A persistent BHC in mice results from the maintenance of the canal initiated by RP. We have first shown that RP is located at a triple tissue boundary (Figure 1) and is thus at the crossroads of several signaling pathways involved in skull base formation, including Shh. We have also shown that abnormal persistence of the BHC in mice is associated with defective Shh signaling and is correlated with anterior midline modifications. In extant and extinct vertebrate species with a normally persistent BHC in adults, we have finally found a distinctive organization of the anterior midline reminiscent of involvement of the Shh pathway.
Here we discuss the evolutionary importance of the position of the endoderm relative to RP. We then provide an overview of the distribution of the BHC in all vertebrate groups, discuss the current hypothesis on the functions of this canal and expose the anatomical relationships between the BHC and the carotid canals.
Endoderm/ectoderm limit and pituitary formation
The absence of endoderm in the hypophyseal placode of mice (Figure 1) is of importance from an evolutionary point of view. It is believed that in the common jawless ancestor of all vertebrates, the hypophyseal placode and the nasal placode were fused into a single median structure called the nasohypophyseal placode [2, 47]. The plesiomorphic state of the nasohypophyseal placode is thus proposed to be a single midline structure. The further steps towards the character state observed in tetrapods are considered to be: separation along the midline into a single midline nasal placode and a single median hypophyseal placode; and duplication perpendicular to the midline of the nasal placode into two paired, lateralized structures [2, 48]. The duplication of the ancestral median nasal placode is believed to have been a major step towards the acquisition of jaws by vertebrates, by allowing the migration of the trigeminal neural crest cells towards the lower parts of the face [49, 50]. Interestingly, in this context the heart-shaped, bilobate outline of RP at E11.5 in mice could represent an evolutionary polarity towards lateral duplication affecting the hypophyseal placode in a similar way as the nasal placode (Figure 2c).
There are only two extant jawless vertebrate groups: hagfish and lampreys. In lampreys, even though the nasal placode is a single midline structure, the development of the hypophyseal region is similar, and probably homologous to that in gnathostomes, except for dorsal shifting of the distal opening of the hypophyseal duct, due to the enlargement of the oral hood .
By contrast, in the case of hagfishes, there had been an ongoing controversy about whether endoderm contributes to the formation of the single midline nasohypophyseal placode, with theories based on the assumption that a posterior displacement of the endoderm boundary may have been associated with the evolutionary dislocation of the nasohypophyseal placode . A recent study on hagfish ontogeny has ruled out any endodermal contribution to the hypophyseal region of both agnathan groups : the absence of endoderm in the nasohypophyseal placode can now be considered as a primitive condition in vertebrates.
Evolutionary overview of the persistence of the buccohypophyseal canal
We have already shown that, in chondrichthyans and sarcopterygians, the persistence of the BHC is associated with a broad midline. In a more general context, the evolution of the BHC raises two issues: the primitive or derived character of its maintenance and the association of its absence or presence with modifications of other midline structures.
Here we propose an overview of the different craniates groups and discuss the BHC-related evolutionary trends (Figure 8).
The nasohypophyseal canal in agnathans (jawless fish)
Living agnathans (cyclostomes) are the sister group of the gnathostomes. Extant groups (lampreys [2, 5, 54] and hagfishes [2, 5, 55]) as well as extinct agnathan groups, such as galeaspids  and osteostracans , possess an anteriorly open nasohypophyseal duct leading from the snout to the hypophysis, with variable pharyngeal posterior opening . This duct corresponds to the invagination of a common nasohypophyseal placode . The formation of this nasohypophyseal duct cannot be easily related to the formation of the BHC by persistence of RP due to its intricate ontogenetic relationships with the nasal airways [2, 48, 53].
The buccohypophyseal canal in placoderms and acanthodians (early jawed fish)
The placoderms, generally referred to as stem gnathostomes and sister group to all modern jawed vertebrates, retain an open BHC (Figure 8). This is evident in arthodires, antiarchs, phyllolepids and acanthothoracids [2, 56]. The morphology of the placoderm parasphenoid varies from a flat, shallow, superficially lying denticulated plate pierced by a BHC opening, to a complicated bony element incorporated into the perichondral bone layer of the cranial base [2, 56].
