ORIGINAL RESEARCH

published: 10 May 2022

doi: 10.3389/fcell.2022.892900

Morphogenesis of the Mammalian

Aortic Arch Arteries
Robert H. Anderson and Simon D. Bamforth *

International Centre for Life, Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle,
United Kingdom

The major vessels in mammals that take blood away from the heart and deliver it to the arms and
the head take their origin from the aortic arch and are derived from the arteries formed within the
embryonic pharyngeal arches. These pharyngeal arch arteries, initially symmetrical, form in a
cranial to caudal sequence within the pharyngeal mesenchyme. They then undergo a complex
process of remodeling to produce the asymmetrical brachiocephalic arteries as seen in the
adult. A complex interaction between the tissues of the pharyngeal arches and the genes they
express is required to ensure that arterial formation and remodeling is able to proceed normally.
If this process is disrupted, life-threatening congenital cardiovascular malformations can occur,
such as interruption of the aortic arch, isolation of individual arteries, or so-called vascular rings.
Here, using state-of-the-art imaging techniques, we describe the morphogenesis of the arteries
in humans and mice and the cardiovascular defects in the Tbx1 mutant mouse model. We
provide details of the process of remodeling, clarifying also the morphogenesis of the external
Edited by:
Wolfgang Johann Weninger,
carotid artery and the so-called “migration” of the left subclavian artery.
Medical University of Vienna, Austria
Keywords: aortic arch artery development, pharyngeal arch, morphogenesis, remodelling, three-dimensional image
Reviewed by: analysis, high resolution episcopic microscopy, micro-computed tomographic imaging, TBX1
Sophie Astrof,
Rutgers Biomedical and Health
Sciences, United States
Irene Zohn, INTRODUCTION
Children’s National Hospital,
United States Malformations involving the outflow tract and brachiocephalic arteries represent a third of all
*Correspondence:
congenital cardiac defects (Thom et al., 2006). Examples include double-outlet right ventricle,
Simon D. Bamforth tetralogy of Fallot, discordant ventriculo-arterial connections, also known as transposition, and
[email protected] interruption of the aortic arch. All of these lesions, often producing cyanosis, are detrimental to
the delivery of oxygenated blood to all parts of the body. In adult mammals, the arteries arising from the
Specialty section: aortic arch are asymmetrically left-sided. They develop, during embryogenesis, from the pairs of
This article was submitted to arteries coursing through the pharyngeal arches which are initially bilaterally symmetrical. The
Morphogenesis and Patterning, pharyngeal arches themselves are a transient series of bulges located along the lateral surface of
a section of the journal the head and neck of the embryo. They appear in a cranial to caudal sequence (Graham and Smith,
Frontiers in Cell and Developmental
2001). Each arch has endodermal, mesodermal, and ectodermal components, along with mesenchyme
Biology
derived from the cells of the neural crest (Chapman et al., 1996). Their boundaries are demarcated by
Received: 09 March 2022
the endodermal pouches and the ectodermal clefts (Veitch et al., 1999). The endodermal component
Accepted: 20 April 2022
gives rise to the pharyngeal glands, specifically the thymus, the parathyroids, and the ultimobranchial
Published: 10 May 2022
bodies. The pharyngeal endoderm, furthermore, has been shown, in a range of species, to provide the
Citation:
cues required for patterning of the arches (Piotrowski and Nusslein-Volhard, 2000; McCauley and
Anderson RH and Bamforth SD (2022)
Morphogenesis of the Mammalian
Bronner-Fraser, 2003; Graham et al., 2005). Disruption of this segmentation is seen when the
Aortic Arch Arteries. expression of pharyngeal endodermal genes, such as Tbx1, is perturbed (Piotrowski and Nusslein-
Front. Cell Dev. Biol. 10:892900. Volhard, 2000; Edlund et al., 2014; Jackson et al., 2014; Hasten and Morrow, 2019). With ongoing
doi: 10.3389/fcell.2022.892900 development, the initially segmented appearance of the pharynx is lost. This, initially, is due to the

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Anderson and Bamforth Aortic Arch Arteries

