Bichir external gills arise via heterochronic shift that accelerates hyoid arch development

In most vertebrates, pharyngeal arches form in a stereotypic anterior-to-posterior progression. To gain insight into the mechanisms underlying evolutionary changes in pharyngeal arch development, here we investigate embryos and larvae of bichirs. Bichirs represent the earliest diverged living group of ray-finned fishes, and possess intriguing traits otherwise typical for lobe-finned fishes such as ventral paired lungs and larval external gills. In bichir embryos, we find that the anteroposterior way of formation of cranial segments is modified by the unique acceleration of the entire hyoid arch segment, with earlier and orchestrated development of the endodermal, mesodermal, and neural crest tissues. This major heterochronic shift in the anteroposterior developmental sequence enables early appearance of the external gills that represent key breathing organs of bichir free-living embryos and early larvae. Bichirs thus stay as unique models for understanding developmental mechanisms facilitating increased breathing capacity.


Introduction
The vertebrate pharynx is composed of a series of repeated embryonic structures called pharyngeal arches (Graham, 2008;Grevellec and Tucker, 2010). In the majority of jawed vertebrates, the first, or mandibular arch contributes to the jaws; the second, or hyoid arch serves as the jaw support, and the more posterior branchial arches typically bear internal pharyngeal gills. Pharyngeal arches form in a highly stereotyped sequence from anterior to posterior, where the contacts between endodermal pouches and surface ectoderm physically separate the mesoderm-and neural crest-derived arch tissues (Graham and Smith, 2001;Shone and Graham, 2014;Choe and Crump, 2015). The progressive development of the pharynx has deep deuterostome origins, as it is characteristic of both cephalochordates and hemichordates (Willey, 1891;Gillis et al., 2012;Koop et al., 2014). In vertebrates, sequential formation of pharyngeal segments represents a fundamental aspect of the metameric organization of the head and face (Piotrowski and Nüsslein-Volhard, 2000;Couly et al., 2002;Choe and Crump, 2015). Any modifications of this well-established anteroposterior differentiation scheme would represent a radical alteration in development of the stereotypic chordate bauplan (Square et al., 2017).
Polypterid bichirs represent the earliest diverged living group of ray-finned (Actinopterygian) fishes (Hughes et al., 2018) and they are often referred to as the most relevant species for studying character states at the dichotomy of ray-and lobe-finned fishes (e.g., Standen et al., 2014). This places bichirs in a unique phylogenetic position among vertebrates, which can be exploited for evolutionary and developmental comparative studies (e.g., Takeuchi et al., 2009;Standen et al., 2014;Minarik et al., 2017). Adult bichirs possess several intriguing characteristics that have been associated with air-breathing during the transition from water to land, such as ventral paired lungs or spiracular openings on the head (Clack, 2007;Coates and Clack, 1991;Graham et al., 2014;Tatsumi et al., 2016). Moreover, bichirs also share several key larval features with lungfishes or amphibians, such as cranial adhesive organs, and larval external gills (Kerr, 1907;Diedhiou and Bartsch, 2009).
The external gills of bichirs represent prominent adaptive structures, and constitute major breathing organs of their free-living embryos and early larvae ( Figure 1A) (Kerr, 1907;Diedhiou and Bartsch, 2009). Strikingly, while external gills of amphibians and lungfishes derive from branchial arches as a rule (Duellman and Trueb, 1994;Witzmann, 2004;Nokhbatolfoghahai and Downie, 2008;Schoch and Witzmann, 2011), those of bichirs have historically been considered as unique hyoid arch derivatives due to their blood supply from the hyoid aortic arch (Kerr, 1907;Goodrich, 1909). Importantly, the external gills of bichir embryos represent the first cranial structures to appear, emerging before the eyes or mouth are evident ( Figure 1B) (Minarik et al., 2017).
Here, we take advantage of an exceptionally complete embryonic series of the Senegal bichir (Polypterus senegalus) to explore the developmental underpinnings of the early formation of their external gills and test their segmental origin. Our results reveal that bichir external gills are definitively derived from the hyoid arch and develop by orchestrated acceleration of tissues of all germ layers of the hyoid segment. Thus, in bichir embryos, the standard anteroposterior differentiation scheme of cranial segments is modified by the unique heterochronic development of the hyoid metamere, allowing early and enhanced development of their external gills.

