Morphology, Development, and Evolution of the Adenohypophysis

Corey S. Johnson^*
*Correspondence: Corey S. Johnson, Department of Biology, University of North Carolina at Chapel Hill, North Carolina, USA, Tel: 9197245429, Email:

Keywords

Morphology • Developmental biology • Adenohypophysis

Introduction

The pituitary gland is a peculiar structure, having several unique features. It is a composite structure made of glandular epithelium, the adenohypophysis (AH), and an extension of the hypothalamus, the neurohypophysis (NH). The AH is responsive to hypothalamic neurohormones transported through a portal system. The evolutionary history of these structures, their function, and their morphological elaboration has become more apparent in recent years as modern developmental gene expression techniques and traditional descriptive embryology has been applied to non-model organisms.

Morphology of the Gnathostome Pituitary

Humans, mammals, and nearly all extant vertebrates are gnathostomes, descendants of jawed fish. The well-characterized physiological functions of the AH in these organisms include control of reproduction, lactation, metabolic function and rate, growth, and pigmentation. Much of this understanding is derived from studies in mammals, although studies in other vertebrates have provided relevant comparative data. The axon terminals of the hypothalamic nuclei are concentrated in two loci of the NH: the pars nervosa (PN) and the median eminence (Figure 1).

In agnathans, chondrichthyans, and some actinopterygian fish, the functionally equivalent region of the NH has been called the proximal neurosecretory contact region [1-3]. Regardless of slight differences in morphology across gnathostome taxa, the hormones of the median eminence regulate the secretions of the AH, while the hormones of the PN find their targets at more distant targets: the blood vessels, kidneys, and mammary glands.

Hypophyseal arteries branch directly or indirectly from the internal carotid arteries. These vessels supply capillary beds to the median eminence [4]. Interestingly, early investigators working in mammalian systems suggested that blood flow was from the AH toward the brain [5]. However, the hypothalamic-hypophyseal portal system was eventually described correctly by Wislocki and King [6]. The capillary blood of the median eminence flows to a secondary capillary bed in the AH by the long portal vessels. While understanding of the venous drainage eluded researchers for some time (reviewed in Page 1982), the modern understanding eventually emerged wherein blood flows into a primary capillary bed in the median eminence to a secondary capillary bed in the AH, and finally is drained via hypophyseal veins to the cavernous sinuses. Comparative studies of portal vasculature among non-mammals have received little attention, but the anatomical arrangement of vessels appears to imply a similar portal circulation in most gnathostomes.

Three subdivisions are found within the gnathostome AH, with three endocrine profiles: the pars distalis, pars tuberalis, and pars intermedia. The pars distalis is the largest and anterior-most region of the AH and contains several hormone-specific cell types: corticotrophs, melanotrophs, somatotrophs, lactotrophs, gonadotrophs, and thyrotrophs. Folliculostellate cells are also present in the AH [7]. Although their role has not been fully understood, they do function in the paracrine regulation of hormone secretion and in immune system interactions with dendritic cells and macrophages of the AH [8,9]. The pars tuberalis is a region that surrounds the infundibulum of the hypothalamus. Like the pars distalis, it contains thyrotropes. However, no other hormone-producing cells appear to be present [10]. Recent evidence has accumulated that implicates the pars tuberalis as a significant site of regulation for circannular rhythm, especially in organisms with distinct seasonal adaptations regarding feeding, metabolism, and reproduction [11]. The pars intermedia (PI) is found adjacent to the PN. The always diminutive PI is comprised of melanotropes that secrete Melanocyte stimulating hormone (MSH). Although found in the human fetus, the PI is absent in postnatal life. Evidence indicates that its cells migrate into the pars distalis and PN [12]. In many other mammalian species, the pars intermedia persists throughout postnatal life. Not all gnathostomes possess all of the regions above, and their absence is considered secondary. For example, birds, cetaceans, and some squamates do not have a pars intermedia; snakes and lungfish lack a pars tuberalis. Such details are beyond the scope of this review, and the reader is directed to Norris and Carr for an excellent review of the literature describing unique examples of variation among gnathostome fish and tetrapods [13].