caudal expansion of the second arch, and then subsequently by the malformations of the fourth arch arteries between different age
internalization of the more caudal arches (Graham et al., 2019). groups of Tbx1+/− embryos (Prism 8.01, San Diego, CA,
The five pairs of arteries formed within the arches are never United States). Groups were considered significantly different
present at the same time. Those within the cranial first two arches are when p < 0.05.
destined to remodel into the craniofacial arteries, whereas the caudal
three arch arteries will remodel to form the aortic arch arteries.
Although the process of arch artery remodeling is known to be RESULTS
conserved between mice and humans (Bamforth et al., 2013), some of
the events that occur, such as formation of the external carotid Using high resolution episcopic microscopy datasets, we first
arteries, and migration of the seventh intersegmental arteries to form compared the pharyngeal arch arteries between human
the subclavian arteries, have yet to be fully described. Here, using (CS11–20) and mouse embryos (Figure 1; Table 1). Selected
state-of-the-art imaging techniques, we have investigated databases were acquired from https://dmdd.org.uk (Wilson et al.,
developmental series of both human and mouse embryos and 2015).
fetuses. This has permitted us to analyze the developmental
remodeling of the pharyngeal arch arteries as they evolve to
become the mature arteries arising from the aortic arch. The Arteries of the First and Second
Pharyngeal Arches
In murine development, prior to embryonic (E) day 9.0, the
MATERIALS AND METHODS ventral aorta arises from the primary heart tube and loops to
connect with the paired dorsal aortas (Hiruma et al., 2002)
Human Samples (Figure 1A). A similar arrangement is seen during human
Human embryos (Carnegie stages 11–20) and fetuses (8 and 11 development at Carnegie Stage (CS) 10, when the embryo is
post conception weeks) were obtained from the MRC/Wellcome- around 22 days post ovulation (Congdon, 1922). The ventral
Trust funded Human Developmental Biology Resource (HDBR) aorta at this stage is also known as the first pharyngeal arch artery
maintained at Newcastle University (www.hdbr.org). All or the primitive aortic arch (Hiruma et al., 2002). By the 19-
specimens collected by the HDBR tissue bank are screened by somite stage the second arch artery is beginning to form
Quantitative Fluorescence Polymerase Chain Reaction for the (Figure 1A). The equivalent stage is reached during human
most common chromosomal abnormalities (13, 15, 16, 18, 21, 22, development at CS11 (Figure 1H). The arteries of the first
and sex chromosomes). All of the human samples used in this two murine arches then undergo significant remodeling before
study had an apparent normal chromosome arrangement. E10.5 (Figures 1C,D), with their distal parts destined to become
the mandibular and hyoid arteries, respectively. The proximal
Mouse Samples parts of these arteries fuse to form the primordium of the external
Wild-type mouse embryos of the NIMR Parkes strain were used carotid artery, with the intervening segments persisting as the
in this study. Tbx+/− mice have previously been described (Jerome capillary networks within both the first and second pharyngeal
and Papaioannou, 2001) and were maintained on a C57Bl/6J arches (Hiruma et al., 2002).
genetic background. All mouse embryos from E9.0–E12.5 were The three caudal pairs of arch arteries are typically named the
staged by somite counting. All studies involving animals were third, fourth, and sixth. We propose, however, that the most
performed in accordance with the United Kingdom Home Office caudal arch artery be re-named as the ultimate artery of the
Animals (Scientific Procedures) Act 1986. pulmonary arch (see Discussion). These arteries are formed by
E10.5 in the mouse (Figure 1D), and CS14 in humans
Imaging (Figure 1K). From E11.5 in the mouse, and CS15 in the
High-resolution episcopic microscopy (HREM), magnetic resonance human, the arch artery system begins to remodel, initially with
imaging (MRI), and micro-computed tomography (µCT) techniques the regression of the right ultimate arch artery (cyan arrows in
were performed as previously described (Schneider et al., 2004; Geyer Figures 1E,L) and the septation of the outflow tract (cyan
et al., 2009; Degenhardt et al., 2010; Bamforth et al., 2012; Weninger arrowheads in Figures 1E,L). The aortic sac develops horns
et al., 2014). Each stack of intrinsically aligned serial images was that direct blood from the aorta into the third and fourth arch
appropriately subsampled and converted into a volume data set. arteries (pink arrowheads in Figures 1E,L). By E11.5 in the
Amira software (ThermoFisher Scientific) was used to create two- mouse, and visible at CS15 in the human, the region of the
and three-dimensional images. The structures studied were manually dorsal aorta between the third and fourth arch arteries, the
segmented using the label field function of Amira, and surface carotid duct, begins to involute and disappear (yellow arrows
rendered to produce the three-dimensional images. To visualize in Figures 1E,F,L,M).
patency of pharyngeal arch arteries at E10.5, embryos were
injected with India ink via the heart with pulled Pasteur pipettes.
The Arteries of the Third Pharyngeal Arch,
Statistical Analysis and Formation of the Carotid Arteries
GraphPad Software was used to calculate 95% confidence The third arch arteries are forming by the middle of E9.5 in
intervals and a two-tailed t test to compare the frequencies of the mouse when the embryo has 24 somites (Figure 1B;

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Anderson and Bamforth Aortic Arch Arteries

FIGURE 1 | Development of the aortic arch arteries in human and mouse embryos. Three-dimensional reconstructions of the arch arteries from a developmental
series of mouse (A–G) and human (H–N) embryos were made from HREM datasets. For mouse, embryonic (E) and Theiler Stage (TS), and for human, Carnegie Stage
(CS) and days post conception (dpc) are given. (A–G) In mouse, at the E9.0 stage, the paired ventral aorta (also known as the first arch arteries) and second arch arteries
have formed (A). The third arch arteries are forming by the 24 somite (s) stage (B) and the fourth arch arteries are forming by 31 s (C). At this stage the first and
second arch arteries have become interrupted. (D) Towards the end of E10.5 the third, fourth and the ultimate (*) arch arteries are symmetrical and of equivalent size, and
the proximal portion of the second arch arteries are maintained. (E) The arch arteries begin to remodel at E11.5 with concomitant septation of the outflow tract (cyan
arrowhead), and thinning of the right ultimate arch artery (cyan arrow) and the carotid duct (yellow arrow). The aortic sac has developed horns that direct blood from the
aorta into the third and fourth arch arteries (pink arrowheads). (F) By E12.5 the arch arteries have further remodelled with thinning of the right dorsal aorta and “migration”
of the primitive subclavian complex anteriorly relative to the position of the heart. (G) By the beginning of the fetal stage of mouse development at E13.5, arch artery
remodelling is complete with the asymmetric appearance of the aortic arch arteries. (H–N) The arch arteries in human embryo development follow the same pattern as in
the mouse. The first and second arch arteries are visible at the CS11 stage (H), with the third and fourth seen at CS12 to CS13 (I,J). The three caudal arch arteries are
symmetrical and of equivalent size by CS14 (K) Remodelling of the human arch arteries is evident at CS15 (L) with septation of the outflow tract (cyan arrowhead),
thinning of the right ultimate artery (cyan arrow), involution of the carotid duct (yellow arrows) and the horns of the aortic sac forming (pink arrowheads). (M) Further
remodelling is evident at CS17 with thinning of the right dorsal aorta. (N) Remodelling is almost complete by CS20. Green text indicates a forming artery, white text a
formed artery, and yellow text a remodelling artery. Abbreviations: ad, arterial duct; ao, aorta; as, aortic sac; bc, brachiocephalic artery; cd, carotid duct; CS, Carnegie
Stage; dpc, days post conception; E, embryonic stage; lcc/rcc, left/right common carotid; lsa/rsa, left/right subclavian artery; pa, pulmonary artery; psc, primitive
subclavian complex; pt, pulmonary trunk; rda, right dorsal aorta; s, somites; TS, Theiler Stage; va, ventral aorta. Scale bars: 100 μm in A–G, 200 μm in H–N.