Results and discussion
External gills of the Senegal bichir are developmentally associated with the hyoid segment In order to examine the origin of bichir external gills, we first followed the morphological development of this structure from the earliest stages of embryogenesis onwards. The first sign of external gill development is a pair of outgrowths situated lateral to the closing neural folds ( Figure 1C). The hyoid origin of these outgrowths is suggested by the expression pattern of the Hoxa2 ( Figure 1D), a selector gene characteristic of hyoid identity in other vertebrates (Rijli et al., 1993;Hunter and Prince, 2002;Baltzinger et al., 2005). Later, at early pharyngula stages, the hyoid outgrowths produce protuberant bulges situated in the pre-otic region on each side of the embryo ( Figure 1E-H), that rapidly increase in size ( Figure 1I), and finally, differentiate into many secondary branches ( Figure 1J-L). This suggests that the prominent external gills of bichir larva ( Figure 1A) initially arise from striking accelerated development of the epidermal outgrowths ( Figure 1B) that are of hyoid segmental origin ( Figure 1F).

Accelerated and predominant hyoid neural crest stream supplies bichir external gills
To gain insights into the accelerated development of the hyoid segment, we focused on the cranial neural crest that arises from the closing neural folds. Cranial neural crest cells emerge in a characteristic pattern and split into mandibular, hyoid, and branchial streams, which in most vertebrates arise in a sequential anteroposterior order of appearance. As a marker for migrating neural crest cells, we used expression of Sox9, a transcription factor critical for their emergence, migration, and differentiation (Cheung and Briscoe, 2003;Mori-Akiyama et al., 2003;Theveneau and Mayor, 2012). In bichir embryos, Sox9 expression pattern reveals that the hyoid neural crest segment is developmentally advanced, as it forms concurrently with the mandibular neural crest segment ( Figure 2A). Sections through the neural folds, however, demonstrate that mandibular neural crest cells still reside within the neuroepithelium ( Figure 2B), while the hyoid neural crest cells have already emigrated from the neural folds ( Figure 2C). This premature emigration of the hyoid neural crest stream correlates with the previously observed external outgrowths of the hyoid area ( Figure 1C). Later in migration, the hyoid neural crest stream remains predominant ( Figure 2D), as it is much larger when compared to the mandibular neural crest stream ( Figure 2E,F). The hyoid neural crest stream still progresses at later stages ( Figure 2G), and as such, the majority of the mesenchyme in the early bichir head appears to arise from this source ( Figure 2H). The Sox9 immunoreactivity further shows that cells of the leading edge of the hyoid stream delaminate from the neural folds prior to the emigration of the mandibular stream ( Figure 2I), and illustrates the voluminous ( Figure 2J) and extended ( Figure 2K) mesenchymal production of the hyoid neural crest segment.
We directly tested whether the hyoid neural crest cell stream contributes to the external gills by performing focal CM-DiI injections into rhombomere 4 ( Figure 2L inset), the source of the prospective hyoid neural crest stream in other vertebrates (Lumsden et al., 1991;Kö ntges and Lumsden, 1996;Minoux and Rijli, 2010;Theveneau and Mayor, 2012). One day after neurulation, the CM-  DiI-positive hyoid neural crest cells are observed all along the proximodistal axis of the external gill primordium ( Figure 2L). Two days later, they occupy the primary branches of the external gills (16/ 21, Figure 2M). After hatching, the CM-DiI-positive cells populate the fully developed and functional external gills ( Figure 2N,O). Thus, our fate mapping experiment confirms that bichir external gills are, indeed, populated by the cells of the hyoid neural crest stream and, implicitly, that they represent hyoid arch derivatives.
The first cranial muscles of bichir embryos support their external gills and are of hyoid segmental origin In vertebrates, cranial neural crest cells are the primary source of craniofacial mesenchyme, but also have a major influence on the differentiation and morphogenesis of the cranial myogenic mesoderm (Ericsson et al., 2004;Tokita and Schneider, 2009). We, therefore, hypothesized that the pattern of cranial muscle differentiation in bichir embryos may be affected by acceleration of the hyoid neural crest segment ( Figure 2). Whole-mount antibody staining against skeletal muscle marker 12/101 revealed that the first muscles differentiate stereotypically from the post-otic somites in the trunk region, as in other vertebrates ( Figure 3A). However, within the cranial region of bichir embryos, the earliest developing muscles form within the hyoid arch and are associated with the external gills ( Figure 3B,C). This first muscle complex (levator and depressor branchiarum, Noda et al., 2017) is situated lateral to the otic vesicle and connects filaments of the external gills to the gill stem ( Figure 3B-D). The premature differentiation of the external gill-associated muscles is further supported by their innervation from the hyo-opercular ramus of the facial nerve, allowing voluntary movement of external gills from the earliest larval stages ( Figure 3E,F). Other cranial muscles fully differentiate only at later larval stages when the external gill muscle complex becomes supplemented by other muscles of hyoid and mandibular origins ( Figure 3G). Thus, bichir embryos display unique heterochrony in the differentiation of the hyoid over the mandibular arch mesoderm, providing muscular supports for their external gills.
