Non-mammalian Vertebrates Research Articles

The Aromatase-Estrogen System in the Testes of Non-Mammalian Vertebrates.

Simple SummaryThe aromatase–estrogen system plays a key role in gonadal sex differentiation in amphibians, reptiles, and birds during development. In adults of seasonal breeding species, aromatase activity and estrogen levels can act as an “on/off” switch for spermatogenesis and can also promote spermiogenesis.Estrogens are important physiological regulators of testicular activity in vertebrates. Estrogen levels depend on the activity of P450 aromatase, the enzyme responsible for the irreversible conversion of testosterone into 17β-estradiol. Therefore, P450 aromatase is the key player in the aromatase–estrogen system. The present review offers a comparative overview of P450 aromatase activity in male gonads of amphibians, reptiles, and birds, with a particular emphasis on the functions of the aromatase–estrogen system in these organisms during their developmental and adult stages. The aromatase–estrogen system appears to be crucial for the sex differentiation of gonads in vertebrates. Administration of aromatase inhibitors prior to sexual differentiation of gonads results in the development of males rather than females. In adults, both aromatase and estrogen receptors are expressed in somatic cells, Leydig and Sertoli cells, as well as germ cells, with certain differences among different species. In seasonal breeding species, the aromatase–estrogen system serves as an “on/off” switch for spermatogenesis. In some amphibian and reptilian species, increased estrogen levels in post-reproductive testes are responsible for blocking spermatogenesis, whereas, in some species of birds, estrogens function synergistically with testosterone to promote spermatogenesis. Recent evidence indicates that the production of the aromatase enzyme in excessive amounts reduces the reproductive performance in avian species of commercial interest. The use of aromatase inhibitors to improve fertility has yielded suitable positive results. Therefore, it appears that the role of the aromatase–estrogen system in regulating the testicular activity differs not only among the different classes of vertebrates but also among different species within the same class.

Early Inflammation Dysregulates Neuronal Circuit Formation In Vivo via Upregulation of IL-1β.

Neuroimmune interaction during development is strongly implicated in the pathogenesis of neurodevelopmental disorders, but the mechanisms that cause neuronal circuit dysregulation are not well understood. We performed in vivo imaging of the developing retinotectal system in the larval zebrafish to characterize the effects of immune system activation on refinement of an archetypal sensory processing circuit. Acute inflammatory insult induced hyperdynamic remodeling of developing retinal axons in larval fish and increased axon arbor elaboration over days. Using calcium imaging in GCaMP6s transgenic fish, we showed that these morphologic changes were accompanied by a shift toward decreased visual acuity in tectal cells. This finding was supported by poorer performance in a visually guided behavioral task. We further found that the pro-inflammatory cytokine, interleukin-1β (IL-1β), is upregulated by the inflammatory insult, and that downregulation of IL-1β abrogated the effects of inflammation on axonal dynamics and growth. Moreover, baseline branching of the retinal ganglion cell arbors in IL-1β morphant animals was significantly different from that in control larvae, and their performance in a predation assay was impaired, indicating a role for this cytokine in normal neuronal development. This work establishes a simple and powerful non-mammalian model of developmental immune activation and demonstrates a role for IL-1β in mediating the pathologic effects of inflammation on neuronal circuit development.SIGNIFICANCE STATEMENT Maternal immune activation can increase the risk of neurodevelopmental disorders in offspring; however, the mechanisms involved are not fully understood. Using a non-mammalian vertebrate model of developmental immune activation, we show that even brief activation of inflammatory pathways has immediate and long-term effects on the arborization of axons, and that these morphologic changes have functional and behavioral consequences. Finally, we show that the pro-inflammatory cytokine IL-1β plays an essential role in both the effects of inflammation on circuit formation and normal axonal development. Our data add to a growing body of evidence supporting epidemiological studies linking immune activation to neurodevelopmental disorders, and help shed light on the molecular and cellular processes that contribute to the etiology of these disorders.

