Pre-mandibular pharyngeal pouches in early non-teleost fish embryos

Abstract

The vertebrate pharynx is a key embryonic structure with crucial importance for the metameric organization of the head and face. The pharynx is primarily built upon progressive formation of paired pharyngeal pouches that typically develop in post-oral (mandibular, hyoid and branchial) domains. However, in the early embryos of non-teleost fishes, we have previously identified pharyngeal pouch-like outpocketings also in the pre-oral domain of the cranial endoderm. This pre-oral gut (POG) forms by early pouching of the primitive gut cavity, followed by the sequential formation of typical (post-oral) pharyngeal pouches. Here, we tested the pharyngeal nature of the POG by analysing expression patterns of selected core pharyngeal regulatory network genes in bichir and sturgeon embryos. Our comparison revealed generally shared expression patterns, including Shh, Pax9, Tbx1, Eya1, Six1, Ripply3 or Fgf8, between early POG and post-oral pharyngeal pouches. POG thus shares pharyngeal pouch-like morphogenesis and a gene expression profile with pharyngeal pouches and can be regarded as a pre-mandibular pharyngeal pouch. We further suggest that pre-mandibular pharyngeal pouches represent a plesiomorphic vertebrate trait inherited from our ancestor's pharyngeal metameric organization, which is incorporated in the early formation of the pre-chordal plate of vertebrate embryos.

1. Introduction

The vertebrate pharynx is a pivotal embryonic structure and a developmental source of our basic body plan. The presence of a pharynx with bulging pharyngeal arches enabled Ernst Haeckel to define the vertebrate pharyngula [1], representing a highly conserved phylotypic stage and a notorious icon of evolution [2,3]. Pharyngeal segmentation forms a fundamental aspect of the metameric organization of the vertebrate head in orchestrating populations of craniofacial mesenchyme [4–6]. Evolutionary modifications of the pharyngeal apparatus have been central to the origin of vertebrates [7–11], and further changes in pharyngeal patterning and morphogenesis have constituted a vital source of vertebrate group diversification [12–16].

Development of the vertebrate pharynx is built around pouching from the primitive gut cavity. At the end of neurulation, the endodermal layer evaginates to make a series of bilateral pharyngeal pouches that contact the corresponding clefts on the surface ectoderm [6,17,18]. These epithelial pockets organize populations of the cranial mesoderm- and neural crest-derived mesenchyme, that all together constitute a series of the pharyngeal arches [18,19]. Segmentation of the pharyngeal region is an intrinsic property of the endoderm. It is inherited from an ancestral deuterostome body plan and well presented in hemichordates, amphioxus and tunicates that lack typical vertebrate novelties [20–23]. In vertebrates, formation of the pharyngeal pouches developmentally precedes the migration of the cranial neural crest cells [16,24–26] and experimental ablation of the neural crest does not prevent pharyngeal pouch formation [4,27], altogether arguing for the ancestral and leading role of pharyngeal segmentation in vertebrate head development.

The genetic background of pharyngeal development is deeply conserved among chordates, even among deuterostomes [11,21]. The Nkx2.1, Nkx2.2, FoxA and Pax1/9 genes are clustered in the genome in the same manner in hemichordates and chordates confirming the ancestrality of the deuterostome pharyngeal region development [28]. The gene regulatory network of Pax-Six-Eya seems to be conserved in the development of the pharynx as it has been shown that expression of these genes is interconnected in the pharynx of mice and that they are co-expressed in the pharynx of many other deuterostome animals [21,29,30]. Namely, transcripts of Pax1/9 were found in the pharyngeal pouches of hemichordates and urochordates [31], in gill slits of amphioxus [32,33], lamprey [34], shark [35], medaka [36], Xenopus [37], chicken [38] and mouse [39]. Expression patterns of Six1 and Eya1, other core parts of the pharyngeal gene cluster, are also shared among deuterostomes, as it was found in the pharyngeal endoderm of hemichordates [21], amphioxus [33], chicken [40,41] and mouse [29].