TABLE 1 | Number of wild-type mouse and human embryos analyzed by HREM described by Hiruma (Hiruma et al., 2002), the external
and μCT in this study. carotid arteries are formed from the proximal parts of the
Mouse Human second arch arteries. Here we show that these vessels rapidly
Stage Somites n Stage n
elongate in dramatic fashion, as the embryo grows in the
anterior-posterior axis and the heart descends from E12.5
E9.5 19–27 4 CS11 1 onwards, thus providing the head with its arterial blood
E10.5 31–40 10 CS12 1 supply (Figure 2I; Figure 3A). Careful interrogation of our
E11.5 41–49 13 CS13 3
E12.5 51–60 6 CS14 3
episcopic datasets shows that the common carotid arteries are
E13.5 — 3 CS15 3 formed from the most proximal segment of the third arch
E15.5 — 2 CS16 2 arteries, and this region elongates extensively with growth of
— — — CS17 1 the embryo (Figure 1F; Figure 2I; Figure 3).
— — — 8 pcw 1
In human embryos, the arteries of the third arch make their
— — — 11 pcw 1
first appearance at CS12, which represents 26 days post
CS, Carnegie Stage; E, embryonic day; pcw, post conception weeks. ovulation (Figure 1I). The second arch arteries are
interrupted by the CS13 stage and begin to elongate as the
future external carotid arteries (Figures 2K,L). Further
Figure 2B). These arteries remain symmetrical and do not remodeling of the arch artery system begins at CS15
substantially remodel until the E12.5 stage when the carotid (Figure 1L; Figure 2M), with septation of the outflow tract
duct involutes (Figure 1F; Figure 2G). This allows the dorsal and involution of the carotid duct. With elongation of the
aorta anterior to the third arch arteries to be incorporated to embryo in the anterior-posterior axis (Figure 3B), the distal
become the distal part of the internal carotid arteries, whereas part of the third arch arteries become the proximal part of the
the majority of the third arch arteries themselves become the internal carotid arteries with the distal part formed from the
proximal part of the internal carotid arteries (Figure 2H). As dorsal aorta anterior to the carotid duct (Figure 2N). As in the

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Anderson and Bamforth Aortic Arch Arteries

FIGURE 2 | Development of the common carotid arteries in the mouse and human. Three-dimensional reconstructions of the arch arteries were made from HREM datasets of
the developing mouse (A–I) and human (J–O) embryo and fetus. In the mouse, the first three arch arteries are formed during the E9.5 stage (A–C) with the first arch arteries
interrupted by the 27 somite (s) stage (C). By E10.5 the fourth arch arteries are forming and the second arch arteries have become interrupted (D). During this stage the proximal part
of the second arch arteries begin to elongate (E). Remodelling of the caudal arch arteries begins at E11.5 with septation of the outflow tract and thinning of the right ultimate arch
artery (*; F). By E12.5 more extensive remodelling is underway with involution of the carotid duct and the third arch arteries become the proximal part of the internal carotid arteries with
the distal part from the dorsal aorta anterior to the carotid duct (G). The second arch arteries are forming the external carotid arteries (F,G). As the embryo begins to elongate in the
anterior-posterior axis, the common carotid arteries also elongate dramatically (H,I). In the human, the first two arch arteries are formed by the CS11 stage with the first arch arteries
already interrupted (J). By CS13 the third and fourth arch arteries have formed, the second arch arteries have become interrupted, and the proximal part begins to elongate (K,L). By
CS15 the caudal arch arteries are remodelling; the outflow tract is septated, the right ultimate arch artery (*) is thinner, and the carotid duct is involuting (M). At CS17 the region that will
become the common carotid artery is elongating as the embryo grows in the anterior-posterior axis (N). Towards the end of the embryonic phase at CS20, the common carotid has
extended and the distal third arch arteries have formed the proximal part of the internal carotid arteries with the distal part formed from the dorsal aorta anterior to the now involuted
carotid duct (O). Abbreviations: ao, aorta; bc, brachiocephalic artery; cc, common carotid artery; cd, carotid duct; eca/ica, external/internal carotid artery; pa, pulmonary artery; psc,
primitive subclavian complex; pt, pulmonary trunk; rsa, right subclavian artery; va, vertebral artery. Scale bar: 100 μm.

mouse, the common carotid arteries are formed from the most The Arteries of the Fourth Pharyngeal Arch
proximal segment of the third arch arteries, and this region The fourth arch arteries are first seen in the mouse around the
also elongates extensively with growth of the embryo E10.25 stage, when the embryo has 30–34 somites (Figure 1C).
(Figure 1M; Figure 2N,O; Figure 3). The caudal third, fourth and ultimate arch arteries appear

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Anderson and Bamforth Aortic Arch Arteries

FIGURE 3 | Mouse and human development. Three-dimensional reconstructions, made to scale, of mouse (A) and human (B) embryo and fetus HREM and µCT
datasets showing the rapid growth in the anterior-posterior axis and the change in location of the developing heart relative to the seventh cervical segment. Arrows
indicate the location of the seventh intersegmental artery at embryonic stages (E10.5–12.5 in mouse, CS14, CS16, CS20 in human) and subclavian arteries at the level of
the seventh cervical vertebra at fetal stages (E13.5, E15.5 in mouse, and 8pcw, 11pcw in human). Scale bars: 1 mm in A, 1 cm in B

equivalent in size to each other by late E10.5 (Figure 1D). In the considered in the overall pantheon of congenital defects, half of
human, the fourth arch arteries are seen forming at CS12 all cases of aortic arch interruption are detected in the setting of
(Figure 1I), and these become equivalent in size to the third the 22q11 deletion syndrome, which is the commonest
arch arteries by CS14 (Figure 1K). As development progresses, microdeletion syndrome (Van Mierop and Kutsche, 1986;
the right fourth arch artery is incorporated as the proximal part of Lewin et al., 1997; Boudjemline et al., 2001). In most cases, a
the right subclavian artery with the distal part of the artery 3 Mb deletion on chromosome 22 removes one copy each of 45
derived from the seventh intersegmental artery (described protein coding genes, including TBX1 (Morrow et al., 2018).
below). If the right fourth arch artery fails to form an aberrant Hemizygosity of the TBX1 gene is believed to be the key player in
right subclavian artery occurs, which may be retro-esophageal or the pathogenesis underlying the cardiovascular defects observed
cervical in origin (Figures 4D,F). The transverse aortic arch is in 22q11 deletion syndrome. Mutation of Tbx1 by genetic
critical to the supply of oxygenated blood to the systemic manipulation in mice is known to cause cardiovascular
circulation. The segment of this vessel between the developmental defects (Jerome and Papaioannou, 2001;
brachiocephalic artery and the left common carotid artery is Merscher et al., 2001). Here we have examined Tbx1 mutant
derived from the left horn of the aortic sac, whilst the segment mice using three-dimensional reconstructions to visualize the
between the origins of the left common carotid and left subclavian cardiovascular defects.
artery is derived from the left fourth arch artery. Should this Mice heterozygous for Tbx1 predominantly develop defects
artery fail to form, or be lost during the remodeling phase, an involving inappropriate formation of the fourth arch arteries
interrupted aortic arch is seen (Figure 4D). Although rare when (Figures 4C,E), with interruption of the aortic arch seen in as few