Prolactin Influences Different Aspects of Fish Biology

Prolactin (PRL) is a 198 amino acid long polypeptide hormone, structurally similar to growth hormone, secreted by adenohypophysis in the vertebrates. The full-length amino acid sequence of PRL has been determined from a variety of teleost and non-teleost fish. Teleost PRL genes are shorter than mammalian. PRL exists in two forms, namely, PRL-1 commonly found in all vertebrates and PRL-2 found in several non-mammalian vertebrates including fish. This hormone binds to transmembrane Prolactin receptor (PRLR) which is a member of the class 1 cytokines receptor superfamily. We know that PRL has over 300 different functions in vertebrates. In fish, PRL plays an important role in migration, osmoregulation, parental behaviour, reproduction and development along with other hormones. Recent studies show prolactin has a role in cell proliferation in the gills of teleost fish. This review highlights diversified role of PRL in fish which may serve as avenues for further researches in near future.

Genetically identified neurons in avian auditory pallium mirror core principles of their mammalian counterparts

In vertebrates, advanced cognitive abilities are typically associated with the telencephalic pallium. In mammals, the pallium is a layered mixture of excitatory and inhibitory neuronal populations with distinct molecular, physiological, and network phenotypes. This cortical architecture is proposed to support efficient, high-level information processing. Comparative perspectives across vertebrates provide a lens to understand the common features of pallium that are important for advanced cognition. Studies in songbirds have established strikingly parallel features of neuronal types between mammalian and avian pallium. However, lack of genetic access to defined pallial cell types in non-mammalian vertebrates has hindered progress in resolving connections between molecular and physiological phenotypes. A definitive mapping of the physiology of pallial cells onto their molecular identities in birds is critical for understanding how synaptic and computational properties depend on underlying molecular phenotypes. Using viral tools to target excitatory versus inhibitory neurons in the zebra finch auditory association pallium (calmodulin-dependent kinase alpha [CaMKIIα] and glutamate decarboxylase 1 [GAD1] promoters, respectively), we systematically tested predictions derived from mammalian pallium. We identified two genetically distinct neuronal populations that exhibit profound physiological and computational similarities with mammalian excitatory and inhibitory pallial cells, definitively aligning putative cell types in avian caudal nidopallium with these molecular identities. Specifically, genetically identified CaMKIIα and GAD1 cell types in avian auditory association pallium exhibit distinct intrinsic physiological parameters, distinct auditory coding principles, and inhibitory-dependent pallial synchrony, gamma oscillations, and local suppression. The retention, or convergence, of these molecular and physiological features in both birds and mammals clarifies the characteristics of pallial circuits for advanced cognitive abilities.

The retinal pigment epithelium: Development, injury responses, and regenerative potential in mammalian and non-mammalian systems.

Diseases that result in retinal pigment epithelium (RPE) degeneration, such as age-related macular degeneration (AMD), are among the leading causes of blindness worldwide. Atrophic (dry) AMD is the most prevalent form of AMD and there are currently no effective therapies to prevent RPE cell death or restore RPE cells lost from AMD. An intriguing approach to treat AMD and other RPE degenerative diseases is to develop therapies focused on stimulating endogenous RPE regeneration. For this to become feasible, a deeper understanding of the mechanisms underlying RPE development, injury responses and regenerative potential is needed. In mammals, RPE regeneration is extremely limited; small lesions can be repaired by the expansion of adjacent RPE cells, but large lesions cannot be repaired as remaining RPE cells are unable to functionally replace lost RPE tissue. In some injury paradigms, RPE cells proliferate but do not regenerate a morphologically normal monolayer, while in others, proliferation is pathogenic and results in further disruption to the retina. This is in contrast to non-mammalian vertebrates, which possess tremendous RPE regenerative potential. Here, we discuss what is known about RPE formation during development in mammalian and non-mammalian vertebrates, we detail the processes by which RPE cells respond to injury, and we describe examples of RPE-to-retina and RPE-to-RPE regeneration in non-mammalian vertebrates. Finally, we outline barriers to RPE-dependent regeneration in mammals that could potentially be overcome to stimulate a regenerative response from the RPE.

Dopamine Modulation of Motor and Sensory Cortical Plasticity among Vertebrates.