The T-box transcription factor Tbx1, regulated by another part of the core pharyngeal gene cluster, FoxA [42], is uniformly expressed in the pharyngeal endoderm and mesoderm in all the animals studied across the chordate clade [43–48]. The lack of Tbx1 expression in the pharynx in hemichordates suggests that its incorporation into the network relates to the later acquisition of mesoderm into the pharyngeal arches in chordates [21].

In tetrapods, transcription factor Ripply3 has been shown to interact with Tbx1 and Pax9 in the development of pharyngeal pouches [49,50]. Most of the actinopterygian fishes have the sequence in their genome except for the Neoteleostei clade (e.g. medaka), which seems to have lost it [51]. In addition, signalling molecules of the Fgf family demark the region of developing pouches in many species across the chordate clade [52–56]. In particular, Fgf3 is expressed in the endoderm, and Fgf8 in both the pouch endoderm and ectoderm of the pharyngeal clefts [53,57]. Finally, Sonic hedgehog (Shh), a potent pleiotropic morphogen, is differentially expressed in the embryonic pharyngeal endoderm and plays multiple roles throughout the development of the pharynx including the repression of Fgf signalling [58]. Mutation of Shh in the sturgeon sterlet resulted in severe pharyngeal phenotypes [59].

In embryos of non-teleost fishes, such as bichirs, sturgeons and gars, the early head endoderm protrudes rostrally into the pre-neural and pre-oral domain [60]. This pre-oral gut (POG) develops by characteristic pharyngeal-pouch-like outpocketing of the primitive gut cavity, which progressively forms similar pouches in the post-oral (pharyngeal) domain. The POG diverticula of the anterior endoderm is considered an ancestral chordate state, as they are also recognized in amphioxus and hemichordates [20,61,62]. In this study, we aimed to determine if POG belongs to the metameric series of pharyngeal pouches and, as such, can be classified as a part of the pharynx. To accomplish this, we examined expression patterns of the genes of the core pharyngeal gene cluster in early embryos of non-teleost fishes, bichirs and sturgeons. We found shared expression patterns in POG and in the post-oral pharyngeal pouches, which indeed supports POG as serial homologues of pharyngeal pouches and allows the classification of POG as a pre-oral or pre-mandibular pharyngeal pouch.

2. Material and methods

(a) . Embryo collection and fixation

Fish were manipulated in accordance with the institutional guidelines for the use of embryonic material and international animal welfare guidelines (Directive 2010/63/EU). Senegal bichir (Polypterus senegalus) embryos were obtained, reared and staged as described [16,60]. The sterlet sturgeons (Acipenser ruthenus) were bred in the Research Institute of Fish Culture and Hydrobiology in Vodnany, University of South Bohemia. Embryos were staged [63,64] and fixed in methanol at −20°C as described [16].

(b) . Histology and three-dimensional visualization

Embryos for histological sectioning were embedded in JB4 (Polyscience) resin. The embedded embryos were sectioned using an RM2155 microtome (Leica, Germany) to 5 µm thick sections. Slides were stained using the Azure B-Eosin kit (SERVA), dried and mounted with DPX (SERVA). Images were taken using the BX51 microscope (Olympus) with DP74 camera (Olympus). The micro-computed tomography scans were taken as described [60]. The data were resegmented to display, in addition to the endoderm, notochord, brain, nasal, optic and otic vesicles using the software AMIRA 6.0.1 (Thermo Fisher Scientific, USA).