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Anderson and Bamforth Aortic Arch Arteries

FIGURE 4 | Cardiovascular development defects in Tbx1-heterozygous mice. Control (A,B) and Tbx1+/− (Tbx1-het; (C–F) mice were examined for fourth arch
artery derived defects by intra-cardiac ink injection at E10.5 (A,C,E) or magnetic resonance imaging at E15.5 (B,D,F). Control embryos at E10.5 have three bilaterally
symmetrical arch arteries, numbered 3, 4 and the ultimate artery (*), which are patent to ink (A). At E15.5 the pharyngeal arch arteries remodel into the mature aortic arch
arteries (B). In Tbx1-het embryos the fourth arch arteries may either be absent or non-patent to ink (C) or hypoplastic (E). An absent fourth arch artery may lead to
defects such as interrupted aortic arch (iaa), cervical origin of the right subclavian artery (c-rsa) (D) and aberrant right subclavian artery (a-rsa) (F). Penetrance of fourth
arch artery defects in Tbx1+/− embryos from published studies (G). Box and whisker plot with mean % defects, and minimum and maximum values, showing all data
points. Significantly fewer mutants with fourth pharyngeal arch artery (4th PAA) derived defects are seen at the fetal and neonate stages compared to those seen at mid-
embryogenesis (E10.5–E11.0). ****p < 0.0001, two-tailed unpaired t-test. Tbx1+/− mutants have fewer left fourth arch artery-derived defects than on the right (H) and
fewer absent fourth arch arteries than thin vessels (I). Abbreviations: ao, aorta; ad, arterial duct; lcc, rcc, left/right common carotid; lsa, rsa, left/right subclavian artery; lv,
rv, left/right ventricle, NP, non-patent. Scale bar: 100 μm in A, C, E, 500 μm in B, D, (F). Figure adapted from Phillips et al., 2019.

TABLE 2 | Penetrance of fourth arch artery defects in Tbx1+/− embryos from and contribute to normal development of the aortic arch, as
published studies. demonstrated in multiple studies (Figure 4G; Table 2) (Lindsay
Stage E10.5-E11.0 E14.5-P2
and Baldini, 2001; Vitelli et al., 2002; Aggarwal et al., 2006; Guris
et al., 2006; Zhang and Baldini, 2008; Calmont et al., 2009;
Studies (n) 7 9 Randall et al., 2009; Ryckebusch et al., 2010; Papangeli and
Tbx1+/− (n) 127 215
Scambler, 2013; Phillips et al., 2019).
4th PAA defects (% mean ± s.d.) 81.7 ± 7.6 28.7 ± 4.9
95% CI 63–100 17–40
From these studies, which used differently generated Tbx1
mutant lines and techniques to visualize the cardiovascular
CI, confidence interval; E, embryonic day; P, postnatal day; PAA, pharyngeal arch artery;
phenotype in heterozygous mutants (Tables 3, 4), it is
s.d, standard deviation.
apparent that when the defects are counted as derived from
either the right or left fourth arch arteries, from 215 fetuses
as 5% of mutants (Figure 4D) (Phillips et al., 2019). More analyzed, 54 (25%) had a right-sided defect, compared to only 21
common is the finding of retroesophageal or cervical origin of (10%) on the left (Figure 4H; Table 3). Moreover, from studies in
the right subclavian artery (Figures 4D,F) (Papangeli and embryos at mid-embryogenesis that recorded the bilateral defects
Scambler, 2013). The abnormal fourth arch arteries seen at as thin-patent or non-patent (n = 4 studies, 50 embryos
mid-embryogenesis, nonetheless, have the capacity to recover analyzed), the fourth arch arteries were bilaterally affected in

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Anderson and Bamforth Aortic Arch Arteries

TABLE 3 | Arch artery defects in Tbx1-heterozygous fetuses and neonates from published studies.

4th PAA-derived defects

Left-sided Right-sided
Study Tbx1 Genetic Method Age n IAA Co-A A-RSA RAA
mouse line used background of analysis

Randall et al. (2009) Lindsay et al. (2001) C57Bl/6 Ink injection E14.5 25 1 0 4 1
Calmont et al. (2009) E15.5 19 2 0 4 0
Papangeli and Scambler, 17 2 3
(2013b)
Vitelli et al. (2002) C57BL/6 x 129SvEv Histology E18.5 41 3 2 10 3
Zhang and Baldini, (2008) Dissection, 29 1 0 8 0
histology
Ryckebusch et al. (2010) Jerome and Papaioannou, C57Bl/6 Dissection E14.5−E18.5 36 1 4 4 1
Guris et al. (2006) (2001) Dissection, E16.5−P2 18 0 1 4 0
histology
Phillips et al. (2019) MRI, dissection E15.5 and P1 25 2 2 12 0
Aggarwal et al. (2006) Merscher et al. (2001) C57BL/6 x FVB Histology E17.5 5 0 0 0 0
x CD1
— Total 215 10 9 46 5
— 21 (10%) 54 (25%)

A-RSA, aberrant right subclavian artery; Co-A, cervical origin of the aortic arch; IAA, interruption of the aortic arch; PAA, pharyngeal arch artery; RAA, right-sided aortic arch.

TABLE 4 | Arch artery defects in Tbx1-heterozygous embryos from published studies.

