Mode of reduction in the number of pharyngeal segments within the sarcopterygians
© Shone et al. 2016
Received: 17 February 2016
Accepted: 18 March 2016
Published: 21 March 2016
Pharyngeal segmentation is a defining feature of vertebrate embryos and is apparent as a series of bulges found on the lateral surface of the embryonic head, the pharyngeal arches. The ancestral condition for gnathostomes is to have seven pharyngeal segments: jaw, hyoid, and five posterior branchial arches. However, within the sarcopterygians, the pharyngeal region has undergone extensive remodelling that resulted in a reduction in the number of pharyngeal segments, such that amniotes have only five pharyngeal arches. The aim of this study is to probe the developmental basis of this loss of pharyngeal segments.
We have therefore compared the development of the pharyngeal arches in an amniote, the chick, which has five segments, with those of a chondrichthyan, the catshark, which has seven segments. We have analysed the early phase of pharyngeal segmentation and we find that in both the most anterior segments form first with the posterior segments being added sequentially. We also documented the patterns of innervation of the pharynx in several vertebrates and note that the three most anterior segments receive distinct innervation: the first arch being innervated by the Vth nerve, the second by the VIIth and the third by the IXth. Finally, we have analysed Hox gene expression, and show that the anterior limit of Hoxa2 aligns with the second pouch and arch in both chick and catshark, while Hoxa3 is transiently associated with the third arch and pouch. Surprisingly, we have found that Hoxb1 expression is spatially and temporally dynamic and that it is always associated with the last most recently formed pouch and that this domains moves caudally as additional pouches are generated.
We propose that the first three pharyngeal segments are homologous, as is the posterior limit of the pharynx, and that the loss of segments occurred between these two points. We suggest that this loss results from a curtailment of the posterior expansion of the pharyngeal endoderm in amniotes at relatively earlier time point, and thus the generation of fewer segments.
Pharyngeal segmentation is a characteristic of all vertebrates. During development it is first seen in the appearance of a series of bulges on the lateral surface of the head, the pharyngeal arches . These primordia consist of epithelia with a mesenchymal filling. Externally, the epithelium is derived from the ectoderm and internally from the endoderm, while the mesenchyme consists of a core of mesoderm surrounded by neural crest cells. Between the arches the ectoderm and the endoderm contact each other, and it is this apposition between these tissues that defines the anterior and posterior margins of each of the pharyngeal arches .
A general feature of gnathostomes is to have seven pharyngeal segments; jaw, hyoid, and five posterior branchial (gill-bearing) arches . This arrangement represents the plesiomorphic state as this situation is observed in many chondrichthyans and all actinopterygians, and this arrangement is also seen in sarcopterygians, such as the coelacanth Latimeria and the lungfishes, the sister group to the tetrapods [3–6]. However, extant tetrapods show a reduced number of pharyngeal segments. Thus, larval amphibians have six pharyngeal segments, while amniote embryos have only five. Moreover, there has also been a loss of pharyngeal segmentation in the adult form as a result of the remodelling of the pharynx, which occurs during metamorphosis in amphibians and embryogenesis in amniotes. The reduction in the number of pharyngeal segments and the loss of explicit segmentation in adult tetrapods clearly reflects the shift from respiration in water via gills to air breathing using lungs.
These alterations to the pharyngeal region that occurred with the evolution of the tetrapods are obviously underpinned by changes to the developmental programme. The loss of overt pharyngeal segmentation is due to the overgrowth of the second arch to cover the more posterior arches, followed by the fusion of the caudal edge of the second arch to the subjacent tissue which results in the internalisation of the posterior arches . However, it is less clear how the reduction in the number of pharyngeal segments has been achieved, although this is likely to involve changes to the early organisation of the pharyngeal endoderm.
The segmentation of the endoderm, which results in the formation of the pharyngeal pouches, is central to the development of the pharyngeal arches . The pharyngeal pouches form at distinct positions along the anteroposterior axis. The two most anterior pouches form first with the more posterior pouches forming subsequently and sequentially [9, 10]. The pouches grow to contact the overlying ectoderm, which invaginates to meet them, generating the pharyngeal clefts, and thus neural crest cells and mesoderm migrate into these preformed units. Significantly, in mutants in which the endoderm fails to segment, the pharyngeal arches fail to form [11, 12]. Consequently, a reduction in the number of pharyngeal segments in sarcopterygians must have involved alterations to the development of the pharyngeal pouches.