(c) . Probes synthesis and in situ hybridization

All probes were prepared as described in [26] using specific primers. For P. senegalus Pax9: 5′-ATGGCCCTCCTCTCACTC-3′ and 5′-CTGAAGCCACCAGCGAATG-3′, Eya1: 5′-TTCCATGACWCCMAATGGCACHGAAG-3′ and 5′-TCTTTAATRGGBGTKGAWGGACTGTG-3′, Shh: 5′-ACRGSYGCKGACMGRCTHATGAC-3′ and 5′-GTCWCKRTCVGAKGTRGTGATRTCAAC-3′, for Fgf8 see [16]. For A. ruthenus Shh: 5′-GCBATYTCDGTRATGAACCA-3′ and 5′-CARSRSSYTGGAGTACCAGT-3′, Six1: 5′-CTSGGHCGNTTTYTBTGGTC-3′ and 5′-CTYCTGTTYTTGAACCAGTT-3′, Tbx1: 5′-AARGCNGGVAGRMGWATGTT-3′ and 5′-GCDGTAAABCKVGTYTCCTC-3′, for Ripply3 see [59], Fgf8: 5′-CAGTCCYCGCCTAATTTTAC-3′ and 5′-CCCTTGGGYAGCCKYTTCAT-3′. For both sturgeon and bichir Pitx2 see [60]. Only Pitx2 for sturgeon was prepared directly from the PCR product. The in situ hybridization was performed as described [60]. Images of the wholemount embryos were taken using a dissection microscope SZX12. For the sectioning, the selected embryos were embedded as described in [26], sectioned (50 µm) on a vibratome VT1200S (Leica, Germany) and counterstained with DAPI. Some of the thick sections needed to be imaged on several planes of focus and combined to the final image using the z-projection in Fiji. To create the magenta channel overlaid with DAPI, the contrast of brightfield image was extremely enhanced, converted to a black and white image and overlaid with the DAPI channel in Fiji.

3. Results

(a) . Early formation of the pre-oral gut and pharynx

Before we analyse gene expression patterns within the pan-pharyngeal region of non-teleost fish embryos, we briefly describe their pharyngeal formation focusing on the rostral-most domain, the POG, and its continuity with the pharyngeal cavity (figure 1).

Figure 1.

Early and late formation of the pre-oral gut (POG) and its connection with the pharynx. Bichir (a,c,e) and sturgeon (b,d,f) embryos, head to the left, showing the development of POG from late neurula stage (a,b), through pharyngula stage (b,c), until cement and hatching gland specification (e,f), endoderm yellow, POG marked by yellow arrowheads. Sagittal plastic sections (a–d) and whole-mount three-dimensional models based on micro-CT data from Minarik et al. [60]. ar, archenteron; ey, eye; fb, forebrain; yo, yolk; n, nose; nt, notochord; ot, otic; pp, pharyngeal pouches; white arrows mark mouth position.

Early embryos of bichir and sturgeon possess an archenteron, which is a primitive gut cavity delineated by a simple endodermal epithelium. Following neurulation, conspicuous paired outpocketings begin to form at the rostral tip of embryos with clearly thickened endodermal epithelium located in front of the forebrain (figure 1a,b). This POG domain represents a rostral expansion of the primitive gut cavity, which further caudally develops similar laterally expanded pouches by outpocketing of polarized epithelia. This is a typical mechanism of pharyngeal morphogenesis [16,60,65].

At the onset of the pharyngula stage, the pouching of POG becomes increasingly evident (figure 1c,d). From this stage onwards, the initially simple pocket-like shape of POG starts to diverge, reflecting specific POG derivatives (figure 1e,f). In free-living embryos and larvae of bichirs, POG forms paired club-shaped projections that prefigure prominent cement glands sited in front of the brain (figure 1e). On the other hand, POG in sturgeons has more of a crescent shape and prefigures the entire rostrum (figure 1f, for detailed three-dimensional morphogenesis see [60]).

Pouches of the POG thus represent a continuity of the primitive gut cavity with the pre-oral domain being separated from post-oral pharyngeal pouches by the forming mouth (stomodeum) (figure 1e,f). To further investigate this process, we next examined and compared expression patterns of the core pharyngeal regulatory network genes in both the pre- and post-oral pharyngeal domains.

(b) . Bichir gene expression patterns

In bichir embryos, we first analysed the expression pattern of Pax9 and Eya1, the two major pharyngeal transcription factors well conserved within the deuterostomes (figure 2; electronic supplementary material, figure S1). Whole-mount view revealed that the Pax9 expression pattern is seen in the rostral part of bichir embryos alongside the just formed neural tube within the entire pan-pharyngeal region prior to the formation of paired cement glands (figure 2a). Within the embryo, Pax9 expression was observed in the pharyngeal domain spanning from the forebrain to forming somites, but also more rostrally in the POG domain situated in front of the forebrain (figure 2b). The pharyngeal epithelium is clearly distinguished with DAPI staining of the nuclei. To precisely localize the gene expression in the epithelial and other structures, we merged a highly enhanced contrast image of the ISH signal with the nuclei fluorescent image (figure 2b). Upon closer inspection of the POG domain, a specific spot of Pax9 expression was detected in the rostral-most outpocketing of the primitive gut cavity (figure 2b′). The POG expression of Pax9 is strongest in the early pouching period of POG development. It disappears later, when POG gets clearly separated into paired pouches, which develops into cement organs (electronic supplementary material, figure S1A–D).