4th PAA defect Bilateral defects

Study Tbx1 Genetic Age n Unilateral Bilateral Th-P/ Th- NP/
mouse line used background Th-P P/NP NP

Lindsay and Baldini, (2001) Lindsay et al. (1999) C57BL/6 x 129SvEv E10.5 48 48 ND
Randall et al. (2009) Lindsay et al. (2001) C57Bl/6 14 6 3 ND
Calmont et al. (2009) 11 3 2 — 1 1
Vitelli et al. (2002) C57BL/6 x 129SvEv 14 5 8 1 5 2
Guris et al. (2006) Jerome and Papaioannou, C57Bl/6 E10.75–11.0 15 9 4 ND
Ryckebusch et al. (2010) (2001) E10.5 17 6 8 5 2 1
Phillips et al. (2019) 8 4 4 3 1 —
Total 127 24 24 9 9 4

Unilateral defects do not state whether the fourth pharyngeal arch artery was thin-patent or non-patent. From the detailed bilateral defects (n = 4 studies), 22 individual fourth arch arteries
are affected: 27 vessels are thin-patent (61%) and 17 are non-patent (39%). Abbreviations: ND, not described; NP, non-patent; PAA, pharyngeal arch artery; Th-P, thin-patent.

22 embryos (44 separate vessels) with 27 (61%) of these vessels injection shows the three caudal vessels are of equivalent size and
mildly affected with a thin-patent artery, and 17 (39%) absent, a patent to ink in the control (Figure 5C), but the Tbx1-null mutant
severe defect where the artery is non-patent to ink and thus only has a second arch artery present, with the first arch artery already
presumed to be absent (Figure 4I; Table 4). interrupted by this stage (Figure 5I). In the absence of the third arch
Mice null for Tbx1, however, display a much more severe arteries, the common carotid arteries appear to take a direct origin
cardiovascular phenotype. At E9.5 the first three pharyngeal from the aortic sac (Figure 5K). Consequently, the defective
arches have developed normally in control embryos displaying the development of the pharyngeal region results in the formation of
characteristic pouches and clefts (Figures 5A,B), but in Tbx1- a common arterial trunk in Tbx1-null embryos (Figures 5K,L). Some
deficient embryos, although the first and second pharyngeal Tbx1-null embryos develop a right-sided aorta (Figure 5K) caused by
arches form, the pharyngeal arches caudal to the second do not the aberrant remodeling of the paired dorsal aorta, where it has
(Figures 5G,H). This failure in caudal arch segmentation results in regressed on the left and persisted on the right. A retro-esophageal
the pharyngeal endoderm resembling a tube rather than the right subclavian artery is also observed (Figure 5L), and this is due to
characteristic pouches and clefts seen in control embryos (Figures an absent right fourth arch artery.
5B,H). Although the caudal arches have failed to segment properly
there is retention of the arteries of the first and second arches (Figures The Subclavian Arteries
5G,H), but the caudal third, fourth and ultimate arteries fail to form The subclavian arteries, and its branches, supply blood to the
(Figures 5I,J). Analysis of the pharyngeal arch arteries at E10.5 by ink arms, head, neck and thorax, and are derived from the seventh

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Anderson and Bamforth Aortic Arch Arteries

FIGURE 5 | Cardiovascular developmental defects in Tbx1-null mouse embryos. Control and Tbx1-null embryos were analyzed by high resolution episcopic
microscopy (A,B,D,E,G,H,J,K), intracardiac ink injection (C,I) and magnetic resonance imaging (F,L). Pharyngeal arches are numbered. Three-dimensional
reconstructions of the aortic and pharyngeal arch arteries (in red) and the pharyngeal endoderm (green). Coronal views and three-dimensional segmentation shows the
typical appearance of the pharyngeal arches, arch arteries and endoderm in control (A,B) and Tbx1-null (G,H) embryos at E9.5. In control embryos the symmetrical
arch arteries are visible (B), along with the segmented appearance of the pharyngeal endoderm. In Tbx1-null embryos only arch arteries 1 and 2 are seen and the caudal
pharyngeal endoderm resembles a tube (H). At E10.5 the third, fourth and the ultimate arch arteries (*) are patent to ink (C) but only one artery patent to ink is seen in
Tbx1-null embryos arrow; (I). In the control embryos, at E11.5 (D) the outflow tract is septated into the aorta and pulmonary trunk and at E12.5 (E) the asymmetric
remodeling of the arch arteries is underway: the left-sided aorta and pulmonary trunk join the left dorsal aorta. The right dorsal aorta (yellow arrow) has regressed and the
primitive subclavian complexes are migrating caudally in relation to the descending heart as the embryo grows. By E15.5 the mature arch artery configuration is seen (F).
In Tbx1-null embryos a common arterial trunk has formed at E11.5 (J) and in the embryo shown at E12.5 the dorsal aorta is right-sided (K). The mature heart by E15.5
displays a common arterial trunk and an aberrant right subclavian artery (L). Somite numbers (s) are indicated. Abbreviations: ad, arterial duct; a-rsa, aberrant right
subclavian artery; ao, aorta; cat, common arterial trunk; lcccc, left/right common carotid artery; lda/rda, left/right dorsal aorta lsa,/rsa, left/right subclavian artery; lv/rv,
left/right ventricle; pa, pulmonary artery; psc, primitive subclavian complex; pt, pulmonary trunk; s, somite number. Scale bar: 100 μm in A-E and G-K, 500 μm in F, L.