To begin to address the route through which the number of pharyngeal segments has been reduced, we have compared the development of the pharyngeal arches in the chick (Gallus gallus), an amniote, which has five segments, to those in the catshark (Scyliorhinus canicula), a chondrichthyan, which has seven segments. We have analysed the formation of pharyngeal pouches, patterns of innervation of the pharyngeal arches, and the expression of Hox genes, which are markers of axial identity in the pharynx, and in particular how these relate to the endodermal pouches. Our data support the view that the three most anterior pharyngeal segments are homologous between different vertebrate classes, and that the reduction in the number of segments must have been achieved by a loss of those lying more caudally. Notably, we find that in both chick and catshark embryos Hoxb1 expression marks the posterior limit of the pharynx. However, this expression pattern is spatially and temporally dynamic, with Hoxb1 labelling the posterior limit of the pharynx at early and late stages irrespective of the number of segments that have been generated. Thus we conclude, that the posterior limit of the pharynx is likely to be homologous across the gnathostomes and that the reduction in the number of pharyngeal segments that accompanied the evolution of the tetrapods will have involved a heterochronic shift such that the posterior expansion of the pharynx was curtailed at a relatively earlier time point and thus less segments were generated.
Materials and methods
Fertile hen’s eggs were incubated at 38 °C to the required stages (HH st)  and the embryos were fixed in 4 % PFA. Scyliorhinus canicula embryos were taken from the egg cases, anaesthetized (MS222), staged  and fixed in 4%PFA. Lamprey embryos were kindly donated by Dr Sebastian Shimeld, University of Oxford.
Previously fixed embryos were washed three times 30 min in PBS/1 % TritonX-100 (PBSTx) before being washed in a blocking solution of 10 % goat serum in PBSTx twice for one hour at room temperature. The relevant primary antibody was diluted in the blocking solution with 0.02 % sodium azide and the embryos incubated at 4 °C for 1–2 weeks. Embryos were then rinsed in blocking solution and washed three times for one hour in blocking solution before adding the secondary antibody diluted in blocking solution with 0.02 % sodium azide. This was incubated at 4 °C for 1–2 weeks. The primary antibodies used were rabbit anti-laminin at 1:100 (Sigma); mouse anti- NFM 1:10000 (Zymed). Secondary antibody was Alexa 488-conjugated goat anti-mouse IgG, and Alexa 568 goat anti-rabbit IgG, both used at 1:1000 (Molecular Probes). For sectioning, embryos were washed into PBS, embedded in gelatin, fixed, and vibratomed at 50 μm slices.
In situ hybridisation
Previously fixed embryos were washed twice in PBST (PBS + 0.1 % Tween-20), dehydrated through a methanol series in PBST, bleached with 6 % H2O2 in methanol for 1 h and then rehydrated. Embryos were then treated with 10 μg/ml Proteinase K in PBST for 20 min, postfixed with 4 % PFA/0.1 % gluteraldehyde and then washed twice in PBST. The embryos were incubated at 70 °C in hybridisation buffer (50 % formamide, 1.3X SSC, 5 mM EDTA, 50 μg/ml tRNA, 100 μg/ml heparin, 0.2 % Tween-20, 0.5 % CHAPS) for 1 h, followed by overnight at 70 °C in digoxigenin-labelled riboprobes. Embryos were washed four times with hybridisation buffer at 70 °C, then three times 30 min with MABT (100 mM maleic acid, 150 mM NaCl, 1 % Tween-20; pH7.5), and blocked with 2 % BBR (Roche Diagnostics)/20 % goat serum/MABT and incubated overnight in anti-DIG-AP antibody (Roche) diluted 1:2000 in the same block. Embryos were washed extensively with MABT and the alkaline phosphatase activity detected using NBT and BCIP in NTMT. The reaction was stopped by washing in MABT and fixing in 4 % PFA.
Sequential generation of pharyngeal segments
Analysis of pharyngeal innervation further identifies the most posterior segments as being reduced in number
HOX gene expression boundaries and their relationship to the pharyngeal arches and pouches in chick embryos
To ascertain which pharyngeal segments/pouches have been lost from extant tetrapods, we need to be able to align the reduced number of posterior pouches of an amniote, in this case the chick, with those of the catshark, which represents the more basal condition. With regard to this issue, Hox genes are useful as they can be used as markers of axial identity. Although the expression patterns of Hox genes in the pharyngeal arches have been well documented in amniotes, these studies have largely reported expression within the mesenchyme of the arches and they have often not explicitly analysed the limits of expression of these genes within the pharyngeal endoderm [17, 18].
Hox gene expression boundaries and their relationship to the pharyngeal pouches in catshark embryos
The overall relationship between the pharyngeal arches and Hox gene expression in catshark (Scyliorhinus canicula) has been described previously and it is broadly similar to the situation observed in amniotes, with Hox genes of paralogous groups (PG) 1–4 being expressed in the pharyngeal region, and Hox gene of paralogous groups (PG) 5–8 not being expressed . There were, however, also some important detailed differences, such as the fact that Hoxd3 expression had shifted posteriorly and was associated with the fourth arch, and that this segment additionally expressed Hoxd1, d2 and d4. The precise relationship between the expression of Hox genes and the pharyngeal pouches has not been scrutinised in any chondrichthyan, but such analysis is vital if we wish to align the pharyngeal pouches across the gnathostomes. The key questions are: Does Hoxa2 expression align with the second pouch in both amniotes and a chondrichthyan, and does Hoxb1 expression which highlights the fourth and last pouch of amniotes align with the fourth pouch in a chondrichthyan, or is this gene expressed by multiple posterior pouches, or indeed only in the sixth and last formed pouch?