Goal-directed learning is a key contributor to evolutionary fitness in animals. The neural mechanisms that mediate learning often involve the neuromodulator dopamine. In higher order cortical regions, most of what is known about dopamine's role is derived from brain regions involved in motivation and decision-making, while significantly less is known about dopamine's potential role in motor and/or sensory brain regions to guide performance. Research on rodents and primates represents over 95% of publications in the field, while little beyond basic anatomy is known in other vertebrate groups. This significantly limits our general understanding of how dopamine signaling systems have evolved as organisms adapt to their environments. This review takes a pan-vertebrate view of the literature on the role of dopamine in motor/sensory cortical regions, highlighting, when available, research on non-mammalian vertebrates. We provide a broad perspective on dopamine function and emphasize that dopamine-induced plasticity mechanisms are widespread across all cortical systems and associated with motor and sensory adaptations. The available evidence illustrates that there is a strong anatomical basis-dopamine fibers and receptor distributions-to hypothesize that pallial dopamine effects are widespread among vertebrates. Continued research progress in non-mammalian species will be crucial to further our understanding of how the dopamine system evolved to shape the diverse array of brain structures and behaviors among the vertebrate lineage.

Research-Relevant Background Lesions and Conditions in Common Avian and Aquatic Species.

Non-mammalian vertebrates including birds, fish, and amphibians have a long history of contributing to ground-breaking scientific discoveries. Because these species offer several experimental advantages over higher vertebrates and share extensive anatomic and genetic homology with their mammalian counterparts, they remain popular animal models in a variety of fields such as developmental biology, physiology, toxicology, drug discovery, immunology, toxicology, and infectious disease. As with all animal models, familiarity with the anatomy, physiology, and spontaneous diseases of these species is necessary for ensuring animal welfare, as well as accurate interpretation and reporting of study findings. Working with avian and aquatic species can be especially challenging in this respect due to their rich diversity and array of unique adaptations. Here, we provide an overview of the research-relevant anatomic features, non-infectious conditions, and infectious diseases that impact research colonies of birds and aquatic animals, including fish and Xenopus species.

Transcription Factor Reprogramming in the Inner Ear: Turning on Cell Fate Switches to Regenerate Sensory Hair Cells.

Non-mammalian vertebrates can restore their auditory and vestibular hair cells naturally by triggering the regeneration of adjacent supporting cells. The transcription factor ATOH1 is a key regulator of hair cell development and regeneration in the inner ear. Following the death of hair cells, supporting cells upregulate ATOH1 and give rise to new hair cells. However, in the mature mammalian cochlea, such natural regeneration of hair cells is largely absent. Transcription factor reprogramming has been used in many tissues to convert one cell type into another, with the long-term hope of achieving tissue regeneration. Reprogramming transcription factors work by altering the transcriptomic and epigenetic landscapes in a target cell, resulting in a fate change to the desired cell type. Several studies have shown that ATOH1 is capable of reprogramming cochlear non-sensory tissue into cells resembling hair cells in young animals. However, the reprogramming ability of ATOH1 is lost with age, implying that the potency of individual hair cell-specific transcription factors may be reduced or lost over time by mechanisms that are still not clear. To circumvent this, combinations of key hair cell transcription factors have been used to promote hair cell regeneration in older animals. In this review, we summarize recent findings that have identified and studied these reprogramming factor combinations for hair cell regeneration. Finally, we discuss the important questions that emerge from these findings, particularly the feasibility of therapeutic strategies using reprogramming factors to restore human hearing in the future.

Characterization of Four Orphan Receptors (GPR3, GPR6, GPR12 and GPR12L) in Chickens and Ducks and Regulation of GPR12 Expression in Ovarian Granulosa Cells by Progesterone.