intersegmental arteries. These arteries take their origin from By CS20, the segment between the seventh intersegmental
the paired dorsal aorta, with each artery supplying blood to artery and the point of bifurcation of the paired dorsal aortas
the somites. The seventh intersegmental arteries are found at is no longer patent, although the remnant of this vessel is still
the point where the paired dorsal aorta comes together to visible (Figure 1N). The right fourth arch artery then provides
form the common dorsal aorta (yellow arrows in Figures the link between the seventh intersegmental artery, which is
6A,D). In the mouse, the right dorsal aorta caudal to the destined to become the right subclavian artery, and the right
origin of the right seventh intersegmental artery begins to horn of the aortic sac which will become the brachiocephalic
regress around E12.5 (Figure 1F; yellow arrow in Figure 5E; artery (Figure 1N; Figure 2O; Figure 7E). When the right
black arrow in Figure 7A). During this period of remodeling, fourth arch artery fails to form, an aberrant right subclavian
the heart descends from its initial location within the neck artery occurs, as seen in Tbx1 mutant embryos (Figure 4F,
region of the embryo, and since the arteries themselves are Figure 5L). In this situation the right dorsal aorta regresses
intersegmental and feed the somites that develop into the caudal to the right seventh intersegmental artery. Both
vertebrae, they must therefore maintain their original relative seventh intersegmental arteries subsequently migrate
position (Figure 3). The right dorsal aorta has disappeared by together to the level of the left subclavian artery in the
E13.5 (Figure 1G; Figure 6B), and the right fourth arch artery fetus, with the right subclavian artery having to cross the
now connects directly to the right seventh intersegmental midline to supply blood to the right arm. Developmental
artery to form the right subclavian artery (Figure 6B). On remodeling of the left subclavian artery in the human is
the left side, the situation is markedly different, with the different than seen in the mouse. In the late embryonic
seventh intersegmental artery, now the left subclavian stage at CS20, the intersegmental artery, now the left
artery, arising from the aortic arch proximal to the level of subclavian artery, is opposite the arterial duct, which is
the arterial duct (white arrow in Figures 6B,C). derived from the left-sided ultimate arch artery
In the human, the right subclavian artery forms in a similar (Figure 6E). By the fetal stage, however, the relative
way to the mouse, from CS14 through to the fetal stage further movement of the left subclavian artery, arising
(Figures 6D–G). At CS14, both dorsal aortas are widely from the dorsal aspect of the left dorsal aorta, “castles”
patent, but by CS17 it is possible to recognize the relative to the ventral union between the arterial duct and
diminution in size of the right dorsal aorta (Figure 1M). the dorsal aorta (Figures 6F,G). This results in there being a

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Anderson and Bamforth Aortic Arch Arteries

FIGURE 6 | Formation of the subclavian arteries in mouse and human development. Three-dimensional reconstructions of HREM (A–E) and μCT (F,G) datasets. In
mouse (A–C) and human (D–G) development the subclavian arteries are initially formed from the seventh intersegmental arteries which emanate from the paired dorsal
aorta at the point of bifurcation (yellow arrow) (A,D). By the end of the embryonic stage, following arch artery remodelling, the insertion of the arterial duct and emergence
of the left subclavian artery are at the same level on the dorsal aorta (white arrow) (B,E) and this is maintained in the mouse into the fetal stage (C). In the human
fetus, however, the origins of these two vessels are separated by a length of dorsal aorta, known as the isthmus (white arrows) (F,G). Note the anomalous origin of the left
vertebral artery (lva) in the 11pcw human fetus (G). Abbreviations: 7th ISA, seventh intersegmental artery; ad, arterial duct; ao, aorta; lcc, left common carotid artery; lsa/
rsa, left/right subclavian artery. Scale bar: 250 µm in A, D; 500 µm in (B,C,E–G).

length of aorta between the origins of the left common carotid cranio-caudal axis then allows for the vertebral arteries to
artery and the left subclavian artery, the so-called isthmus, extend upwards (Figure 3 Figures 7C,F). The first rib, and
which is not seen in the mouse. manubrium of the sternum, can now be recognised, with the
subclavian arteries lying above this bone (Figures 7C,F) and
The Vertebral Arteries underneath the clavicle (not shown).
The vertebral arteries supply blood to the upper part of the
spinal cord and the brain and originate from the seventh The Arteries of the Ultimate Arch
intersegmental arteries. They are clearly visible at E12.5 in the The arteries within the final, and most caudal, of the pharyngeal
mouse (Figure 7A) and at CS17 in the human (Figure 7D). As arches can be seen during E10.5 in murine development, where they
they form, the arteries course up towards the head in close connect the caudal part of the aortic sac to the paired dorsal aorta
apposition to the forming cervical vertebrae, which have not (Figure 1D). Although the arteries of the ultimate arches are the last
fully ossified at this stage (Figures 7A,D). By E13.5 in the to form, they are the first to remodel. In the mouse, during the E11.5
mouse (Figure 7B), and CS20 in the human (Figure 7E), the stage, the aortic sac has been septated to join the separate
vertebrae have progressed in their development. The vertebral intrapericardial components of the aorta and pulmonary trunk,
arteries are then seen to enter the transverse foramen of the formed by the distal part of the outflow tract (Figure 1E). The
sixth cervical vertebrae, and can be traced through each rotation of the outflow tract that subsequently occurs causes the
foramen until they exit from the atlas to join and form the right ultimate arch artery to lengthen and thin distal to the origin of
midline basilar artery. On the right side, as the right the right pulmonary artery, and eventually regresses completely
subclavian artery has not completed its full remodelling (Figures 1E,F). The artery of the left ultimate arch expands in
process in relation to the vertebrae, the right fourth arch diameter and functions as the arterial duct, or “ductus arteriosus”
artery takes a downward kink to accommodate the origin of (Figure 1G; Figures 6B,C).
the developing subclavian artery at the level of the seventh During human development, the ultimate arch arteries are
cervical vertebra (Figures 7B,E). By the fetal stages, E15.5 in formed by CS14 (Figure 1K). By CS15 the right ultimate arch
mouse and 11pcw in human, the heart has descended further artery is beginning to regress distal to the origin of the right
into the thoracic cavity. The extension of the fetus in the pulmonary artery (Figure 1L). By CS17 the artery of the right

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Anderson and Bamforth Aortic Arch Arteries

