e) is not as prominent as before, and appears to have shifted towards more peripheral layers. An adjacent section stained for h-caldesmon (f), however, shows a segment of the arch (arrow) which lacks expression of this protein in its outer media cell layers, in contrast to more up- and downstream regions. Scale bar=100 µm. (g,h) Light micrographs of sections through the aortic arch (Ao) of a neonatal mouse stained for {gamma}-actin (g) and vinculin (h), respectively. {alpha}-SM-actin, {gamma}-actin, and caldesmon still revealed irregularities in the B-segment (arrows) of the aortic arch, demonstrated by absence of immunoreactivity in outer media cell layers. Vinculin (h) is apparently as much expressed in the aberrant vascular ring as it is in the rest of the aortic media. VN, vagal nerve; ACV, anterior cardinal vein. Scale bar=150 µm.

 
Similar results for {gamma}-actin (Fig. 4g), caldesmon, and {alpha}-actin (not shown) were obtained in neonatal mice. As in earlier stages, vinculin stained both actin/caldesmon-negative and positive cells in the vessel wall, but was more prominent in the latter (Fig. 4h).

3.4 Adult: prolongation of distinct morphology of 4th arch derived vessel segments
In adult mice, the fully developed segment B of the aortic arch and the proximal right subclavian artery still revealed characteristics in which they differed from all other adjoining vessels. At low magnification the segments were clearly distinguishable by a significantly lower level of staining for {alpha}-actin and vinculin, compared with other vessel segments (Fig. 5a–f). Further scrutiny revealed that the majority of segment B medial cells do express actin, but that the relative cellular volume/matrix ratio was much smaller than elsewhere (Fig. 5g,h). Elastic lamellae appeared thicker and more numerous in the B-segment than in adjacent aortic segments, although there was no detectable thickening of the aortic wall of this segment, and B-segment cells were generally smaller. Resorcin-fuchsine histological staining for elastin showed that the dorsal aspect of the B-segment contained 10–12 distinct lamellae, but only seven to nine laminae were present in the adjoining C-segment and thoracic descending aorta (Fig. 5i,j).


Figure 5
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  		Fig. 5 (a,b) Light micrographs of two adjacent sections through an adult murine aortic arch (Ao), stained for {alpha}-SM-actin (a) and vinculin (b), respectively. The dotted lines indicate the boundaries of an aortic segment which differed in morphology from adjoining vessels segments. BA, brachiocephalic artery; LA, ligamentum arteriosum; LCA, left carotid artery. Scale bar=500 µm. (c,d) Higher magnifications of the aortic vessel wall at the outer curvature of the arch (c, actin; d, vinculin). The aortic B-segment between the dotted lines is clearly aberrant in showing optically lower staining levels caused by reduced cellular volume of SMCs and increased elastic matrix. Scale bar=200 µm. (e,f) A similar phenomenon as was detected in the aortic arch was also present in the proximal part of the adult right subclavian artery (RSA). A circular segment (between dotted lines) just downstream from the bifurcation of the brachiocephalic artery into the right carotid artery (RCA) and subclavian artery, clearly revealed reduced SM actin-positive tissue (e). Somewhat further downstream (f) the subclavian artery shows homogeneous actin expression along its entire circumference. Scale bars=200 µm. (g,h) SM-actin expression patterns in the aortic wall at segment B (g) and segment C (h). Note that the relative amount of actin-expressing tissue is much lower in the B-segment than elsewhere in the arch. B-segment cells also appear to be smaller. A small number of cells in the B-segment failed to express detectable levels of actin (arrows). A, adventitia; M, media. Scale bars=50 µm. (i,j) Resorcin/fuchsine staining revealed differences in elastic matrix composition between the aortic wall at segment B (i) and at segment C (j). Elastic laminae in the B-segment are more numerous and not as neatly organised as elsewhere in the aortic arch. Scale bars=50 µm.