The three structurally related orphan G protein-coupled receptors, GRP3, GPR6, and GPR12, are reported to be constitutively active and likely involved in the regulation of many physiological/pathological processes, such as neuronal outgrowth and oocyte meiotic arrest in mammals. However, the information regarding these orphan receptors in nonmammalian vertebrates is extremely limited. Here, we reported the structure, constitutive activity, and tissue expression of these receptors in two representative avian models: chickens and ducks. The cloned duck GPR3 and duck/chicken GPR6 and GPR12 are intron-less and encode receptors that show high amino acid (a.a.) sequence identities (66–88%) with their respective mammalian orthologs. Interestingly, a novel GPR12-like receptor (named GPR12L) sharing 66% a.a. identity to that in vertebrates was reported in the present study. Using dual-luciferase reporter assay and Western blot, we demonstrated that GPR3, GPR6, GPR12, and GPR12L are constitutively active and capable of stimulating the cAMP/PKA signaling pathway without ligand stimulation in birds (and zebrafish), indicating their conserved signaling property across vertebrates. RNA-seq data/qRT-PCR assays revealed that GPR6 and GPR12L expression is mainly restricted to the chicken brain, while GPR12 is highly expressed in chicken ovarian granulosa cells (GCs) and oocytes of 6 mm growing follicles and its expression in cultured GCs is upregulated by progesterone. Taken together, our data reveal the structure, function, and expression of GPR3, GPR6, GPR12, and GPR12L in birds, thus providing the first piece of evidence that GPR12 expression is upregulated by gonadal steroid (i.e., progesterone) in vertebrates.

HIC1 Represses Atoh1 Transcription and Hair Cell Differentiation in the Cochlea.

SummaryAcross species, expression of the basic helix-loop-helix transcription factor ATOH1 promotes differentiation of cochlear supporting cells to sensory hair cells required for hearing. In mammals, this process is limited to development, whereas nonmammalian vertebrates can also regenerate hair cells after injury. The mechanistic basis for this difference is not fully understood. Hypermethylated in cancer 1 (HIC1) is a transcriptional repressor known to inhibit Atoh1 in the cerebellum. We therefore investigated its potential role in cochlear hair cell differentiation. We find that Hic1 is expressed throughout the postnatal murine cochlear sensory epithelium. In cochlear organoids, Hic1 knockdown induces Atoh1 expression and promotes hair cell differentiation, while Hic1 overexpression hinders differentiation. Wild-type HIC1, but not the DNA-binding mutant C521S, suppresses activity of the Atoh1 autoregulatory enhancer and blocks its responsiveness to β-catenin activation. Our findings reveal the importance of HIC1 repression of Atoh1 in the cochlea, which may be targeted to promote hair cell regeneration.

Broad-Spectrum Robust Direct Bactericidal Activity of Fish IFNφ1 Reveals an Antimicrobial Peptide-like Function for Type I IFNs in Vertebrates.

Type I IFNs (IFN-Is) play pivotal roles in host defense against viral infections but remain enigmatic against bacterial pathogens. In this study, we recombinantly expressed and purified intact grass carp (Ctenopharyngodon idella) IFNφ1 (gcIFNφ1), a teleost IFN-I. gcIFNφ1 widely powerfully directly kills both Gram-negative and Gram-positive bacteria in a dose-dependent manner. gcIFNφ1 binds to LPS or peptidoglycan and provokes bacterial membrane depolarization and disruption, resulting in bacterial death. Furthermore, gcIFNφ1 can efficiently protect zebrafish against Aeromonas hydrophila infection and significantly reduce the bacterial loads in tissues by an infection model. In addition, we wonder whether antibacterial IFN-I members exist in other vertebrates. The amino acid compositions of representative IFN-Is with strong positive charges from Pisces, Amphibia, reptiles, Aves, and Mammalia demonstrate high similarities with those of 2237 reported cationic antimicrobial peptides in antimicrobial peptide database. Recombinant intact representative IFN-I members from the nonmammalian sect exhibit potent broad-spectrum robust bactericidal activity through bacterial membrane depolarization; in contrast, the bactericidal activity is very weak from mammalian IFN-Is. The findings display a broad-spectrum potent direct antimicrobial function for IFN-Is, to our knowledge previously unknown. The results highlight that IFN-Is are important and robust in host defense against bacterial pathogens, and unify direct antibacterial and indirect antiviral bifunction in nonmammalian jawed vertebrates.