Development of the Gnathostome Adenohypophysis

The pre-placodal ectoderm (PPE) is a region of placode and neural crest forming tissue that forms along the anterior border of the anterior neural plate. It is from this region that five placodes form: the single median adenohypophyseal placode and paired bilateral olfactory and lens placodes [14-16]. Later in embryogenesis, the adenohypophyseal placode is found forming among cells of the oral ectoderm, the stomodeum. Invagination of the placode towards the developing brain forms Rathke's pouch. Various transcription factors are known to be important to the specification of the adenohypophyseal placode and Rathke's pouch, including Six1/2, Eya, and Pitx [17]. In close proximity, the hypothalamus forms in the ventral floor of the diencephalon [18]. A portion of the developing hypothalamus, the infundibulum, is responsible for the induction of Rathke's pouch from the stomodeal ectoderm through its release of BMP and FGF signals [19]. The infundibulum and Rathke's pouch remain in close contact throughout their continued development. The neuroectoderm-derived infundibulum forms the hypothalamus, and its axons project to the eventual sites of hormone release in the median eminence and PN.

Rathke's pouch develops several distinct lobes. The posterior portion in contact with the hypothalamus forms the pars intermedia. Two lateral lobes form the pars tuberalis, and the anterior lobe forms the pars distalis [20,21]. Depending on the species and developmental stage, the lumen between the pars intermedia and pars distalis may be lost or persist as a narrow cleft. In normal human development, the connection to the stomodeum is lost, although remnants of Rathke's pouch commonly persist outside the AH proper as a pharyngeal hypophysis in the roof of the nasopharynx. The cell types present have been determined to be the same as that of the pars distalis [22].

Chordate Development

Jawed vertebrates, along with the jawless cyclostomes, make up the living vertebrates. The wider group to which vertebrates belong is the chordates. There are morphological, developmental, and physiological features among the non-vertebrate chordates that may provide clues to the evolution of the vertebrate condition. All chordates (cephalochordates, urochordates, vertebrates) at some time in their development possess a notochord, for which they are named. They also possess a post-anal tail, a dorsal hollow nerve cord, and pharyngeal slits. Model chordates include the tunicates (Halocynthia and Ciona) and amphioxus. Amphioxus is a filter- feeding cephalochordate with a highly conserved genomic structure and morphology [23,24]. It superficially resembles the lamprey ammocoetes larva as well as Pikaia gracilens, a putative chordate from the Cambrian [25,26]. The model tunicate species are filter- feeding urochordates that have a motile larval stage and metamorphose into sessile adults. Although tunicates do not share a strong resemblance to vertebrates and cephalochordates, they do share some vertebrate-like features of early development. Unlike amphioxus, some tunicates have a migratory cell population arising from the margins of the neural tube during its development. Like neural crest cells in vertebrates, these cells form melanophores [27]. Aspects of tunicate development, however, are quite divergent from what is found in vertebrates and amphioxus. As reviewed by Holland, tunicates have evolved a more mosaic paradigm of development based on the use of operons [28]. Despite the limited morphological similarity between urochordates and vertebrates, they are considered the most likely sister group to vertebrates [29]. Although more distantly related, cephalochordates such as amphioxus have more in common with vertebrates morphologically, making them essential models for understanding the roots of the chordate and vertebrate lineages.

Amphioxus has an evagination of its rostral gut, Hatschek's pit, that is in contact with the ventral brain. Such an arrangement has, for obvious reasons, given the impression that these are the rudiments of a primitive hypothalamus-AH association, beginning with Berthold Hatschek himself and extending to modern times [28,30-32]. From a morphogenetic standpoint, the question of homology between Hatschek's pit and the vertebrate AH is complicated due to the convoluted embryological formation of the cephalochordate structure. As reviewed in Glardon, et al. (1998), the developmental events that lead to Hatschek's pit formation begin in the neurula and are not completed until metamorphosis. From an evagination of the left rostral gut, Hatschek's left diverticulum (HLD) is formed. Separating from this layer, HLD fuses with an invagination of the ectoderm to form the preoral pit. The preoral pit, with its ectodermal and HLD- derived components, establishes continuity with the pharyngeal cavity. During metamorphosis, it is from the derivatives of the preoral pit that Hatschek's pit evaginates dorsally. Accurate lineage tracing will be required to discern which germ layer(s) form Hatschek's pit and provide definitive evidence for homology.