 
The width of the aortic segment varied along the circumference, and interestingly, the B-segment directly adjoined the ligamentum arteriosum at the inner curvature of the arch. This is illustrated by a graphic reconstruction, based on vinculin and actin staining, in Fig. 6.


Figure 6
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  		Fig. 6 (a) Graphic reconstruction of an adult murine arterial tree (seen from a left oblique angle) delineating the unique segments in the aortic arch and subclavian artery as histologically revealed by actin and vinculin stained sections (see Fig. 5). The gross morphological appearance is slightly distorted because of dissection and fixation procedures. Note that the aortic B-segment (B) (designated based on its aberrant morphology) varies in thickness along the circumference and adjoins the ligamentum arteriosum (LA) at the inner curvature of the arch. (b) Diagram of the late foetal murine thoracic arterial tree. The shaded areas correspond with the alleged 4th and 6th arch artery derivates. Note that the shaded segment B (B) adjoins the ductus arteriosus (DA) at the inner curvature of the aortic arch. See also Fig. 1 for comparison with traditional representation of the 4th arch artery derivates. Ao, aorta; BA, brachiocephalic artery; LCA, left carotid artery; LSA, left subclavian artery; PA, pulmonary arteries; PT, pulmonary trunk; RCA, right carotid artery; RSA, right subclavian artery.

 
There was a small number of cells in the aortic B-segment that did not stain with either actin or vinculin. These ‘nonmuscular’ cells appeared throughout the entire thickness of the media. Some of these non-SMCs, particularly those in the outer media, were shown to be associated with neurofilament-M staining in adjacent sections (Fig. 7a,b). In adult mice, NF-M staining detected nerve fibres in the aorta and subclavian artery in the aforementioned segments. Fibres and, occasionally, positively-stained cells were preferentially located at the media–adventitia interface, never penetrating very deeply into the media. Entire circumferential innervation of the segments, as was apparent at embryonic stages, could not be established. The vascular autonomic nervous system, including intramural fibres, also stained for S-100 protein. However, in addition to marking those cells and fibres that show NF-M expression, the S-100 protein could be detected in a much greater area in the B-segment with occasional staining of medial cells (Fig. 7c). Remarkable numbers of S-100 positive cells were also observed in that part of the aortic wall which is attached to the ligamentum arteriosum (not shown).


Figure 7
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  		Fig. 7 (a,b) Adjacent sections through the adult aortic wall at segment B. Some of the actin-indigent cells (a; arrowheads) were shown to react with the RMO-270 antibody (b) against neurofilament-M protein, indicating the relation to vascular innervation at this particular site in the arch. Scale bar=50 µm. (c) S-100 reactivity was found in cells that stained with RMO-270 and, in addition, in a number of other cells of the outer media at segment B (arrowheads). The nature of these cells is still unclear. Scale bar=50 µm.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
4.1 Vascular heterogeneity: distinct aortic and subclavian segments
Although marked differences between artery types have long since been recognised, there are no previous reports which designate a certain aortic arch segment as being intrinsically different. The current nomenclature of segments A, B, and C, used to divide the human aortic arch, was based on the aortic branching pattern (see Fig. 1) and served as a tool to describe the location of aortic anomalies, for example stenoses and interruptions [31]. With respect to the nomenclature of aortic segments we chose to call the unique aortic segment, ‘segment B’, because of its position between the left carotid and left subclavian arterial orifices at the outer curvature of the arch. At the inner curvature, however, the histologically distinguishable segment directly adjoined the ligamentum arteriosum (Fig. 6), implicating that there would be no room for a circumferential segment A, between the left subclavian artery and the ligamentum arteriosum. The B-segment as defined by Celoria and Patton [31] is not apposed to the ligamentum arteriosum. In part, this difference with the human situation may be explained by the absence of a true isthmus (or A-segment) in the mouse, as the left subclavian orifice and ductal entry point were too close to each other in the mice we examined. Similarly, a distinct isthmus is also difficult to delineate in some human cases. The apposition of the 4th arch-derived segment B to the 6th arch-derived ductus arteriosus is, however, in agreement with their relative position during early development when they jointly meet the dorsal aorta [14,20].