Figure 2.

Bichir—gene expression patterns. Left column: whole-mount embryos, lateral views, head to the left. Mid column: sagittal sections showing POG (arrowheads) and pharynx (brackets), magenta shows ISH signal (see methods), white is DAPI. Right column: details of the original ISH signal in POG, dashed white line outlines the endodermal epithelium based on the segregation of nuclei visible with DAPI staining. fb, forebrain; som, somites; yo, yolk; asterisks point to primary gut cavity; arrowhead shows POG, arrows indicate placodal expression.

The expression pattern of Eya1 can be observed within the cranial part of the embryo in the pan-pharyngeal region, similarly to the previous Pax9 expression. However, the Eya1 expression further extends into the forming somites of the trunk region (figure 2c,d; electronic supplementary material, figure S1E–SG). Enlarged view on the POG domain reveals, again, a specific rostral expression in the pouching endoderm, in addition transcripts of Eya1 are also seen in the ectoderm situated in front of the forebrain representing the adenohypophyseal placode (figure 2d′). Notably, in both Pax9 and Eya1 later expressions, a conspicuous pattern is evident within the forming outer gills (electronic supplementary material, figure S1B–D,F,G), which in bichir embryos represent a prominent pharyngeal pouching structure [16].

The Fgf8 gene plays a crucial role in the developing pharynx by encoding one of the key signalling molecules. This gene is commonly found in both the endodermal and ectodermal epithelial components of the forming pouches [53]. Previous studies have shown that Fgf8 is observed in the lateral portions of endodermal pharyngeal outpocketings [16]. Our recent analysis has revealed an additional prominent area of expression in the rostral-most region of the POG pouching endoderm. This area is located in front of the forebrain and shows an apparent overlap with Pax9 and Eya1 expression (figure 2b′,d′,f′).

SHH is an overall important signalling molecule and a key regulator of many developmental processes including formation of the pharyngeal pouches [59]. In early bichir embryos, Shh expression can be seen in the axial tissues of the entire body (electronic supplementary material, figure S1J–M). Specifically, within the cranial region, it can be observed in the notochord, pharyngeal pouches, as well as in the anteriormost endoderm (figure 2g,h). Moreover, within the POG domain, a separate endodermal Shh expression appears as the rostral-most endoderm, in an iterative pattern with the pharyngeal pouches, in front of the forebrain (figure 2h′).

The Pitx2 gene, generally known for its placodal expression, has been previously found in later development of bichir cement glands [60]. The whole-mount expression pattern of Pitx2 prefigures the pan-placodal region and later the position of cement glands (figure 2i,j; electronic supplementary material, figure S1N–P). The inside view reveals that the early Pitx2 expression is broadly present in the anteriormost endoderm epithelium that delineates the entire POG domain (figure 2j′). A weak signal in the adenohypophyseal placode is visible as well.

(c) . Sturgeon gene expression patterns

In sturgeons, we have identified distinct patterns of gene expression in two developmental stages: an early stage of post-neurulation embryos with a very prominent POG domain similar to bichir's (figure 3), and later stages, in which POG modifies its shape, but the post-oral pharyngeal pouches are already well developed (figure 4).

Figure 3.

Sturgeon—early gene expression patterns. Left column: whole-mount embryos, dorsal views, head to the top. Mid column: medial sections, head to the left, magenta shows ISH signal (see methods), white is DAPI. Right column: details of the original ISH signal in POG, dashed white line outlines the endodermal epithelium based on the segregation of nuclei visible with DAPI staining. fb, forebrain; yo, yolk; asterisks point to primary gut cavity; arrowhead shows POG, arrows indicate placodal expression.

Figure 4.