Transient Hoxb1 expression marks the caudal limit of the pharynx
In the present study, we have attempted to address the route through which the reduction in the number of pharyngeal segments that occurred within the sarcopterygians, and which is evident in extant tetrapod embryos. To do this, we compared the development of the pharyngeal segments in an amniote, the chick, with that in a chondrichthyan, the catshark. We show that in both the overall trend in the formation of the pharyngeal pouches is similar, with the most anterior forming first and then the posterior segments forming sequentially. We further analysed the patterns of innervation and the expression profiles of Hox genes in the forming arches, and in particular how these relate to the pharyngeal pouches, which dictate the number of segments formed. Our results show support for the first three anterior segments being homologous across the gnathostomes. Each of these had a distinct innervation, and both the mesenchyme of these arches and the corresponding pouches expressed the same repertoire of Hox genes in both chick and catshark. To gain insights into how the posterior pouches align between chick and catshark, we analysed the expression of Hoxb1, which is expressed in the most posterior pouch in amniotes. Somewhat unexpectedly, we found that Hoxb1 exhibits a dynamic expression profile during the formation of the pharyngeal segments, with the expression domain of this gene progressively moving caudally such that at any given stages it is expressed in the last formed pouch. This leads us to suggest that the posterior limit of the pharynx is homologous between chick and catshark, and that a reduction in the number of pharyngeal segments has been achieved by the earlier termination of the caudal expansion of the pharyngeal endoderm. Thus the caudal limit of the pharynx is established at a relatively more anterior position and therefore fewer pharyngeal pouches form and correspondingly fewer pharyngeal segments.
While our work gives an insight into the possible mechanism that underpinning the reduction in arch number that occurred within the sarcopterygians, the morphological data form fossils also provides a very useful complement that give us insights into when the reduction in the number of pharyngeal segments started . Thus while extant and fossil coelacanths have five branchial arches, it has been reported that Gogonasus and Eusthenopteron, which are tetrapodomorph fish, have lost the fifth arch. Thus it has been suggested that the absence of the fifth branchial arch is a derived feature of advanced sarcopterygians.
Importantly, other studies suggest that the mode of arch reduction that we have identified, in which deletion occurs between a Hox1-defined posterior limit of the pharynx and more anterior segments, is likely to extend beyond events within the sarcopterygians. An analysis of Hox gene expression in the lamprey, Lethenteron japonicum, found that the Hox1 gene, LjHox1w, in this this species also marks the caudal limit of the pharynx, although it is unclear whether LfHox1w displays a similar temporally and spatially dynamic pattern of expression . Perhaps even more significantly, an analysis of pharyngeal gill slit formation in a hemichordate, Saccoglossus kowalevskii, which concludes that this process is homologous to the formation of the pharyngeal segments of vertebrates, also reported spatially and temporally dynamic expression of Hox1 . In that study, the authors noted that at Hox1 expression was restricted to the posterior boundary of the pharyngeal endoderm at the three gill-slit stage and that it was still restricted to the posterior boundary of the pharyngeal endoderm at the four gill-slit stage. Collectively, our work and those of others therefore suggest that a possible role for Hox1 genes in defining the caudal limit of the pharynx extends beyond the vertebrates, and is likely to have evolved much earlier with the emergence of the deuterostomes.
This is also significant in that it could help us understand how the number of pharyngeal segments has been modified across the deuterostomes. Thus while enteropneust hemichordates and cephalochordates have numerous pharyngeal gill slits [24, 25], which extend significantly along the length of the body, those in vertebrates are fewer in number and are focussed just caudal of the mouth. Thus, the number of pharyngeal segments could be decreased by terminating the posterior expansion of the pharynx prematurely and so decreasing the distance between the Hox1 expression domain and the last formed anterior segment. Correspondingly, an increase in the number of pharyngeal segments, Thus the number of pharyngeal segments could be increased, such as is seen in the extinct jawless vertebrate Endeiolepis which had up to thirty pairs of gill slits , by allowing the posterior of the pharynx to extend for a relatively longer time and thus increase the region between the Hox1 expression limit and the anterior.
Our results support the view that the three most anterior pharyngeal segments are conserved across the vertebrates and that the caudal limit of the pharynx is also conserved. Thus, within the sarcopterygians, the segments that were lost were those form the “branchial”/posterior region which lie between the third arch and the caudal limit. Furthermore, our results would suggest the reduction in the number of pharyngeal segments was achieved as a result of the premature termination of the posterior extension of the pharyngeal endoderm which in turn would result in the generation of fewer segments. Finally, we suggest that such a mechanism may also account for the variability in the number of pharyngeal segments seen across the vertebrates and other deuterostomes.
We would like to thank the Anatomical Society for support for part of this work and Dr Seb Shimeld for lamprey embryos. We would like to thank Clemens Kieckers for comments on the manuscript.
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