We here show that, in adult mice, a segment located at the B-site is histologically different from the remainder of the arch. It presented to be distinct in both its cellular and elastic matrix organisation. Like elsewhere, SMCs in the B-segment expressed SM-{alpha}-actin, {gamma}-actin, caldesmon, and vinculin, but apparently did so at lower levels. In addition, B-segment cells in the adult were generally smaller compared with SMCs in adjacent segments, whilst being embedded in more numerous and thicker elastic laminae.

Presence of medial cells that lack all typical SMC characteristics was previously observed in the adult bovine pulmonary artery [32]. In our study, the aortic and subclavian segments of the adult mouse also presented a number of ‘nonmuscular cells’. Some of these were shown to be related to the vascular autonomous nervous system, the innervation pattern of which was observed to be closely related to the specific segments (see below). Another small number of nonstaining cells, however, may represent vascular dendritic cells (VDCs), as was suggested by occasional staining of S-100 throughout the media, although their location in the media does not correspond with that of S-100+ VDCs within the aortic intima as previously described [33,34].

We have not been able to trace the progeny of the early group of cells that lacked SMC marker expression in the 4th arch arteries. Their relative numbers appeared to decrease with time and they shifted towards the periphery of the media. The absence of NF-M staining makes it unlikely that these cells belong to the nervous system, but their association to vascular innervation is supported by tight association of nerve fibres with them. Thus, it is not yet clear whether the occurrence of embryonic ‘nonmuscular cells’ represents early segregation of SMCs and other cells types (e.g. baro/chemoreceptor cells) – presumably all of neural crest origin [1,2,35] – or whether it represents a specific group of primordial SMCs that is much delayed in its differentiation by yet unknown factors.

4.2 Aberrant segments derive from the fourth pharyngeal arch arteries: relation to neural crest
The embryonic pharyngeal arterial system, which is clearly bilaterally segmented, is subject to major remodelling in all mammals including man, making it extremely difficult to delineate which final positions will be occupied by which embryonic segments. The importance of tracing embryonic vessel segments into adulthood lies in its possible consequences for vascular morphology and susceptibility to congenital malformations and vascular pathology.

Cells derived from the cardiac neural crest in the chick, have been reported to populate the pharyngeal arches and to finally contribute to the vast majority of arterial SMCs, ‘nonmuscular cells’, and fibroblasts of vessels derived from the arches [1,2]. In doing so, neural crest cells (NCCs) comply to positional cues which results in a distribution pattern with sharp border lines. Previous reports from this laboratory have shown in chicks that these sharp boundaries between NC-derived vessels and vessels of sole mesodermal origin, often coincide with marked changes in vascular morphology [2]. Combined with in vitro studies, which showed differences between NC and non-NC SMCs in response to TGF-β [36], this supports a concept of origin-related vascular morphology and physiology. In addition, scrutiny of NCC patterning in the circulatory system is of great value to understand cardiovascular anomalies that are associated with disorders of the neural crest which, amongst others, include vitamin A deficiency [37] and the DiGeorge syndrome [38].

Intriguingly, the aberrant aortic and subclavian segments that we recognised in adult mice exactly colocalise with the areas in the thoracic arterial tree that are reported to be derived from the fourth pharyngeal arch arteries [20]. The present evaluation of SMC differentiation in the fourth arch arteries throughout embryonic development suggests that the relationship between adult aberrant segments and the 4th arches is indeed present, as media formation differed from other arch arteries from a very early stage (E11.5) onwards. It is remarkable that Congdon’s considerations [20] of 4th arch positioning match so well with our findings, as his work was not primarily supported by histogenesis and immunohistochemistry of the arterial tree, but rather depended on anatomical observations. It should be emphasised, however, that proof to confirm the hypothesis of 4th arch derivation can only be obtained by lineage studies in this highly plastic system.