Among acanthodians, now considered as an extinct paraphyletic group at the base of osteichthyans (for example, Acanthodes, Figure 8) and chondrichthyans , the BHC is persistent in the few species in which the basicranium is described [2, 57, 58]. These data on stem gnathostomes suggest that an open BHC is a plesiomorphic condition for crown jawed vertebrates, that is, chondrichthyans and osteichthyans (Figure 8).
The buccohypophyseal canal in chondrichthyans (cartilaginous fish)
The fossil chondrichthyans that possess an open BHC (see Results section The buccohypophyseal canal and the cranial base in chondrichthyans and Figure 8) are resolved as stem chondrichthyans in recent phylogenetic studies [37, 38]. By contrast, crown chondrichthyans, such as the living neoselachians and holocephalans, display a closed BHC. Moreover, basal relatives of these two modern clades (for example, the Cretaceous neoselachian Synechodus; the Carboniferous holocephalan Iniopera) also possess a closed canal.
The different conditions found in stem chondrichthyans and in crown chondrichthyans suggest that an open BHC is a primitive feature for chondrichthyans, and that the closure of the canal occurred in more advanced chondrichthyans.
As the closure of the canal is correlated with modifications in the midline (Figures 6 and 8), this evolutionary pattern supports the hypothesis that the transition between groups with a persistent BHC and groups with a closed BHC may have occurred by modulations in the expression territories of Shh pathway-related genes.
The buccohypophyseal canal in actinopterygians (bony ray-finned fish)
Within modern actinopterygians, the distribution of an open BHC does not seem to carry a strong phylogenetic signal, as the canal can be either open or closed in closely related species (for instance within the Galaxioidea ). Nevertheless, a persistent BHC is probably an ancestral character for actinopterygians; Paleozoic stem actinopterygians, such as Mimia and Kansasiella[62, 63] possess a persistent BHC (Figure 8). Polypterus, which is considered as an extant example of the most basal actinopterygians, retains - to various extents - a persistent canal [2, 5]. Furthermore, an open canal is retained in the teleost Elops, regarded as a plesiomorphic taxon of that group . It is noteworthy that the development of the adenohypophysis in teleosts does not involve a RP, but instead an unfolded epithelial invagination , which has no major bearing on the question of the persistence of a BHC.
The buccohypophyseal canal in sarcopterygians (bony lobe-finned fish)
Paleozoic stem sarcopterygians such as Guiyu have a persistent BHC. The same holds for ancestral dipnoans and coelacanths [2, 44]. As seen above (see Results section The buccohypophyseal canal and the cranial base in a sarcopterygian), the braincases of stem actinistians and dipnoans display an open BHC with an oral opening, whereas in modern forms there is no oral opening of the BHC (Figure 9). A persistent BHC with an opening on the oral side of the parasphenoid thus appears to be the ancestral condition in sarcopterygians and actinistians (Figure 8).
The lungfishes (or dipnoans) form with the porolepiformes (a poorly diversified Paleozoic group) a clade named Dipnomorpha. LAs the coelacanths, the lungfish, like coelacanths, have a long and complex evolutionary history (more than 400 million years). Their extant forms are rare and considered as relics. The lungfishes show similar anatomical BHC-related modifications of the midline structures of the palate to the coelacanths and chondrichthyans. More precisely, in porolepiforms and early dipnomorphs such as Powichthys, Youngolepis and Diabolepis, the palatoquadrates are widely separated from each other by a long parasphenoid, and a BHC clearly opens in the latter. In primitive lungfish (for example, Uranolophus, Dipnorhynchus), a BHC opens in the anterior part of a short parasphenoid fused to two massive dental plates (in relation with the palatoquadrates). By contrast, crown lungfish usually show massive dental plates located close to each other along the midline and the absence of a BHC . Of note, lungfish palatoquadrates are fused to the neurocranium, although this autostyly is distinct from that of holocephalans and tetrapods. Interestingly, some early porolepiforms (for example, Heimenia) and early dipnomorphs (for example, Youngolepis, Powichthys) show the coalescence of the grooves for the two branches of the internal carotid arteries inside a large hypophysial fossa [68, 69], as seen in some fossil chondrichthyans and placoderms (see Discussion section Relationship between the buccohypophyseal canal and the carotid canals).