Sexual Dimorphic Distribution of Hypothalamic Tachykinin1 Cells and Their Innervations to GnRH Neurons in the Zebrafish

Substance P (SP) and neurokinin A (NKA), encoded by TAC1/Tac1 gene are members of the tachykinin family, which exert their neuromodulatory roles in vertebrate reproduction. In mammals, SP and NKA have been shown to regulate gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion via kisspeptin neurons. On the other hand, the role of SP/NKA in the regulation of reproduction in non-mammalian vertebrates is not well known. In the present study, we first localized expression of tac1 mRNA in the brain of male and female zebrafish, Danio rerio. Next, using an antibody against zebrafish tachykinin1 (Tac1), we examined the neural association of SP/NKA neural processes with GnRH3 neurons, and with kisspeptin (kiss2) neurons, in the brains of male and female zebrafish. In situ hybridization showed an apparent male-dominant tac1 expression in the ventral telencephalic area, the anterior and posterior parts of the parvocellular preoptic nucleus, and the suprachiasmatic nucleus. On the other hand, there was female-dominant tac1 expression in the ventral periventricular hypothalamus. Confocal images of double-labeled zebrafish Tac1 and GnRH3 showed associations between Tac1-immunoreactive processes and GnRH3 neurons in the ventral telencephalic area. In contrast, there was no apparent proximity of Tac1 processes to kiss2 mRNA-expressing neurons in the hypothalamus. Lastly, to elucidate possible direct action of SP/NKA on GnRH3 or Kiss2 neurons, expression of SP/NKA receptor, tacr1a mRNA was examined in regions containing GnRH3 or Kiss2 neurons by in situ hybridization. Expression of tacr1a mRNA was seen in several brain regions including the olfactory bulb, preoptic area and hypothalamus, where GnRH3 and Kiss2 cells are present. These results suggest that unlike in mammals, Tac1 may be involved in male reproductive functions via direct action on GnRH3 neurons but independent of kisspeptin in the zebrafish.

The gut-brain axis in vertebrates: implications for food intake regulation.

The gut and brain are constantly communicating and influencing each other through neural, endocrine and immune signals in an interaction referred to as the gut-brain axis. Within this communication system, the gastrointestinal tract, including the gut microbiota, sends information on energy status to the brain, which, after integrating these and other inputs, transmits feedback to the gastrointestinal tract. This allows the regulation of food intake and other physiological processes occurring in the gastrointestinal tract, including motility, secretion, digestion and absorption. Although extensive literature is available on the mechanisms governing the communication between the gut and the brain in mammals, studies on this axis in other vertebrates are scarce and often limited to a single species, which may not be representative for obtaining conclusions for an entire group. This Review aims to compile the available information on the gut-brain axis in birds, reptiles, amphibians and fish, with a special focus on its involvement in food intake regulation and, to a lesser extent, in digestive processes. Additionally, we will identify gaps of knowledge that need to be filled in order to better understand the functioning and physiological significance of such an axis in non-mammalian vertebrates.

Characterization of four urotensin II receptors (UTS2Rs) in chickens

Urotensin II receptor (UTS2R) is suggested to mediate the actions of urotensin II (UTS2) and UTS2-related peptide (URP, also called UTS2B) in mammals. However, the information regarding the gene structure, functionality and tissue expression of UTS2/URP receptor remains largely unknown in non-mammalian vertebrates including birds. In this study, using RACE-PCR, we cloned the full-length cDNAs of four chicken UTS2/URP receptors and designated them as cUTS2R1, cUTS2R2, cUTS2R3 and cUTS2R5 respectively, according to their evolutionary origin. The cloned cUTS2R1, cUTS2R2, cUTS2R3 and cUTS2R5 are predicted to encode 7-transmembrane receptors of 382, 343, 331 and 363 amino acids respectively, which show 50–66 % amino acid sequence identity with human UTS2R. Using cell-based luciferase reporter assays and Western blot, we demonstrated that chicken UTS2Rs expressed in HEK293 cells could be effectively activated by synthetic chicken UTS2−12, UTS2−17 and URP peptides, and their activation can elevate intracellular calcium concentration and activate MAPK/ERK signaling cascade, indicating that the four UTS2Rs are functional and capable of mediating UTS2/URP actions in chickens. Quantitative real-time PCR revealed that the four receptors are widely, but differentially, expressed in adult chicken tissues, while cUTS2 and cURP are highly expressed in the hindbrain and spinal cord, and moderately/weakly expressed in other tissues examined including the spleen and gonads. Taken together, our data provide first piece of evidence that all four UTS2Rs are functional in an avian species and help to reveal the conserved roles of UTS2R signaling across vertebrates.