Early expansion of the hyoid endoderm triggers the formation of bichir external gills Interestingly, the accelerated development of the external gill rudiments is also reinforced by the morphogenesis of the hyoid pharyngeal segment ( Figure 4A-J). Reconstruction of the endodermal epithelium of the bichir pharynx using micro-CT imaging (Minarik et al., 2017) reveals that the pharyngeal endoderm forms two pairs of early outpocketings ( Figure 4B). Whereas the rostral pair represents the embryonic precursor of the cement glands ( Figure 4A-D,F-I) (Minarik et al., 2017), the posterior paired outpocketings constitute primordia of the external gills ( Figure 4A-D). These posterior outpocketings belong to the hyoid segment, as the first pharyngeal pouch (mandibulo-hyoid, or spiracular) is situated rostrally ( Figure 4C,D, white arrowhead) and the second pharyngeal pouch (hyoid-branchial) more caudally ( Figure 4H, black arrowhead). Transverse sections confirm that these hyoid endodermal outpocketings constitute a substantial proportion of the external gill primordium ( Figure 4E). At later stages, these outpocketings further transform into pocket-like structures ( Figure 4G,H,J) that become supplemented with mesenchymal cells of the hyoid neural crest stream ( Figure 2L-N). Thus, while ectoderm covers the entire external gill primordium, the endodermal outpocketing constitutes a considerable portion of the developing external gill ( Figure 4E).
We sought to explore a possible role of the hyoid endodermal outpocketings in controlling development and morphogenesis of the bichir external gills. Morphogenesis of the pharyngeal pouches is critically regulated by factors from many signaling pathways (Graham and Smith, 2001;   Graham, 2008), among which alterations in Fibroblast growth factor (Fgf) signaling lead to defects in proper endodermal pouch development and pharyngeal segmentation (Jandzik et al., 2014;Abu-Issa et al., 2002;Crump et al., 2004;Walshe and Mason, 2003). To assess the possible role of Fgf signaling during bichir external gill development, we scored expression of the Fgf8 ligand and the readouts of Fgf signaling activity. Fgf8 expression is present in endodermal outpocketings and becomes confined to their lateral portions (Figure 4-figure supplement 1). These portions of endoderm in fact constitute the outgrowing tips of the prospective external gill ( Figure 4K). Expression of Dusp6 and Pea3 (the Fgf signaling pathway readouts) and antibody localization for activated MAPK (marker of active Fgf signaling) are present in the external gill mesenchyme adjacent to the outgrowing endodermal tips or both in the mesenchyme and the endodermal tips (Figure 4L-N; Figure 4-figure supplement 2). The topographical relation of endodermal outpocketings and the direction of Fgf signaling within the external gill primordium thus suggest that the endodermal epithelium signals to the adjacent mesenchyme through Fgf signaling to regulate outgrowth of the external gill ( Figure 4O).
To test the possible role of signaling events, we treated bichir embryos with SU5402, a collective Fgf and Egf signaling inhibitor, at early neurulation and scored the phenotypes at later pharyngula stages. In contrast to control embryos displaying well-developed hyoid endodermal outpocketings and external gill primordia (18/18, Figure 4P-Q), disrupting Fgf signaling perturbs morphogenesis of the hyoid endodermal outpocketings and leads to the loss of the external gill primordia (14/15, Figure 4R-S) possibly due to the loss of expression of downstream genes ( Figure 4T-U). These results support a central role of the pharyngeal endoderm in triggering early development of bichir external gills. The pharyngeal origin of the external gill primordia is surprising given that the external gills are commonly considered as outer surface structures composed of ectoderm (Takeuchi et al., . Asterisk, cement gland; am, adductor mandibulae; ah/ao, complex of adductor hyomandibulae and adductor operculi; b, brain; ba, branchial arches; bm, branchiomandibularis; cd,constrictor dorsalis; cement gland; e, eye primordium; lb/db, complex of levator branchiarum and depressor branchiarum; hh, hyohyoideus; ih, interhyoideus; im, intermandibularis; ot, otic vesicle; pf, pectoral fin; y, yolk; V., nervus trigeminus; VII., nervus facialis. DOI: https://doi.org/10.7554/eLife.43531.004  Diedhiou and Bartsch, 2009). However, our finding of an endodermal component in the early formation of bichir external gills reveals an unanticipated similarity with the true, internal gills of vertebrates, which typically form as pharyngeal endodermal structures (Warga and Nüsslein-Volhard, 1999;Gillis and Tidswell, 2017). Pharyngeal morphogenesis might thus represent a central developmental component of vertebrate gill breathing organs irrespective of their actual topographic position.