In the tunicate, like in amphioxus, a pair of tissues are juxtaposed in a similar developmental and spatial relationship. From the rudiment of the central nervous system, an evagination develops that proceeds toward the stomodeum. As contact is made with the stomodeum, the two structures fuse, and their lumens become continuous. In time, the neural component differentiates to form the neural gland. The oral component maintains its continuity with the stomodeum and contributes to the ciliated funnel (buccal funnel) found at the stomodeal roof. Together, the tunicate neural complex is likely an organ for osmotic balance and was implicated by early investigators as containing the AH homolog [33-36].

While revealing no definitive evidence of endocrine function, Hatschek's pit and the ciliated funnel are suggestive of early homologs to the AH. Despite not having the same endocrine structures as vertebrates, tunicates and amphioxus have a variety of individual cells located elsewhere in their epidermal tissues with the characteristics one would expect in the evolutionary precursors to the hypothalamic-AH axis. That is, some cells act as chemoreceptors to respond to the environment, and other cells secrete substances to affect internal systems [37]. Evidence suggests these functions are linked such that stimuli within the environment can affect internal physiology [38,39]. More investigation into a putative endocrine function within these organisms is needed to complement our growing developmental understanding of these organisms. Thus, the morphological forerunners of the hypothalamus-AH are present, and the sensory-secretory building blocks of an endocrine seem to be present, but these features are not found within the same part of the cephalochordate or urochordate body as they are in vertebrates.

A comparison of the development of Hatschek's pit and ciliated funnel with the AH has another notable difference. The developmental forerunner of Rathke's pouch, the adenohypophyseal placode, is absent from non-vertebrate chordates. In fact, there are no placodes of any kind in the development of these organisms. In the early stages of vertebrate development, the PPE expresses Six1/2, Eya, and Pitx gene family members. These genes play a role in defining the anterior placode-forming region, including the adenohypophyseal placode [40-42]. Amphioxus does not exhibit Six1/2 and Eya expression in this region, and Pitx is present only briefly [37]. These results are consistent with the lack of placode formation in amphioxus since these are transcription factors associated with placode and cell shape changes. However, at later stages of development, Six and Eya family members are found in HLD. Still later, during metamorphosis Six, Eya, and Pitx genes are found in Hatschek's pit [28]. Despite differences in developmental timing, the same network of genes does seem to be involved in the formation of Hatschek's pit and the vertebrate AH. This evidence is only correlative, yet it does provide an intriguing possibility of either direct homology of structures or the co-option of the gene network.

In tunicates, like amphioxus, placodes and the discrete gene expression subdomains that would prefigure them are not observed. Examination of embryos prior to neural gland formation has shown that an ectodermal developmental regulatory domain containing Six1/2 and Eya genes is present in tunicates [43]. This "proto- placodal ectoderm" is found directly anterior to the tunicate neural plate, just as is the case with the vertebrate PPE. Although it does not form placodes, it does bear the characteristic regulatory network of the vertebrate PPE, and its cells exhibit gene expression characteristic of vertebrate placodal cell populations. Portions of the proto-placodal ectoderm later form the ciliated funnel, which continues to express Six1/2 and Eya genes as well as Pitx [32,44,45]. These data indicate that the genetic regulatory network that compartmentalizes and then specifies the vertebrate AH was present in the common ancestor of tunicates and vertebrates, even if placodes per se were not present. The steps that would result in the gathering of ectoderm-derived neurosecretory or sensory cells into placodes would only occur after the split between tunicates and their last common ancestor with the vertebrates.

