Background: The pharyngeal arches are a series of bulges found on the lateral surface of the head of vertebrate embryos, and it is within these segments that components of the later anatomy are laid down. In most vertebrates, the post-otic pharyngeal arches will form the branchial apparatus, while in amniotes these segments are believed to generate the larynx. It has been unclear how the development of these segments has been altered with the emergence of the amniotes. Results: In this study, we examined the development of pharyngeal arches in amniotes and show that the post-otic pharyngeal arches in this clade are greatly diminished. We find that the post-otic segments do not undergo myogenesis or skeletogenesis, but are remodelled before these processes occur. We also find that nested DLX expression, which is a feature of all the pharyngeal arches in anamniotes, is associated with the anterior segments but less so with the posterior arches in amniotes. We further show that the posterior arches of the mouse embryo fail to properly delineate, which demonstrates the lack of function of these posterior segments in later development. Conclusion: In amniotes, there has been a loss of the ancestral "branchial" developmental programme that is a general feature of gnathostomes; myogenesis and skeletogenesis This is likely to have facilitated the emergence of the larynx as a new structure not constrained by the segmental organisation of the posterior pharyngeal region.
Keywords: Pharyngeal segmentation; Pharyngeal pouch; Pharyngeal arch; DLX; Amniote evolution; Larynx
The development of the pharyngeal arches is underpinned by the generation of the pharyngeal pouches, outpocketings of the endoderm, which contact the overlying ectoderm, with the other constituents of the arches, the neural crest and mesoderm, migrating into these preformed segmental units [[
In post-metamorphic amphibia and in amniotes, however, the branchial skeleton is lost and the larynx develops [[
Yet, there have been few studies focussed on the development of the posterior arches and it is unclear whether such a correspondence exists or how the development of the posterior arches was modified to allow for the emergence of the larynx. Moreover, the situation in amniotes is more complex than that in other vertebrates, since the posterior pharynx is segmentally organised for only a transient period. Once all the arches have been formed, the second arch expands disproportionately to cover the posterior arches before fusing and enclosing them [[
Graph: Fig. 1 Segmental and post-segmental stages of pharyngeal development in human embryos. In this figure High-Resolution Episcopic Microscopy (HREM) data from the human embryos from the DMDD website (https://dmdd.org.uk/) is shown. a At Carnegie Stage (Cs) 13, the first three most anterior pharyngeal arches have formed and are morphologically evident. b By Cs 15 stage, a further fourth arch has formed. c However, by Cs16 the segmental nature of the pharyngeal region has been lost as the second arch has overgrown the posterior arches, covered and subsumed them. Scale bar = 0.25 mm
In this study, we assessed the early development of the posterior pharyngeal arches and their subsequent envelopment by the second arch. We further document the relationship between the pharyngeal segments and the formation of the muscular and skeletal derivatives. We find that while there is a clear correspondence between these events and the anterior arches, this is not the case for the posterior segments. Thus, while the processes of myogenesis and chondrogenesis are underway at several sites in the embryo, they are not a feature of the posterior pharyngeal region. We further show that, although the anterior pharyngeal segments exhibit nested DLX gene expression, the expression of these genes is greatly reduced in the posterior arches, suggesting that the need to regionalise the neural crest along the proximodistal axis in these segments is less in amniotes. We note that the reduction in DLX expression is more extensive in the mouse than in the chick, and this prompted us to further investigate differences between the posterior segments in these species. Interestingly, we find that while the 4th pharyngeal pouch contacts the ectoderm in chick and human embryos, it does not do so in mice. Consequently, the posterior pharyngeal segments are not fully delineated in this species, which further underscores their lack of significance for later events.