Conclusions
The sequential formation of pharyngeal segments during embryonic development has deep deuterostome origins (Willey, 1891;Koop et al., 2014;Gillis et al., 2012) and it is well conserved among vertebrates, where all the embryonic cranial segments typically follow the sequential anteroposterior order during development (Quinlan et al., 2004;Grevellec and Tucker, 2010;Schilling, 2008;Santagati and Rijli, 2003). Bichir embryos diverge from this common scheme by the profoundly accelerated development of the second, hyoid segment, with earlier and orchestrated formation of the endodermal, mesodermal, and neural crest tissues ( Figure 5). This unique heterochronic shift in the anteroposterior sequence constitutes a developmental basis for the early appearance of external gills that represent key breathing organs of bichir free-living embryos and early larvae.
Bichir external gills significantly differ from the external gills of amphibian and lungfish larvae that characteristically supplement the post-hyoid, branchial arches (Duellman and Trueb, 1994;Witzmann, 2004;Nokhbatolfoghahai and Downie, 2008;Schoch and Witzmann, 2011). The hyoid segmental origin represents a major developmental dissimilarity and suggests an independent evolution of bichir external gills. Developmentally, bichir external gills likely correspond to opercular structures that in ray-finned fishes typically form as caudal expansions of the hyoid arch to cover the gill-bearing branchial arches, and that persist in amniotes as early embryonic opercular flaps (Richardson et al., 2012). In bichirs, the opercular flap forms directly from the base of their external gills, and it progressively expands during early larval stages while external gills become reduced (Diedhiou and Bartsch, 2009). Interestingly, the hyoid arch-derived external gills and opercular flaps are both engaged in breathing and gill ventilation in bichir larvae. Moreover, in adult bichirs, the hyoid domain also contributes to air-breathing by forming paired spiracular chamber with openings located on the dorsal surface of the skull . Bichirs thus seem unique across recent vertebrates in enhancing breathing capacity through the development of several structures associated with the hyoid cranial segment.

Embryo collection
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 Cuvier, 1829) embryos were obtained, reared and staged as previously described (Minarik et al., 2017;Diedhiou and Bartsch, 2009). Embryos were dechorionated manually, fixed in 4% PFA in 0.1 M PBS at 4˚C overnight, and then gradually dehydrated through a series of PBS/methanol mixtures and finally stored in 100% methanol. In situ hybridization and fate mapping Whole-mount in situ hybridization with probes against Hoxa2 (GenBank accession number: MK630352), Sox9 (GenBank accession number: MK630350), Fgf8 (GenBank accession number: MK630353), Pea3 (GenBank accession number: MK630351), and Dusp6 (GenBank accession number: MK630349) was performed as described (Minarik et al., 2017). Selected specimens were embedded in gelatine/albumin solution with glutaraldehyde, sectioned and counterstained with DAPI. Fate mapping experiments were carried out as described (Minarik et al., 2017). CM-DiI was injected into the neural fold of the prospective rhombomere 4 ( Figure 2L). To confirm correct localisation of the tracking dye, some embryos were fixed immediately after injection, sectioned, and observed under the fluorescent stereomicroscope in order to confirm proper localization of the cell tracking dye. The rest of the specimens were incubated until the desired stage and then fixed in 4% PFA in 0.1 M PBS.

Scanning electron microscopy (SEM) and MicroCT imaging
Samples for SEM were fixed in modified Karnovsky's fixative (Mitgutsch et al., 2008). For direct visualization of cranial neural crest streams, the epidermis was removed using tungsten needles as described . Specimens for MicroCT analysis were treated with phosphotungstic acid following the protocol developed by Metscher (2009) and scanned with a MicroXCT (X-radia) at the Department of Theoretical Biology, University of Vienna. Images were reconstructed in XMReconstructor (X-Radia), and virtual sections were analyzed in Amira (FEI Software).

Pharmacological treatments
For inhibition of pharyngeal outpocketing morphogenesis, embryos were treated with 50 mM SU5402 in DMSO (Sigma Aldrich) from stage 20 until stage 26. Treatments were performed in E2 medium (Brand et al., 2002). Controls were reared in E2 medium with the equivalent DMSO concentrations.