Cyclostome Development

The curious developmental sequence, the physical proximity of the AH and NH, and the portal circulation beg for an explanation of the evolutionary roots and adaptive value of this system. To understand the earliest vertebrate patterns of development and evolution, studies in cyclostomes have been very insightful. The two extant lineages include the Myxini (hagfishes) and the Petromyzontiformes (lampreys). Although vertebrates, these are jawless fish with many simple anatomical features that have been elaborated in the gnathostome lineages: the Chondrichthyes (cartilaginous fish), the Actinopterygii (ray-finned fish), and the Sarcopterygii (lobe-finned fish and tetrapods; Figure 2). The morphological features of the hagfish are, in many ways, more simplistic than that of the lamprey. However, the most recent developmental and genomic evidence suggests that the cyclostomes are indeed monophyletic [46-48].

Although extant animals with unique specializations of their own, the early embryos of hagfish and lampreys are particularly useful indicators of the anatomical and developmental patterns common to their clade. Several factors have historically lead researchers to speculate that there was no functional or morphological homology of the hagfish AH to that of higher vertebrates. Morphologically, the region first identified as the AH in hagfish was viewed as a simple plate of tissue, and the hypothalamic region that would regulate its activity was separated from the AH by a thin connective tissue; no portal circulation had been identified in these organisms. Developmentally, early studies indicated that Rathke's pouch arises from endoderm in hagfish, unlike the stomodeal (ectodermal) origin common to lampreys and other vertebrates [49]. Further, there was no known hormone identified in the hagfish AH until 2003, when a gonadotropin was detected in the AH [50]. This finding was an important piece of evidence that homology may exist between the hagfish AH and that of extant vertebrates.

A recent detailed study of cyclostome embryology has resolved the apparent discrepancies between hagfish and the lamprey [47]. Anatomically, hagfish and lampreys have a single naris that forms from the invagination of a single nasohypophyseal placode. From the ectoderm, the nasohypophyseal placode invaginates, and olfactory and adenohypophyseal placodal components separate to form discrete organs, although they remain close together within the nasohypophyseal duct. Confusion regarding the germ layer origin of the AH in hagfish has a simple explanation. Oisi et al. found that the hagfish has a peculiar secondary oropharyngeal membrane that forms anterior to the oropharyngeal membrane proper [47]. The earlier investigation claimed the nasohypophyseal invagination formed posterior to the oropharyngeal membrane; thus, it was deemed to be of endodermal origin. In fact, the nasohypophyseal invagination was merely posterior to the secondary oropharyngeal membrane, yet anterior to the oropharyngeal membrane proper. Developmental gene expression studies of cyclostome nasal and hypophyseal placodal gene expression by the same investigators further supported the homology between the AH of hagfish and that of other vertebrates.

Like many morphological features of the hagfish, the AH and NH are simpler in appearance than those in gnathostomes or lampreys. The hagfish AH appears not to have subdivisions. It has no pars intermedia nor its typical hormone, MSH (discussed below). The NH is thin and does not appear to have a PN. On the other hand, the lamprey condition is much more typical of gnathostome fish in some respects. There are rostral and caudal divisions of the pars distalis, and they have a distinct pars intermedia in physical contact with a PN (Figure 3). As in higher vertebrates, the pars intermedia is innervated directly with hypothalamic neurons, although innervation may be lost in post-metamorphic lampreys [51]. If hagfish and lampreys are understood to be monophyletic, the relatively simplistic morphology of the AH and NH in hagfish is the result of evolutionary simplification. The inferred condition of the last common ancestor of cyclostomes and gnathostomes is then clear: the first vertebrates possessed an AH with defined subdivisions, yet it lacked a portal system as well as some of the hormones found in gnathostomes.

Hormones of the Gnathostome Adenohypophysis

The gnathostome AH produces three families of hormones. The Pro-opiomelanocortin (POMC) hormones include several cleavage variants of a single peptide, including adrenocorticotropin (ACTH), which contains α-melanotropin (also called α-melanocyte-stimulating hormone (α-MSH)). Additionally, β-MSH, -MSH, and β-endorphin are contained within the POMC peptide. Uniquely, Chondrichthyans have an additional, MSH [52]. The single-protein hormones include growth hormone (GH) and prolactin (PRL). The glycopeptides FSH, LH, TSH, and thyrostimulin are dimers with α and β subunits. FSH, LH, and TSH share a common α subunit, GpA1, and they are differentiated from one another by the presence of a unique β subunit: FSHβ, LHβ, or TSHβ. Thyrostimulin has unique α and β subunits, GpA2/GpB5.