We conducted a detailed analysis of the period covering the formation of the post-otic pharyngeal segments and their subsequent envelopment by the second arch in chick. At stage 17 (HH17), PAX1 staining highlights the formed, and forming, pharyngeal pouches and it is apparent that the first three arches are delineated, but that the more posterior segments are not clearly defined (Fig. 2a). By HH21, however, the full complement of four pouches and five arches—numbered 1,2,3,4 and 6—have formed (Fig. 2b). In amniotes, the most posterior pharyngeal arch is termed the sixth, even though this is numerically the fifth arch, due to the long held, but erroneous, belief that a transient fifth arch formed between this segment and the fourth [[
Graph: Fig. 2 The generation and remodelling of the pharyngeal segments. a Side view of a HH17 chick embryo, the three fully formed anterior arches can be seen while at HH21 (b) the full complement of arches have formed. PAX1 highlights the position of the intervening pouches. (c) Longitudinal section of a HH21 chick embryo, DLX2 expression shows the neural crest component of the arches. Noticeably, the caudal edge of arch 6 has no clear definition; highlighted by the black arrow while the anterior limit of this arch is delineated by the pouch of contact between the fourth pouch and the overlying ectoderm, indicated by the black arrowhead. d Longitudinal section of a HH21 embryo, TBX1 expression demonstrates the relative positions of the mesoderm components of each of the arches as well as the pharyngeal endoderm and pouches. A sizeable mesodermal population can be seen in arches 1, 2, and 3. Arch 4 by comparison only has a reduced mesoderm component; indicated by the white arrow. At HH20 (e) the second arch is beginning to expand and by HH24 (f) it has overgrown the posterior arches. The arches are numbered in all panels. Scale bar = 0.1 mm
It has been widely documented that in anamniotes myogenesis and skeletogenesis occur within the segmental framework of the pharyngeal arches [[
Graph: Fig. 3 Myogenesis and chondrogenesis at segmental and post-segmental pharyngeal stages (a) MYOD expression in a HH21 chick embryo. Myogenesis can be seen to be occurring in the first two arches but not the more posterior arches. The migratory hypoglossal myoblasts, which are somite derived can be seen to migrate through the ventral pharyngeal midline region – white arrow. b MyoD expression in a TS16 mouse embryo. Myogenesis can be seen to be occurring in the first two arches, but not the more posterior arches. The somite derived hypoglossal myoblasts can be seen to be migrating around the caudal aspect of the pharyngeal arches and along the ventral pharyngeal midline, indicated by the white arrow. c MYOD expression in chick embryo at HH25. Ongoing myogenesis can be seen to be occurring in the somites and within the limb buds. There is some myogenesis apparent in the extended second pharyngeal arch, but not in the posterior pharynx. d Longitudinal section through the pharyngeal region of a HH25 chick embryo. MYOD expression within the second arch is apparent, but there is no expression in the more posterior pharyngeal region. MYOD expression is also seen in the somites. The position of the notochord (N) is marked. e MyoD expression in mouse TS17 embryo. Myogenesis is associated with the somites, developing limb buds and anterior pharyngeal arches, but is absent from the posterior pharyngeal region, except for expression in the hypoglossal myoblasts, indicated by the white arrow. f COL2A expression in HH25 chick embryo. Ongoing chondrogenesis can be seen to be occurring in the somites and limb buds. There is some chondrogenesis apparent in the extended second pharyngeal arch but not in the posterior pharynx. g Longitudinal section through the pharyngeal region of a chick embryo at HH25. COL2A expression within the second arch is apparent, as is expression around the notochord (N) but there is no expression in the more posterior pharyngeal region. h Col2a expression in mouse TS17 embryo. Chondrogenesis is associated with the somites, developing limb buds and anterior pharyngeal arches, but is absent from the posterior pharyngeal region
We further analysed muscle and cartilage differentiation at later stages while the second arch is covering the posterior arches. In chick at HH25, MYOD staining can be seen in the first and second arch, but not in the more posterior pharynx (Fig. 3c, d), even though these segments have a resident mesodermal population (Fig. 2). However, it is also clear that myogenesis is well underway in other areas of the embryo including the myotome of the somites and myoblasts migrating into the limbs. Similarly, in the mouse at TS17, myogenesis is evident in the first two arches, in the myotome and in the myoblasts populating the forelimb. Yet, there is no myogenesis within the posterior arches at this stage, bar the migratory hypoglossal myoblasts (Fig. 3e). We also used COL2A staining to reveal sites of chondrogenesis at these later stages. We find that, while there is extensive chondrogenesis in the somites and the limbs, in the chick at HH25, there is little chondrogenesis in the pharynx. COL2A staining is evident in the first and second arches, but not in the more posterior arches (Fig. 3g). In the mouse at TS17, we also found chondrogenesis underway in the somites and limbs, and while there was some staining in the first two arches, the posterior was devoid of COL2A expression (Fig. 3h). Thus, we find that the posterior pharyngeal segments do not entertain myogenesis or skeletogenesis during their period of definition.