Sturgeon—late gene expression patterns. First column: whole-mount embryos with expression in POG, dorsal views, head to the top. Second column: medial sections, head to the left, magenta shows ISH signal (see methods), white is DAPI. Third column: details of the original ISH signal in POG, dashed white line outlines the endodermal epithelium based on the segregation of nuclei visible with DAPI staining. Fourth column: whole-mount embryos of slighter later stage showing expression in pharyngeal arches. fb, forebrain; pp, pharyngeal pouch; yo, yolk; asterisks point to primary gut cavity; arrowhead shows POG.

During the post-neurulation stages (figure 3), the expression of Shh is strongly seen in the forming axial tissues of sturgeon embryos, with a prominent broad expression pattern in front of the forebrain (figure 3a). Sections revealed that Shh marks the entire POG domain (figure 3b) and that its expression is restricted to the endoderm epithelium of the primitive gut cavity (figure 3b′). In later stages, Shh expression includes the axial tissues and pharyngeal pouch endoderm in the posterior head (electronic supplementary material, figure S2A–D). Notably, the foremost Shh activity in the POG is maintained during later stages and appears to prefigure the rostral expansion of the sturgeon head (figure 1f; electronic supplementary material, figure S2A–D). Expression patterns of Shh during sturgeon pharyngeal development are in accord with recently described Shh mutant phenotypes lacking both pharyngeal pouches and POG [59].

During the post-neurulation stages of sturgeon embryos, the Pitx2 gene is expressed broadly in the rostral pan-placodal field, but also faintly in the early rostral endoderm (figure 3c,d). A detailed view of the early POG domain further reveals that the majority of Pitx2 expression is restricted to the early placodal epithelium above the primitive gut cavity (figure 3d′).

Six1, another part of the conserved deuterostome pharyngeal transcription network, shows in early sturgeon embryos a similar placodal-like expression pattern as Pitx2 (figure 3e,f). Furthermore in later development, Six1 is also expressed in forming posterior pharyngeal pouches (electronic supplementary material, figure S2E–H). In front of the forebrain, within the POG domain, Six1 is broadly expressed in the pan-placodal epithelium above the pouching primitive gut cavity, but also in the rostral-most pouching endoderm (figure 3f′).

During later stages of the sturgeon embryonic development, the POG domain forms a crescent-shaped pocket in front of the brain, and the post-oral pharyngeal pouches begin to appear (figure 4). The expression pattern of Pax9 prefigures endoderm pouch formation in the entire pan-pharyngeal region (figure 4a–c). Pax9 expression is first seen in the endoderm layer of POG and the first two forming post-oral pharyngeal pouches (figure 4a,b,b′). Whereas later, Pax9 expression is not detected in the POG domain anymore but it strongly marks all post-oral pharyngeal pouches (figure 4c).

Tbx1, a chordate-specific pharyngeal transcription factor, has the expression pattern similar to the Pax9 gene in sturgeon embryos following the rostro-caudal progression of the forming pan-pharyngeal region (figure 4d). A detailed view of the POG domain shows Tbx1 to be focally expressed in the rostral endoderm pouch, just like Pax9 (figure 4b,b′,e,e′). However, in later stages, when the Pax9 expression marks post-oral pharyngeal pouches, Tbx1 expression shifts to the posterior pharyngeal mesoderm instead (figure 4c,f; electronic supplementary material, figure S2M–P).

In sturgeon embryos, Ripply3 has a very distinct expression pattern within the pouching epithelium of the entire pan-pharyngeal region, and its early whole-mount pattern visualizes POG as a crescent-shaped pocket in front of the brain (figure 4g). A detailed view on the POG domain reveals a focal expression pattern of Ripply3 shared with Pax9 and Tbx1 in the rostral-most pouching of the primitive gut cavity (figure 4b,b′,e,e′,h,h′). Ripply3 expression also persists during sequential formation of the more post-oral pharyngeal pouches in later sturgeon embryos (figure 4i; electronic supplementary material, figure S2Q–T).

In later stages of sturgeon embryonic development, when Tbx1 expression is confined to the pharyngeal arch mesoderm and in between the pouch endoderm (electronic supplementary material, figure S2P), Tbx1 expression in the pouch endoderm seems to be replaced with Ripply3, a well-known Tbx repressor [50], (compare figure 4d,g, and figure 4f,i).