The regulation of all hormones of the AH occurs primarily through releasing and inhibiting hormones of the hypothalamus. Such hypothalamic hormones include corticotropin-releasing hormone, growth hormone-releasing hormone, somatostatin, dopamine, gonadotropin-releasing hormone (GnRH), and thyrotropin-releasing hormone. Extensive paracrine activity and some direct innervation play a role in the regulation of AH hormone secretion [53-55].

Chordate Hormones

The homologs to vertebrate hormones in chordates are few in number. Genes encoding forms of the hypothalamic hormone GnRH and GnRH receptors have been identified in the amphioxus and tunicate genomes [56-61]. It is important to note, however, that GnRH does not function exclusively to control gonadotropins; it has many well known neural functions independent of its control over the AH. An initial report that amphioxus has a gonadotropin localized to Hatschek's pit provided intriguing evidence for the functional homology of Hatschek's pit to the vertebrate AH [62]. Likewise, PRL immunoreactivity in Hatschek's pit has been reported. However, these antibody-based approaches have since been refuted by more definitive genomic studies indicating that there are no adenohypophyseal hormones in chordates. Additionally, there is no definitive evidence for GH family peptides [58,63,64]. Also, not present are genes encoding POMC hormones or TSH [56,58,63,64]. The lack of a POMC gene or sequences for its subcomponents is somewhat surprising. Several invertebrate groups have proteins with high sequence similarity to the gnathostome POMC. Leech and mollusk genomes contain the POMC gene components in the same sequential order as in gnathostomes. The absence of these genes in chordates is puzzling. It opens the door to the possibility of horizontal gene transfer from parasite to host, or less parsimoniously, the chordate inheritance of the gene, followed by its loss in both urochordate and cephalochordate lineages after the divergence of the vertebrate lineage.

Although there is no gonadotropin in either chordate genome, the α and β components of Thyrostimulin have been identified [23]. It remains to be determined whether GnRH has influence over Thyrostimulin in amphioxus, or whether its expression is localized to Hatschek's pit or the ciliated funnel. The remarkable scarcity of hormones in chordates indicates that POMC family hormones and single-protein hormones like GH are vertebrate innovations. GnRH and Thyrostimulin are the sole representatives of vertebrate hypothalamic-AH hormones that are found among non-vertebrate chordates.

Cyclostome Hormones

Until recent decades, it was unknown whether the morphologically simplistic hagfish AH had any functioning hormones. Studies have since revealed that all three gnathostome hormone families have at least a single representative in cyclostomes. These include a glycopeptide hormone (GpH) with gonadotropin function. This hormone is comprised of GpA1/GpHβ subunits, which contain the α subunit common to FSH, LH, and TSH of higher vertebrates but having a unique β subunit [65,66]. Lampreys likewise have a gonadotropin with a homologous GpHβ subunit but do not possess the hagfish and gnathostome GpA1 subunit. Instead, lamprey gonadotropin utilizes the same α subunit as in thyrostimulin, GpA2 [67]. Interestingly, the thyrostimulin subunits (GpA2/GpB5) have not been identified in hagfish, but they are present in gnathostomes, lampreys, as well as in non-vertebrate chordates [23,56,68]. One evolutionary scenario would place GpA1, GpA2, and GpB5 at the last common ancestor of all vertebrates, with the subsequent loss of GpA1 from the lamprey lineage and GpA2/GpB5 from the hagfish lineage. Neither cyclostome group has the β subunits for TSH, FSH, or LH. These are gnathostome novelties that have likely arisen from gene duplication and divergence events from their respective hormone families.