A DNA methylation age predictor for zebrafish.

Changes in DNA methylation at specific CpG sites have been used to build predictive models to estimate animal age, predominantly in mammals. Little testing for this effect has been conducted in other vertebrate groups, such as bony fish, the largest vertebrate class. The development of most age-predictive models has relied on a genome-wide sequencing method to obtain a DNA methylation level, which makes it costly to deploy as an assay to estimate age in many samples. Here, we have generated a reduced representation bisulfite sequencing data set of caudal fin tissue from a model fish species, zebrafish (Danio rerio), aged from 11.9-60.1 weeks. We identified changes in methylation at specific CpG sites that correlated strongly with increasing age. Using an optimised unique set of 26 CpG sites we developed a multiplex PCR assay that predicts age with an average median absolute error rate of 3.2 weeks in zebrafish between 10.9-78.1 weeks of age. We also demonstrate the use of multiplex PCR as an efficient quantitative approach to measure DNA methylation for the use of age estimation. This study highlights the potential further use of DNA methylation as an age estimation method in non-mammalian vertebrate species.

Sex determination, gonadal sex differentiation, and plasticity in vertebrate species.

A diverse array of sex determination (SD) mechanisms, encompassing environmental to genetic, have been found to exist among vertebrates, covering a spectrum from fixed SD mechanisms (mammals) to functional sex change in fishes (sequential hermaphroditic fishes). A major landmark in vertebrate SD was the discovery of the SRY gene in 1990. Since that time, many attempts to clone an SRY ortholog from nonmammalian vertebrates remained unsuccessful, until 2002, when DMY/dmrt1by was discovered as the SD gene of a small fish, medaka. Surprisingly, however, DMY/dmrt1by was found in only 2 species among more than 20 species of medaka, suggesting a large diversity of SD genes among vertebrates. Considerable progress has been made over the last 3 decades, such that it is now possible to formulate reasonable paradigms of how SD and gonadal sex differentiation may work in some model vertebrate species. This review outlines our current understanding of vertebrate SD and gonadal sex differentiation, with a focus on the molecular and cellular mechanisms involved. An impressive number of genes and factors have been discovered that play important roles in testicular and ovarian differentiation. An antagonism between the male and female pathway genes exists in gonads during both sex differentiation and, surprisingly, even as adults, suggesting that, in addition to sex-changing fishes, gonochoristic vertebrates including mice maintain some degree of gonadal sexual plasticity into adulthood. Importantly, a review of various SD mechanisms among vertebrates suggests that this is the ideal biological event that can make us understand the evolutionary conundrums underlying speciation and species diversity.

Exogenous Oestrogen Impacts Cell Fate Decision in the Developing Gonads: A Potential Cause of Declining Human Reproductive Health.

The increasing incidence of testicular dysgenesis syndrome-related conditions and overall decline in human fertility has been linked to the prevalence of oestrogenic endocrine disrupting chemicals (EDCs) in the environment. Ectopic activation of oestrogen signalling by EDCs in the gonad can impact testis and ovary function and development. Oestrogen is the critical driver of ovarian differentiation in non-mammalian vertebrates, and in its absence a testis will form. In contrast, oestrogen is not required for mammalian ovarian differentiation, but it is essential for its maintenance, illustrating it is necessary for reinforcing ovarian fate. Interestingly, exposure of the bi-potential gonad to exogenous oestrogen can cause XY sex reversal in marsupials and this is mediated by the cytoplasmic retention of the testis-determining factor SOX9 (sex-determining region Y box transcription factor 9). Oestrogen can similarly suppress SOX9 and activate ovarian genes in both humans and mice, demonstrating it plays an essential role in all mammals in mediating gonad somatic cell fate. Here, we review the molecular control of gonad differentiation and explore the mechanisms through which exogenous oestrogen can influence somatic cell fate to disrupt gonad development and function. Understanding these mechanisms is essential for defining the effects of oestrogenic EDCs on the developing gonads and ultimately their impacts on human reproductive health.

The Role of the Thyroid Axis in Fish.