Fgf8 expression can be seen in both the endoderm and ectoderm of the forming pouches in sturgeon embryos (figure 4j–l). Initially, expression is detectable in the POG domain and forebrain (figure 4j). Subsequently, Fgf8 expression also appears in the post-oral pharyngeal pouches, and also in the corners of the forming mouth (figure 4l; electronic supplementary material, figure S2V,W), where it resides until much older stages (electronic supplementary material, figure S2X). A detailed view of the POG domain shows early focal expression in both the ectoderm and endoderm in the rostral-most domain, but transcripts were also found in the rostral brain centre, similar to bichir embryos (figure 4k,k′).

In summary, all genes analysed within the pan-pharyngeal region of sturgeon and bichir embryos are expressed similarly in both POG and the post-oral pharyngeal pouches, supporting POG as a part of the pharynx (figure 5).

Figure 5.

Serial homology of the pre-oral gut (POG) with pharyngeal pouches, and POG as an ancestral pharyngeal module in deuterostome embryos. (a) A cartoon of embryos with early primary gut cavity (endoderm, yellow) and later formation of pharyngeal pouches that share morphogenesis and gene expression profile in both the pre-oral and post-oral pharynx. (b) Late embryos with POG derivatives in clearly a pre-oral position, that disappear in later stages similar to the post-oral pharyngeal pouches. (c) POG-diverticula as stomochord (S) in hemichordates, as Hatschek's diverticula/club-shape gland (C) in amphioxus, with similar pre-oral pharyngeal domain supposedly also present early vertebrate yunnanozoan. M, mouth, G, gills.

4. Discussion

(a) . Serial homology of POG with vertebrate pharyngeal pouches

In the previous study, we identified a paired anterior outgrowth of the foregut into the pre-oral head domain in early non-teleost fishes [60]. This POG is characterized by the expression of Otx2, Sox17 and FoxE4 transcription factors as an endoderm-derived structure with an apparent positional and transcriptional relatedness to the hemichordate stomochord and amphioxus club-shaped gland [60–62]. Given that all these structures represent outgrowths of the pharynx, we decided to test the pharyngeal nature of POG in early non-teleost fish embryos by examining the expression patterns of candidate genes typically expressed in vertebrate pharyngeal pouches.

Expression of all analysed genes (Pax9, Eya1, Six1, Tbx1, Ripply3, Fgf8, Shh) was clearly found during POG development. In the case of bichir embryos (figure 2), the expression of pharyngeal pouch genes (Pax9, Eya1, Fgf8, Shh) takes place during an earlier stage of POG formation (figure 1a). In sturgeons, we identified two stages of pharyngeal gene expression profiles in POG: Six1, Shh, Pitx2 expression during an earlier POG stage (figures 1b and 3), similar to the situation in bichir, and expression of Pax9, Ripply3, Tbx1, Fgf8 during the more pronounced POG pouching (figures 1d and 4).

Segmentation of the pharynx is deeply conserved among deuterostomes and is orchestrated by pouching of the endoderm under the Pax-Six-Eya regulatory elements [21,28]. Expression of all of these genes was clearly detected in POG of bichir and sturgeon embryos. Pax9 expression is restricted to POG, pharyngeal endoderm and later pharyngeal pouches in both studied species. Bichir Eya1 and sturgeon Six1 expression was found within the early POG endoderm but also in the pre-placodal ectoderm covering POG, corresponding to their roles as placodal transcription factors [66]. A similar pattern was observed in the expression of Pitx2, which was previously discovered within the forming bichir cement glands [60]. Here, we show the early presence of Pitx2 transcripts in the POG endoderm of bichir and also faintly in the early POG of sturgeon, even though sturgeon POG does not develop into cement glands. Interestingly, Pitx2 expression can also be observed in POG of early shark embryos [35].