Among piscine tetrapodomorphs, both Eusthenopteron and Tiktaalik have a BHC that opens into the oral roof through the parasphenoid . Within the tetrapod crown group, this character is lost, except in lissamphibians at larval stages . In fact, no canal is found in any extant adult tetrapod species, but paleontological data indicate that this character state is derived: the early tetrapod Ichthyostega had an open BHC, but the parasphenoids of younger Palaeozoic tetrapods (for example, anthracosaurs and temnospondyl labyrinthodonts) show no evidence of opening in the midline.
In summary, it appears that the BHC was open in most early vertebrates and in the stem taxa of all the major groups we examined, mainly before the Mesozoic (Figure 8). Furthermore, this overview confirms the link between the presence of the BHC and platybasic skulls and supports the potential role of Shh in the maintenance of the BHC throughout phylogeny. Interestingly, variations in the expression domains of Shh have previously been related to other types of phenotypic changes between related vertebrate species or between variants of a single species. In the teleost cavefish Astyanax mexicanus, the extension of the expression domains of Shh accounts for the evolution of eye regression . Similarly, the adaptive radiation of the lower jaw in cichlid fish (teleosts), namely the difference in craniofacial anatomy between Labeotropheus trewavasae and Metriaclima mbenjii, two closely related rock-dwelling cichlid genera with very different feeding behavior, relies on modifications in expressions of genes belonging to the Shh pathway . In both cases, modulations in the Shh pathway were related to eco-morphologic shifts and the phenotype change had a clear functional significance [71, 72]. In the case of the BHC, the functional aspect of its maintenance is not well understood.
Functional hypothesis on the buccohypophyseal canal
While we have provided further insight into the mechanisms leading to BHC maintenance, the function of this canal remains a mystery. Mechanosensory osmoreceptors have been described in the pituitary of the few teleosts that retain an open BHC [61, 64, 73] and could subsequently indicate involvement of an open BHC in osmoregulation and adaptation to the salinity of the water . The persistence of this structure might suggest special environmental conditions such as permanently inconstant salinity levels in the Paleozoic basins inhabited by vertebrates . Changes in the environment to the modern-type aquasphere and water salinity regime might have resulted in the obliteration of the canal independently in the evolution of various vertebrate groups. For instance, in chondrichtyans, the functions of the BHC might have been replaced by the direct osmotic regulation of urea content in blood via the epithelium of the orobranchial cavity. The canal might have become redundant after moving to other environments, such as fully marine or fully freshwater ones with more stable salinity.
Interestingly, Devonian porolepiformes and primitive lungfishes (with an open BHC) were found in marine deposits whereas almost all post-Devonian lungfishes with a closed BHC are considered as freshwater forms . Furthermore, the closure of the BHC in lower tetrapods might be related to the transition from gill-breathing in water to air-breathing in amphibians. Nevertheless, the lack of an obvious environment-related distribution of species that retain an open BHC within current teleosts  does not provide obvious support to the osmoregulatory role of this canal.
Relationship between the buccohypophyseal canal and the carotid canals
The BHC in placoderms is often combined with the canals for the internal carotids . The occasional fusion of the BHC with the internal carotid canals results from their anatomical relationship, where they penetrate the cranial base in close proximity to each other and are bordered laterally by the paired trabeculæ cranii. Different types of fusions and occlusions of these three canals are observed within different groups at the time of cranial base formation [5, 78].
Remarkably, within chondrichthyans, the blood supply on the cranial base follows a transition related to the anatomy of the BHC in modern groups. In neoselachians and holocephalans the dorsal and lateral dorsal aortas are free below the neurocranium, whereas in some basal chondrichthyans the dorsal aorta is enclosed in the basicranial canal (for example, Cobelodus) and in all basal chondrichthyans the lateral dorsal aortas run into the basicranial canals (for example, Cladodoides). In addition, in neoselachians the posterior part of the basicranial arterial system forms a bell-shaped circuit due to the laterally curving position of the lateral dorsal aortas posteriorly and the V-shaped disposition of the internal carotid arteries anteriorly , whereas in basal chondrichthyans the basicranial circuit is narrower, more elongated and lacks the posterior bell-shaped curve. In recent holocephalans, as well as in some primitive holocephalans (for example, Iniopera), the internal carotid arteries abort during ontogeny . Knowing that Shh expression is necessary for the formation of the branchial arch arteries, the transition in the structure of these arteries observed within chondrichthyans can potentially relate to a modification in Shh expression domains  and parallel the closure of the BHC.