Although patterning studies in the chick show that the arch arteries are ensheathed along their entire length with NCCs of certain AP-axis levels [2,39], alleged Hox gene-imprinted information may only be present in the dorsal half of an arch artery as is suggested by transient HOX 2.1 (HOXB5) staining in the 6th arch [40] (Bergwerff et al., unpublished observations, 1998). In the chick, it is only the dorsal half of the 6th arch arteries, that develops into the ductus arteriosus (DA). The DA is unique in its differentiation [15–19,41], which differs markedly from adjoining other NC-derived vessels, and it is tempting to speculate that an important embryonic regulatory gene as Hoxb5 is involved in instigation of this specific DA differentiation. Likewise, the fourth branchial arches are reported to express a combinatorial Hox code including genes of the 4th (Dfd) paralogous group, i.e. Hoxa4, Hoxb4, Hoxc4, and Hoxd4 [6,7, and references therein], but lack members of the 5th (Src) paralogous group. Patterns of expression have never been scrutinised within these arches, or in the primary SMCs that constitute the arch arteries’ walls. Kirby et al. [13] have shown that vascular identity can indeed be influenced by interfering with Hox expression in premigratory cardiac NCCs.

Differences in ventral and dorsal aspects of the early pharyngeal arches or of the arch arteries within them are illustrated by the aforementioned HOXB5 expression in the sixth arch, and are reminiscent of the early angiogenic processes during which the arch arteries are formed by a ventral and a dorsal component, namely a ventrally protruding sprout from the dorsal aortae and a dorsal sprout from the aortic sac [14,20]. The unique adult aortic and subclavian segments may, therefore, derive from only the dorsal part of the original 4th arch arteries, as is also suggested by nascent actin expression in this study.

Firm conclusions on NCC distribution and arch artery remnants can not yet be made in mammalian species. Experimental access to premigratory mammalian NCCs, followed by long term cell lineage studies, is technically unresolved. In the chick, however, precise NC patterning has been established, but remodelling of the avian pharyngeal arterial system differs from that of mammals. By homology to the chick, the SMCs, adventitial fibroblasts, and nonmuscular cells that comprise the aberrant aortic and subclavian segments in the mouse are likely to be neural crest-derived [1–3].

According to current definitions, regions in the aortic arch upstream from any vessel segment that is designated as arch artery-derived, will be designated as aortic sac-derived, whereas downstream regions would be remnants of the dorsal aortae. If basic NCC distribution in the chick [2] is to be superimposed on the mammalian arterial tree, aortic sac-derived structures (ascending aorta upto segment B, and brachiocephalic artery) would only have a small NCC contribution, whereas the descending aorta is probably completely devoid of NCCs. This would imply that, in the case of the 4th and the 6th arch arteries, dominance of neural crest cells is somehow reflected in the specific differentiation that these vessels undergo, compared to non-NC and mixed vessels.

4.3 Relation to vascular innervation
In this study, we show that the development of the aortic and subclavian segments is closely related with the presence of nerve branches within the embryonic media. Nerve fibres were observed in the specific segments from as early as E12.5 onwards. In chicks, similar results were obtained with localisation of vagal and recurrent branches from stage HH29 onwards [3]. The observed nerve fibres represent abundant sensory afferent innervation related to baro- and chemoreceptor function, as was previously established at these particular sites in adult mammals [42]. The ring-shaped area that these branches occupy, which was most apparent during embryonic and foetal stages, was described earlier by Thievent and Cornat [43], who observed calcitonin gene-related peptide-positive fibres, in a ring-formed pattern in late foetal rat aortas.