One of the earliest known markers of pharyngeal endoderm segmentation is FGF signalling [53,57]. In bichir and sturgeon embryos, Fgf8 expression was revealed in the endoderm of the forming pouches and the ectoderm of the pan-placodal domain. Furthermore, in the POG of sturgeon, the expression of Tbx1 and Ripply3 was found. Tbx1 has been supposedly acquired to pharyngeal development in chordates together with the acquisition of mesoderm in the pharyngeal arches, and it is expressed in both the pharyngeal endoderm and mesoderm [21]. In early sturgeon embryos, Tbx1 expression appears in both the POG and post-oral pharyngeal pouch endoderm, but during the later stages, it can be solely observed in the core mesoderm and non-pouching endoderm of the posterior pharyngeal arches (compare figure 4f,i; electronic supplementary material, figure S2M–P), further pointing to the apparent lack of mesoderm in the POG domain. The expression of Tbx1 in the endoderm is later replaced by Ripply3, which is expressed in the POG domain even later (figure 4g,h,h′; electronic supplementary material, figure S2). The similarities in expression patterns of the developmental regulators between the developing pharyngeal pouches and POG described are further highlighted by the phenotypes of recently produced sturgeon Shh mutants that had severely reduced both POG and anterior pharyngeal pouches [59].

In conclusion, in both bichir and sturgeon embryos, POG and post-oral pharyngeal pouches share their developmental origin from the endodermal epithelium of the archenteron cavity, together with identical pouching morphogenesis [60]. It is noteworthy that POG-derived cement glands of bichir embryos disappear during later larval stages [64], and POG-derived ventral rostrum in sturgeon fishes is later occupied by sensory fields of an apparently placodal origin [60,67]. Thus, POG and post-oral pharyngeal pouches in vertebrates also share their transient fate—after opening of the gill slits and formation of the adult pharyngeal apparatus they are no longer recognizable [17,18,30]. Here, we show that POG and post-oral pharyngeal pouches also share the expression profile of the core pharyngeal regulatory network genes (figure 5a). Taken together, this strongly argues for the pharyngeal pouch identity of POG, and for its serial homology with post-oral pharyngeal pouches. It will be interesting to further test functional similarity of POG and post-oral pharyngeal pouches in pharyngeal cartilage induction, however, a role for POG in patterning the cranial-most neural crest cells has already been suggested [26].

(b) . Anterior outgrowths of endoderm as an ancestral module in deuterostome and chordate embryos

POG and similar anterior gut diverticula are well known from many deuterostome embryos (figure 5c). In hemichordate acorn worm juveniles, an anterior outgrowth of the pharynx into the proboscis, called stomochord or buccal diverticulum, expresses many typical pharyngeal genes like hh (the homologue of Shh) or SoxE [61,68]. Stomochord, having a clearly pre-oral position, is also formed in indirectly developing pterobranch hemichordates, and was even described in a tornaria larva (figure 5c) [69–71]. Additionally, FoxE and Ptx (amphioxus homologue of Pitx) are also expressed in the club-shaped gland of larval amphioxus [62,72], which arises as a rostral pouch from the pharyngeal endoderm in late embryos, and disappears during metamorphosis [73,74]. Importantly, an amphioxus club-shaped gland and hemichordate stomochord are classically considered homologous as the rostral diverticula of the tripartite deuterostome body plan organization [75–77].

In Cambrian yunnanozoans, that supposedly possess an ancestral vertebrate pharyngeal arch condition [78], the rostral-most head is occupied by a pharyngeal domain extending into a prominent snout in a clearly pre-oral position (figure 5c), reminiscent of the situation in early non-teleost fish embryos (figure 5b). The ancestral vertebrate condition, as revealed in yunnanozoans, is also consistent with the conventional hypothesis that every pharyngeal arch was once equipped with gills as a typical branchial arch [7,8,12,78], which has recently been supported by findings of rudimentary mandibular gills in cartilaginous and teleost fishes [79,80]. Apparently, during early vertebrate evolution, fully pharyngeal metameric organization of our ancestors became reduced, in concert with extensive changes specifically in the pre-chordal part of the head, including emancipation of the mouth and jaw apparatus, and formation of the rostral brain [9–11]. Developmental transition from the branchiomeric segmentation of the chordate animal to the New vertebrate head might be seen as a major evolutionary transition during early stages of vertebrate evolution.