In all vertebrates, the thyroid axis is an endocrine feedback system that affects growth, differentiation, and reproduction, by sensing and translating central and peripheral signals to maintain homeostasis and a proper thyroidal set-point. Fish, the most diverse group of vertebrates, rely on this system for somatic growth, metamorphosis, reproductive events, and the ability to tolerate changing environments. The vast majority of the research on the thyroid axis pertains to mammals, in particular rodents, and although some progress has been made to understand the role of this endocrine axis in non-mammalian vertebrates, including amphibians and teleost fish, major gaps in our knowledge remain regarding other groups, such as elasmobranchs and cyclostomes. In this review, we discuss the roles of the thyroid axis in fish and its contributions to growth and development, metamorphosis, reproduction, osmoregulation, as well as feeding and nutrient metabolism. We also discuss how thyroid hormones have been/can be used in aquaculture, and potential threats to the thyroid system in this regard.

SLC2A12of SLC2 Gene Family in Bird Provides Functional Compensation for the Loss ofSLC2A4Gene in Other Vertebrates

Avian genomes are small and lack some genes that are conserved in the genomes of most other vertebrates including nonavian sauropsids. One hypothesis stated that paralogs may provide biochemical or physiological compensation for certain gene losses; however, no functional evidence has been reported to date. By integrating evolutionary analysis, physiological genomics, and experimental gene interference, we clearly demonstrate functional compensation for gene loss. A large-scale phylogenetic analysis of over 1,400 SLC2 gene sequences identifies six new SLC2 genes from nonmammalian vertebrates and divides the SLC2 gene family into four classes. Vertebrates retain class III SLC2 genes but partially lack the more recent duplicates of classes I and II. Birds appear to have completely lost the SLC2A4 gene that encodes an important insulin-sensitive GLUT in mammals. We found strong evidence for positive selection, indicating that the N-termini of SLC2A4 and SLC2A12 have undergone diversifying selection in birds and mammals, and there is a significant correlation between SLC2A12 functionality and basal metabolic rates in endotherms. Physiological genomics have uncovered that SLC2A12 expression and allelic variants are associated with insulin sensitivity and blood glucose levels in wild birds. Functional tests have indicated that SLC2A12 abrogation causes hyperglycemia, insulin resistance, and high relative activity, thus increasing energy expenditures that resemble a diabetic phenotype. These analyses suggest that the SLC2A12 gene not only functionally compensates insulin response for SLC2A4 loss but also affects daily physical behavior and basal metabolic rate during bird evolution, highlighting that older genes retain a higher level of functional diversification.

Evolutionary history of histamine receptors: Early vertebrate origin and expansion of the H3-H4 subtypes

Histamine receptors belonging to the superfamily of G protein-coupled receptors (GPCRs) mediate the diverse biological effects of biogenic histamine. They are classified into four phylogenetically distinct subtypes H1-H4, each with a different binding affinity for histamine and divergent downstream signaling pathways. Here we present the evolutionary history of the histamine receptors using a phylogenetic approach complemented with comparative genomics analyses of the sequences, gene structures, and synteny of gene neighborhoods. The data indicate the earliest emergence of histamine-mediated GPCR signaling by a H2 in a prebilaterian ancestor. The analyses support a revised classification of the vertebrate H3-H4 receptor subtypes. We demonstrate the presence of the H4 across vertebrates, contradicting the currently held notion that H4 is restricted to mammals. These non-mammalian vertebrate H4 orthologs have been mistaken for H3. We also identify the presence of a new H3 subtype (H3B), distinct from the canonical H3 (H3A), and propose that the H3A, H3B, and H4 likely emerged from a H3 progenitor through the 1R/2R whole genome duplications in an ancestor of the vertebrates. It is apparent that the ability of the H1, H2, and H3-4 to bind histamine was acquired convergently. We identified genomic signatures suggesting that the H1 and H3-H4 shared a last common ancestor with the muscarinic receptor in a bilaterian predecessor whereas, the H2 and the α-adrenoreceptor shared a progenitor in a prebilaterian ancestor. Furthermore, site-specific analysis of the vertebrate subtypes revealed potential residues that may account for the functional divergence between them.