The co-development of locally aberrant vascular histology and extensive media innervation described here raises pivotal questions as to the relation between the two. Firstly; could the aberrant segments have differentiated in their unique fashion under influence of local vascular innervation? Or, was ingrowth of nerve branches established by local stimuli of an embryonic vessel segment which was already intrinsically different from its surroundings? A third option consists of a combination of both hypotheses.

By studying aortic wall development with the early SMC marker, 1A4, we have shown that primary media formation in the early 4th arch artery already differed from other arteries, very early in development (E11.5), at which time nerve branches from vagal, recurrent, or aortic nerves had not yet formed near the arteries. Although media formation was delayed in proximity to the vagal nerves, the affected primary media spread out to areas at reasonable distance from the vagal nerve as well. Moreover, other vessels in close association with the vagal nerves, like the 6th arch arteries, do not develop the ‘nonmuscular’ ring of cells in their media. It therefore seems most likely that the fourth aortic arch arteries are intrinsically different and signal the main adjacent nerves to send branches into the media.

4.4 Susceptibility to interruption
The recognition of a unique identity of the fourth pharyngeal arch arteries and their derivates will be of great importance in understanding the pathogenesis of aortic arch interruptions (AAI) and the anomalous origin of the right subclavian artery (ARSA). Kutsche and Van Mierop [25] previously noted a correlation between type-B interruptions and anomalous right subclavian arteries (often arteria lusoria). Their study showed 14 cases of ARSA of 21 infants with type B-AAI. High incidence of ARSA in type B-AAI was also reported in earlier clinical studies [24,44]. They furthermore illustrated that obliteration of the embryonic left 4th arch artery results in interruption of the B-segment, whereas degeneration of the right interrupts the normal origin of the right subclavian artery.

More recently it was shown that in almost half the number of cases of type B-AAI, deletions of chromosome 22q11 were present [26]. Deletion of 22q11 is often involved in DiGeorge syndrome and Velocardiofacial syndrome, and appears to be related to disorders in neural crest function and/or migration [45]. Although neural crest-related disorders involve malformations in multiple cardiovascular structures, interruption of neural crest-derived vessels is by far most common in the aortic and subclavian segments described here [26,46]. In this respect, the right aortic arch with a retro-oesophageal vascular ring and the cervical/high left aortic arch (anomalies found either isolated, or associated with Tetralogy of Fallot and in 22q11 deleted patients [47,48, and references therein]) may also correlate with a fourth arch artery defect. Like in the type-B interruption, the left fourth arch artery may also be interrupted in the right aortic arch-vascular ring and cervical aortic arch phenotypes.

The apparent genetic predisposition towards interruption or severe hypoplasia of the fourth arch artery has also been illustrated by several gene targeted mouse models. Homozygous null mutant mice for Mesenchyme Fork Head-1 (MFH-1) [21], endothelin-A receptor (ETA) [23], and endothelin converting enzyme-1 (ECE-1) [22] showed malformations similar to those found in the DiGeorge syndrome. Fourth arch anomalies (interruption or tubular hypoplasia) rated 83% (MFH-1), >44% (ETA), and >58% (ECE-1), respectively. Although all three null mutant mice show anomalies similar to those found in 22q11 deletions, the respective genes do not map to this chromosomal region. Yet, genetic disruptions in MFH-1, ETA, ECE-1, and 22q11 may lead to 4th arch interruption (and other malformations) by affecting the same signalling pathways. Although embryonic expression patterns of the three genes are not confined to the fourth arch, but rather appear equally distributed among the arches, deletions of the genes specifically cause observable anomalies in arch 4, but not arches 3 or 6. This is yet another indication that the fourth pharyngeal arch is an embryonic entity of its own, derived from the neural crest, with a unique morphogenesis and predisposition towards interruption.

Time for primary review 30 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
The authors gratefully acknowledge the technical support by Jayne Reader and Bert Wisse and graphic aid by Jan Lens and Bas Blankevoort. This work was financially supported by a research grant of the Netherlands Heart Foundation: grant number 93.111.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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