The single-protein family member GH is found in the lamprey genome and evidence supports its presence in hagfish [69,70]. PRL is not present, except in gnathostomes. The third hormone family, the hormones, is present in parts. Rather than being found as a single gene sequence as in gnathostomes, the hormone-encoding sequences are found on two genes, pro-opiocortinand (POC) and pro-opiomelanotropin (POM) [71]. Genetic data from hagfish is lacking. Immunohistochemical binding assays indicate the likelihood of a pro-opiocortin similar to that of lampreys, but there is little evidence to suggest the presence of MSH-like molecules [70,72]. The hypothalamic control of the identified cyclostome hormones, GpH, thyrostimulin, ACTH, and GH, has not been thoroughly investigated. Nonetheless, initial evidence indicates the presence of GnRH and corticotropin-releasing hormone [73,74]. These hypothalamic hormones may regulate glycopeptides and POC, respectively. A hypothalamic regulator of GH in hagfish has not been identified.

Research in lampreys has shed light on the regulation of gonadal and thyroid function. Interestingly, exogenous GnRH raises both thyroxine and estradiol levels, indicating that both GpH and Thyrostimulin may be under the control of the same hormone [75,76]. In lampreys, reproductive function and increased metabolic activity are coincidental with metamorphosis; thus, the overlapping regulation of ovarian and thyroid endocrine activity is not surprising. In the course of gnathostome evolution, FSH and LH would functionally replace the single gonadotropin, the regulation of the gonadotropins and thyrostimulin would become independent, and TSH would be added to the AH-thyroid axis of control.

Evolutionary Connections and Conclusions

Examining the origins of the AH among the non-vertebrate chordates shows that the building blocks of these systems are present. First, although there are few hormones are present in chordates, homologs to those that would form a hypothalamic-AH axis are present within the genome. The expression patterns of GnRH and thyrostimulin in cephalochordates or urochordates are unknown. Secondly, Hatschek's pit and the ciliated funnel appear to be homologs to the AH of vertebrates; that is, they form like the vertebrate AH and from the expected tissue layers, even if they do not share common function. Lastly, it is also clear that chordates possess homologous cell types, sensory cells sensitive to olfactory, gustatory, or otherwise chemoreceptive stimuli in the environment. Also, chordates possess secretory cells capable of releasing chemical messengers to the blood for long-range communication. The juxtaposition of two such cell types in the ancestors of vertebrates would allow important physiological coordination in response to environmental cues.

If cyclostome ontogeny is indicative of an ancestral morphological condition, the single nasohypophyseal placode was the anlagen of both chemoreceptive and secretory adenohypophyseal cells. These cells would have had a close functional relationship, much like the enterocytes and enteroendocrine cells of the intestinal wall in higher vertebrates. How the hormones came to be expressed in the sensory and secretory cell types, and how the secretory cell types organized into placodes and became incorporated into evaginating stomodeal ectoderm, remains a central question in the evolution of vertebrates.

However, it should be noted that the separation of the olfactory and AH placodes found in the course of vertebrate development and evolution is only apparent. Cells originating in the olfactory placode migrate during ontogeny to take up residence in hypothalamic nuclei, not far from the AH. These migratory cells from the olfactory placode become GnRH-secreting hypothalamic neurons [77-79]. From this destination in the hypothalamus, GnRH neurons were sufficiently close to control the AH in a diffusional manner as seen in cyclostomes or an endocrine manner as in gnathostomes. Such an evolutionary step, where GnRH secreting cells undergo epithelial- mesenchymal transition and migrate to retain their developmental and ancestral proximity to the AH, would allow for the rearrangement and expansion of tissues in the head. Freeing the olfactory epithelium from the AH would have been an essential step in permitting the evolution of morphologically diverse patterns of tissues within the head.

Despite minor differences in overall morphological proportions of their components, all gnathostome vertebrates retain similar developmental and morphological relationships between the hypothalamus and the AH. These similarities extend to the regulation of the AH by way of the portal circulation. Portal circulations are found in birds and reptiles, amphibians, lobe-finned fish and cartilaginous fish [80-84]. In apparent deviation from the norm, teleost fish, a highly derived clade of actinopterygians, have an extensive innervation of the AH by hypothalamic neurons. Although extensive, the innervation of the AH in teleosts is not completely without a portal circulation [85]. Thus, the regulation of the AH in all examined vertebrate groups involves a portal system.