FIGURE 7 | Formation of the vertebral arteries in mouse and human development. Three-dimensional reconstructions of HREM (A–E) and μCT (F) datasets.
Arteries are coloured in red, vertebrae in green. In the mouse embryo, the vertebral arteries emanate from the dorsal surface of the seventh intersegmental arteries and
course anteriorly towards the head, abutting the forming vertebrae (A). The thinning right dorsal aorta is indicated (black arrow). In the mouse fetus the vertebral arteries
insert into the transverse foramen of the sixth cervical vertebra (B,C). In the human the same process of vertebral artery development occurs in equivalently stage-
matched specimens (D–F). The thinning right dorsal aorta at CS17 is indicated (D; black arrow). Note the human fetus at 11pcw with the aberrant origin of the left
vertebral artery from the aortic arch (F; left view). The vertebral artery anomalously enters the transverse foramen of the fourth cervical vertebra. Abbreviations: C, cervical
vertebra; E, embryonic day; pcw, post conception weeks; rda, right dorsal aorta; lsa/right; left/right subclavian artery; T, thoracic vertebra; va, vertebral artery. Scale bar:
500 µm.

ultimate arch has disappeared, leaving the pulmonary arteries arteries (Congdon, 1922), we have used state-of-the-art
arising directly from the caudal part of the aortic sac (Figure 1M). imaging techniques to produce three-dimensional models of
The left ultimate arch artery has developed into the arterial duct the developing pharyngeal arch arteries, and other associated
by CS20 (Figure 1N; Figures 6E–G). arteries, as they remodel into the aortic arch arteries in humans in
mice. We clarify the similarities and differences between human
and mouse arch artery development, as well as the origin of the
DISCUSSION common carotid artery from the proximal part of the third arch
artery (Figure 8).
In this study, in the centenary year of the seminal work produced Various interpretations of the fate of the third arch arteries
by Congdon on the development of the human aortic arch are to be found in the literature. For human development, the

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Anderson and Bamforth Aortic Arch Arteries

data also suggests that the external carotid arteries are

formed from the elongation of the proximal parts of the
second arch arteries after they have become interrupted
around E10.5 in the mouse and CS13 in the human. That
the third arch artery is critical for formation of the common
carotid artery has been demonstrated in genetically altered
mice. In the absence of the third arch arteries in Hoxa3-
deficient mice, the common carotid arteries do not form
(Kameda et al., 2002; Kameda, 2009). In mice lacking Pax9
the third arch arteries collapse through lack of smooth muscle
cell investment resulting in persistence of the carotid duct and
the internal and external carotid arteries arising separately
from the aortic arch itself (Phillips et al., 2019).
Analysis of ten published studies that investigated the
cardiovascular defects in Tbx1-heterozygous mice
confirmed that there is recovery of the fourth arch artery
between mid-embryogenesis and the fetal stage, as fewer
defects are observed than expected. The mechanism of
recovery is unknown, but is likely to be linked to a
hypoplastic artery further developing into a more normal
sized vessel between E10.5 and the fetal stage. Indeed some
studies have looked at this intermediate stage (Lindsay and
Baldini, 2001; Ryckebusch et al., 2010; Papangeli and
Scambler, 2013). Collectively, they found that two-thirds of
Tbx1-heterozygous embryos had an abnormal fourth arch
artery. This is intermediate between the incidences of four-
fifths for E10.5, and three-tenths at fetal stages.
The genetic background was fairly consistent between all
the Tbx1-heterozygous studies examined, with the mice used
either being backcrossed to C57Bl/6, or containing a
FIGURE 8 | Cartoon showing the fate of the originally bilaterally
proportion of this strain on a mixed background. The
symmetrical pharyngeal arch arteries. Each pharyngeal arch artery is
numbered, with asterisks indicating the arteries of the pulmonary, or ultimate, methods of analysis to visualize the defects in fetuses and
arch. The arch arteries that are seen in the remodeled system are neonates were by dissection, combined with ink injection in
labelled. Abbreviations: AD, arterial duct; Ao, aorta; BC, brachiocephalic some studies, histology, and magnetic resonance imaging. It is
artery; CCA, common carotid artery; CD, carotid duct; ECA, external carotid possible that retroesophageal, or cervical origin, of the right
artery; ICA, internal carotid artery; ISA, intersegmental artery; LDA, left dorsal
subclavian artery may be missed in some studies. The
aorta; PA, pulmonary artery; PT, pulmonary trunk; RDA, right dorsal aorta; VA,
vertebral artery. investigation employing magnetic resonance imaging for
analysis identified the highest percentage of embryos with
an aberrant right subclavian artery (Phillips et al., 2019). This
third arch arteries have been reported to form only the suggests that imaging and three-dimensional reconstructions
common carotid arteries (Rana et al., 2014) or the internal are better suited to detect complex cardiovascular
carotid arteries (Padget, 1948; Moffat, 1959). Other studies in malformations in mouse models than histology or direct
humans, however, describe that the proximal parts of the third visualization at dissection (Schneider et al., 2004; Bamforth
arch arteries form both the common carotid artery and the et al., 2012).
distal part forms the proximal segment of the internal carotid Our analysis of the published data from Tbx1-heterozygous
artery (Congdon, 1922). In the mouse, Hiruma showed that embryos demonstrates that malformations in the fetal and
the proximal parts of the third arch arteries did, indeed, form neonatal stages more frequently involve the right rather than
the common carotid arteries, with fusion of the proximal parts the left fourth arch artery. Left side-derived defects, such as
of the first and second arch arteries contributing to the origin interruption of the aortic arch, are lethal in the neonatal
of the external carotid artery, and the distal part of the third period if not surgically corrected, whereas those defects
arch arteries form the basal part of the internal carotid arteries affecting the right fourth arch artery, such as aberrant right
(Hiruma et al., 2002). Our own reconstructions agree with this subclavian artery, may be asymptomatic. Given that these
interpretation, although we show it is the most proximal part vessels form symmetrically in the embryo, and it is the absence
of the third arch arteries, caudal to the origin of the external of either vessel that will cause the respective phenotypes, it is
carotid arteries, that form the common carotid arteries, and difficult to speculate on a mechanism that explains the higher
this initially short vessel dramatically elongates with the frequency of problems with the right fourth arch artery. It is
growth of the embryo in the anterior-posterior axis. Our possible that, in future, some as yet unrecognized asymmetry