We further assessed the degree to which the pharyngeal arches are regionalised as they develop. The patterning of the neural crest and its skeletal derivatives along the proximodistal axis of the arches involves the nested expression of members of the DLX family of transcription factors. DLX genes are organised as linked pairs—DLX1/2, DLX3/4 and DLX5/6—with DLX 1/2 being expressed throughout the ectomesenchyme of the arches, DLX 3/4 at the distal portion and DLX 5/6 from the mid region distally. This situation is believed to be a general feature of gnathostomes [[
Graph: Fig. 4 DLX expression in the pharyngeal arches in chick and mouse embryos. a Side view of a HH21 chick embryo showing DLX2 expression throughout the dorsoventral extent of all the pharyngeal arches. b Longitudinal section through the arches showing DLX2 expression in the ectomesenchyme of the arches. c Side view of a TS16 mouse embryo showing pronounced expression of Dlx2 in the first two arches, but much reduced expression in the posterior arches. d Side view of a HH21 chick embryo showing DLX6 expression in the mid region of the three most anterior arches but not the most posterior arches. e Longitudinal section through the arches showing DLX6 expression in the ectomesenchyme of the three most anterior arches. f Side view of a TS16 mouse embryo showing high levels of Dlx6 in the mid region of the first two arches, but much reduced expression in the posterior arches. g Side view of a HH21 chick embryo showing DLX3 expression in the more distal region of the two most anterior arches but not those lying posteriorly. h Longitudinal section through the arches showing DLX3 expression in the ectomesenchyme of the two most anterior arches. f Side view of a TS16 mouse embryo showing high levels of Dlx3 in the distal region of the anterior two arches, but much reduced expression in the posterior arches. Scale bar = 0.1 mm
Graph: Fig. 5 Pharyngeal pouch morphology in mouse and human embryos. a Confocal section through the pharyngeal region, at the segmental phase, of a TS17 (E10.5) mouse embryo. Neural crest (Wnt1:cre+) structures are labelled with membrane GFP, while non-neural crest tissues express membrane RFP. It is noticeable that the fourth pouch, highlighted by an arrow, does not contact the ectoderm. b HREM section through the pharyngeal region, at the segmental phase, of a CS15 human embryo. The fourth pouch, highlighted by an arrow, does contact the ectoderm. Scale bar = 0.25 mm
These differences in DLX expression in the posterior arches between chick and mouse prompted us to further analyse the underlying organisation of the arches. Central to the organisation of the pharyngeal arches is the establishment of the contact between the pharyngeal pouches and the overlying ectoderm. This serves to define the anterior and posterior limits of the arches and to segregate the mesenchymal populations of the different arches. We have previously documented the formation of the pharyngeal pouches and arches in detail in chick [[
A key observation from this study is that the development of the posterior pharyngeal arches in amniotes is markedly different from that of other vertebrate clades. In anamniotes, the posterior arches generate muscular and skeletal structures, exhibit nested expression of DLX linked pairs and these segments form the blueprint for the organisation of the branchial apparatus [[
Finally, it should also be noted that while the skeletal components of the branchial apparatus of anamniotes are generally neural crest derived [[
Chick embryos were staged according to Hamburger and Hamilton [[
Embryos were fixed in in 3.7% (v/v) formaldehyde in PBS and in situ hybridisation carried out as previously described [[
CCSFE labelling was carried out as previously described [[
The project was conceived by AG. The experimental work was carried out by SP, JR, AS, AD, KL and AG. The paper was written by AG and SP with JR, AS, AD and KL commenting and modifying the draft. All authors read and approved the final manuscript.
All embryo collection was carried out as prescribed by the UK Animals (Scientific Procedures) Act 1986.
All authors consent to publication.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We would like to thank Dr. Tom Mohun, Crick Institute London, for help with the HREM analysis of the Human and mouse embryos. We thank Caroline Formstone, Clemens Kiecker, Malcolm Logan and Jill Sales for comments on the text.
This work was funded by a BBSRC grant (BB/R006199/1) to AG.
All data and material are available from the corresponding author. anthony.graham@kcl.ac.uk.
By Subathra Poopalasundaram; Jo Richardson; Annabelle Scott; Alex Donovan; Karen Liu and Anthony Graham
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