The portal circulation that most gnathostomes depend upon for direct hormonal communication between the median eminence and the AH is not present in cyclostomes. Instead, the hypothalamus of cyclostomes exerts its influence over the AH by paracrine signaling. Studies in hagfish have demonstrated that substances injected into the third ventricle can diffuse quickly to the AH [86]. Dyes and even the large protein, horseradish peroxidase, eventually travel to the AH through the intervening connective tissue. Although there is no portal system, the anatomical proximity of the hypothalamus and AH allows the physiologically relevant exchange of substances. It should be noted that in one hagfish species, several small blood vessels have been identified within the connective tissue between AH and the hypothalamus. The authors conjecture that these vessels may allow a more efficient communication in the manner of a primitive portal system [87]. No studies have yet been conducted to indicate the precise anatomical arrangement of these vessels nor the direction of blood flow. The lack of similar vessels in other hagfish species and lampreys suggests their appearance may be an evolutionary novelty if indeed the vessels function in a manner analogous to the portal system found in higher vertebrates.

If paracrine factors can efficiently diffuse from the NH to the AH, one might wonder why a portal system is necessary. Lampreys, with many of the same neuroendocrine signaling systems seen in gnathostomes, are seemingly not limited by the lack of a hypothalamic-hypophyseal portal system. The morphology of the cyclostome AH provides a clue. Its thin, flat appearance may be an optimization for surface area. In order for paracrine communication from the NH to be efficient, the responding cells must be within proximity. The evolutionary acquisition of jaws and a more active predatory lifestyle coincide with the increase in body size in gnathostome fishes [88]. The pituitary gland would be expected to enlarge with vertebrate body size, and perhaps a reliance upon paracrine signaling became size-limiting for vertebrates.

A more significant explanation for the portal circulation may lie within the morphological changes that occurred in the vertebrate head throughout evolution. As has been discussed, cyclostomes possess a single nasohypophyseal duct. Fossil agnathans such as the anaspids also exhibit a single nasohypophyseal duct but the Galeaspids appears to be one of the earliest lineages of vertebrates to have bilateral nasal organs [89]. In these well preserved endocranial specimens, it is evident that the hypophyseal duct retains its connection to the exterior environment. Unlike cyclostomes, which have an anterio-dorsally located opening to their duct, galeaspids have a hypophyseal duct that opens ventrally within the oral cavity. Gai et al. suggest that these changes removed the barrier to the expansion of tissues to form the upper jaw in the ancestors of gnathostomes [90]. This evolutionary repositioning of the adenohypophyseal duct and the formation of bilateral nasal sacs was a significant rearrangement of tissues in the head. Such changes may have played a role in distancing the AH from the hypothalamus in gnathostome ancestors. A portal circulation would ensure continuity of function between these organs, despite changes in their proximity. While it is impossible to know whether a functional portal system in galeaspids was present, their fossils indicate the presence of hypophyseal arteries, branches of the internal carotid arteries that serve the hypothalamus and AH in extant organisms that possess portal circulations [90].

Connections drawn from physiology, development, and morphology of extinct and extant organisms reveal a picture of the evolutionary cohesion of endocrine cells of the AH and their GnRH- secreting regulators. This morphological and functional relationship has not been identified among modern urochordates or cephalochordates, but it must have formed among the ancestors of vertebrates [91]. While their physical proximity is present embryologically in cyclostomes, the apparent separation of olfactory and AH cells during development would leave the AH without its physiological regulator. The migration of a subset of olfactory cells to take up residence in the hypothalamus allows continuity of function despite evolutionary changes to the relative position of olfactory organs and the AH [92]. Further changes early in the vertebrate lineage would include the expansion of the head region and the increase of body size. Concomitantly, the position of the adenohypophyseal placode underwent a significant ventral relocation. Together, these factors may have been responsible for further distancing the AH from its regulators in the hypothalamus [93]. The acquisition of a portal circulation allowed for the maintenance of a physiological connection despite the morphological distancing that occurred throughout the evolution of gnathostome ancestors.

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