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Anderson and Bamforth Aortic Arch Arteries

in gene expression between the left and right sides may channels, however, have been considered by some to represent
provide an explanation. the enigmatic arteries of the fifth arches. We believe that this is
The development of the left subclavian artery in humans and incorrect, since the channels do not connect the aortic sac with
mice is very similar during the embryonic stages. There is a the dorsal aorta, as they would had they been true arch arteries.
difference, however, in the final position of the left subclavian The persistence of such channels, nonetheless, provides an
artery apparent in the fetal stages. In the mouse the left subclavian excellent explanation for the lesion known as “double
artery emerges from the dorsal aorta at the same level as the barrelled aorta”. The many alternative lesions described on
insertion of the arterial duct. In the human, although the left the basis of persistence of a putative fifth arch artery, in
subclavian artery is at an equivalent level in the embryo, there is contrast, are all well explained on the basis of alternative
further movement as development progresses resulting in the left remodelling of the initially bilateral system of the third and
subclavian artery emerging from the aortic arch more cranially to fourth arch arteries, along with the horns of the aortic sac
the arterial duct. This “castling” maneuver occurs as the fetus (Gupta et al., 2014; Anderson et al., 2018). It is also the case,
grows substantially in the anterior-posterior axis and creates a however, that we initially described a vessel traversing through a
segment of the aorta known as the isthmus. In the human fetus segment of pharyngeal mesenchyme towards the aortic sac in a
the isthmus is referred to as an “arterial watershed” with a human embryo as a fifth arch artery (Bamforth et al., 2013). As
complex hemodynamic physiology from different left and this vessel did not make contact with the aortic sac, we now
right ventricular cardiac outputs (Tynan et al., 2016). The interpret this as a transient collateral entity, rather than an
isthmus is a common site for coarctation of the aorta (Kenny artery of a true fifth pharyngeal arch.
and Hijazi, 2011). Although this condition is relatively common Although the Human Developmental Biology Resource
in human neonates, it is not widely reported in mouse models collects many specimens at multiple stages of development,
(Gessler et al., 2002; Quintero-Rivera et al., 2015). Whether the these are processed in a wide variety of ways to accommodate
differences in aorta anatomy are the reason for this disparity the multiple users of this biobank. Also, the availability of the
remains to be elucidated. younger embryo stages is limited. We were fortunate to have
The “traditional” approach to remodelling of the arteries of access to a total of 16 specimens processed for HREM or µCT
the pharyngeal arches has been to number the five pairs of imaging, covering the periods from 3.5 to 11 weeks
arteries as one through to four, but with the final pair being subsequent to conception. For a number of these stages,
described as the sixth. This non-sequential system of however, only one specimen was available for analysis
numbering has been attributed to the basic plan adopted for (Table 1). Due to the limited availability of intact human
evolutionary distant vertebrates (Kardong, 2008), or to the embryos for research, some published studies do rely on using
presumed disappearance during development of a fifth pair of one embryo per stage, making the assumption that these are
pharyngeal arches, along with their arteries (Congdon, 1922). representative of normal (Hikspoors et al., 2022) or make
Unequivocal evidence has recently been provided to refute the comparisons to other databases (Rana et al., 2014). The
existence of the fifth arch arteries during normal development human embryos used in our study were all genotyped and
(Graham et al., 2019). Our own investigations of murine and found to be karyotypically normal, and with the expected
human development, now supported by the data presented in morphology when compared to published studies of human
this study, endorse these findings (Anderson et al., 2020). It developmental anatomy (Congdon, 1922; Rana et al., 2014).
would create huge confusion, nonetheless, if the ultimate
arches, and their arteries, were now labelled in logical
fashion as being fifth in number. One solution to this DATA AVAILABILITY STATEMENT
dilemma, therefore, is to describe the arches as being
ultimate, or terminal. An alternative is to follow the The datasets presented in this study can be found in online
precedent of Congdon, and to consider the arches as being repositories. The names of the repository/repositories and
pulmonary (Anderson et al., 2020; Congdon, 1922). It can be accession number(s) can be found below: This study
argued, however, that this may add further confusion, since the reanalyzed existing high resolution episcopic microscopy
pulmonary arteries themselves develop within the pharyngeal datasets from https://dmdd.org.uk. MRI and micro-CT
mesenchyme. They take their origin, nonetheless, from the datasets of mouse and human fetuses are openly available at
arteries that are formed within the ultimate arches, albeit that https://doi.org/10.25405/data.ncl.19313939.
only the artery of the left arch normally persists, becoming the
arterial duct. Our preference, therefore, is to follow the
suggestion of Congdon, and to describe the arteries as ETHICS STATEMENT
belonging to the pulmonary arches (Figure 8).
The issue of the potential presence of arteries representing The studies involving human participants were reviewed and
alleged fifth arches then requires additional consideration. It is approved by North East—Newcastle and North Tyneside 1
well recognized that transient collateral channels connect the Research Ethics Committee. The patients/participants provided
dorsal parts of the arteries of the third and fourth arches during their written informed consent to participate in this study. The
their embryonic development (Congdon, 1922; Lorandeau et al., animal study was reviewed and approved by the Animal Welfare
2011; Geyer and Weninger, 2012; Bamforth et al., 2013). These Ethical Review Board, Newcastle University.

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Anderson and Bamforth Aortic Arch Arteries

AUTHOR CONTRIBUTIONS Biology Resource (www.hdbr.org) is funded jointly by the

Medical Research Council and the Wellcome Trust (MR/
RA analysed the data and wrote the manuscript. SB conceived the R006237/1).
project, acquired funding, analysed the data, created the figures,
and wrote the manuscript.
ACKNOWLEDGMENTS
FUNDING
We thank Janet Kerwin, Timothy Mohun and Jürgen Schneider
This work was funded by the British Heart Foundation (FS/08/ for the production of μCT, HREM and MRI datasets, and Gemma
016/24741 and PG/16/39/32115). The Human Developmental Price for the schematic of the developing arch arteries.

Graham, A., Okabe, M., and Quinlan, R. (2005). The Role of the Endoderm in the
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Associated with Bicuspid Aortic Valve, Aortic Coarctation and Patent potential conflict of interest.
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ddv004 Publisher’s Note: All claims expressed in this article are solely those of the authors
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Frontiers in Cell and Developmental Biology | www.frontiersin.org 14 May 2022 | Volume 10 | Article 892900