(c) . POG as an ancestral pharyngeal module in the pre-chordal vertebrate head

Anterior gut diverticula seemingly represent a deeply shared part of the pharyngeal metameric organization of vertebrate ancestors. All these structures develop at the rostral-most tip of deuterostome and chordate embryos (figure 5c). In early vertebrate embryos, the extreme anterior domain is typically seen as occupied by the primary mouth formation, with the stomodeal invagination rostrally restraining the expansion of the foregut [81,82]. However, in most vertebrate embryos the prospective mouth area becomes displaced to the ventral side due to the anterior growth of the forebrain, and a flexure of the antero-posterior axis [83–86]. As a result, the vertebrate mouth does not represent the rostral end of the endoderm but rather opens ventrally, leaving enough space for the earlier rostral expansion of POG at the prospective mouth roof (figures 1 and 5).

This was well recognized by older authors, and POG has been classically described in many early vertebrate embryos as the endoderm at the roof of the anterior end of the alimentary canal, which joins the pharynx at the level of the oral plate, but which soon thereafter disappears [87–90]. More recently, POG was identified in early lamprey and shark embryos as a flattened endodermal process extending from the roof of the archenteron beneath the forebrain to the rostral embryonic tip [85,91,92]. POG was observed to disappear early in the development accompanying morphogenesis of the pre-chordal plate, and its developmental role has never been clarified [85,91,92]. However, the transient appearance of POG within the pre-chordal plate formation is well documented in many vertebrate embryos [85,88,92–97], further suggesting that POG is a pan-vertebrate embryonic feature inherited from our ancestor's pharyngeal metameric organization [60].

POG seems commonly misidentified as a part of the pre-chordal plate mesoderm, which, however, appears as a new, vertebrate-specific acquisition of the rostral head, and forms as an axial cell mass anterior to the notochord [98]. By contrast, POG typically forms by lateral enterocoelic pouching of the endodermal epithelial lining of the archenteron cavity, it shares an expression pattern with posterior pharyngeal pouches, and as such it belongs to pharyngeal segmentation with a deep pre-vertebrate origin. POG also appears earlier and rostral to the formation of the so-called head cavities, which are epithelial mesodermal coeloms of the vertebrate head [7,8,92,99]. Importantly, the pre-chordal plate was classically described to be intimately connected with the roof of the archenteron at the early rostral embryonic pole [92,95–97,100]. This implies that the so-called pre-chordal plate might be of a dual embryonic origin composed of a posterior true axial mesoderm part and an anterior endodermal part, which does not seem to express mesodermal genes and soon disappears [35]. Thus, we speculate that this anterior pre-chordal plate might in fact represent a rudiment of POG in all vertebrates. Given the fundamental role of the pre-chordal plate as the early head organizer in establishing the vertebrate body plan, this suggests that POG, as the rostral-most pharyngeal pouch, might represent a part of ancient pharyngeal metameric organization incorporated in the rostral, pre-chordal part of the New vertebrate head.

Ethics

Fish were manipulated in accordance with the institutional guidelines for the use of embryonic material and international animal welfare guidelines (Directive 2010/63/EU).

Data accessibility

All data analysed during this study are included in the manuscript and associated files. All sources are cited in the Methods section.

Supplementary material is available online [101].

Declaration of AI use

We have not used AI-assisted technologies in creating this article.

Authors' contributions

A.H.: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, validation, visualization, writing—original draft, writing—review and editing; A.P.: data curation, investigation, methodology, project administration; J.S.: data curation, investigation, methodology, writing—review and editing; M.M.: resources, software, visualization, writing—review and editing; D.J.: methodology, resources, supervision, validation, visualization, writing—review and editing; R.C.: conceptualization, data curation, formal analysis, funding acquisition, investigation, project administration, writing—original draft, writing—review and editing.

All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Conflict of interest declaration

We declare we have no competing interests.

Funding

This study was supported by the Czech Science Foundation GACR 19-18634S (to R.C.), the Charles University grant SVV 260685/2023 and by the Charles University project GA UK 1172420 (to A.H.). D.J. was supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 751066 and by the Scientific Grant Agency of the Slovak Republic VEGA grant no. 1/0450/21.