Photoperiodic Modulation of Circadian Clock and Reproductive Axis Gene Expression in the Pre-Pubertal European Sea Bass Brain

The acquisition of reproductive competence requires the activation of the brain-pituitary-gonad (BPG) axis, which in most vertebrates, including fishes, is initiated by changes in photoperiod. In the European sea bass long-term exposure to continuous light (LL) alters the rhythm of reproductive hormones, delays spermatogenesis and reduces the incidence of precocious males. In contrast, an early shift from long to short photoperiod (AP) accelerates spermatogenesis. However, how photoperiod affects key genes in the brain to trigger the onset of puberty is still largely unknown. Here, we investigated if the integration of the light stimulus by clock proteins is sufficient to activate key genes that trigger the BPG axis in the European sea bass. We found that the clock genes clock, npas2, bmal1 and the BPG genes gnrh, kiss and kissr share conserved transcription factor frameworks in their promoters, suggesting co-regulation. Other gene promoters of the BGP axis were also predicted to be co-regulated by the same frameworks. Co-regulation was confirmed through gene expression analysis of brains from males exposed to LL or AP photoperiod compared to natural conditions: LL fish had suppressed gnrh1, kiss2, galr1b and esr1, while AP fish had stimulated npas2, gnrh1, gnrh2, kiss2, kiss1rb and galr1b compared to NP. It is concluded that fish exposed to different photoperiods present significant expression differences in some clock and reproductive axis related genes well before the first detectable endocrine and morphological responses of the BPG axis.European Community [222719 - LIFECYCLE]; Foundation for Science and Technology of Portugal (FCT) [SFRH/BPD/66742/2009, PEst-C/MAR/LA0015/2011]; Valencian Regional Goverment [Prometeo II/2014/051]; Spanish Ministry of Science and Innovation (MICINN) [CSD 2007-0002]info:eu-repo/semantics/publishedVersio

Mapping the pattern of essential neuroendocrine cells related to puberty and VA opsin expression provides further insight in the photoreceptive regulation of the BPG axis in Atlantic salmon (Salmo salar)

In Atlantic salmon (Salmo salar), seasonal photoperiod is shown to regulate the onset of sexual maturation, yet which brain region(s) is involved, and how light information impacts the neuroendocrine system are still not fully understood in teleosts. Detailed knowledge about the photoperiodic regulation of maturation in fish is still missing. In birds, it is shown that gonadotropin-releasing hormone (Gnrh) is located in the same neurons as vertebrate ancient (VA) opsin, suggesting a direct photoreceptive regulation for the onset of sexual maturity. This study presents a comprehensive topographic mapping of gnrh2, gnrh3, kisspeptin 2 (kiss2), gonadotropin-inhibiting hormone (gnih), and VA opsin using in situ hybridization on mature Atlantic salmon brains. Neurons positive for gnrh3 are expressed in the olfactory bulb and ventral telencephalon, while gnrh2-positive neurons are located dorsally in the midbrain tegmentum. Gonadotropin-inhibiting hormone (Gnih)-expressing cell bodies are present in the ventral thalamus and extend caudally to the hypothalamus with kiss2-expressing cells appearing in a lateral position. VA opsin-positive cells are present in the telencephalon, the rostro-dorsal ring of the left habenula, the ventral thalamus, and the midbrain tegmentum. The results show no similar co-location as found in birds, hypothesizing that the photoreceptive modulation of Gnrh in salmon may interact through neuronal networks. The topography analyses of the essential neuroendocrine cells related to sexual maturation in the Atlantic salmon brain show that diencephalic (thalamus, hypothalamus) and midbrain (tegmentum) regions seem central for controlling sexual maturation.acceptedVersio

Localization of three forms of gonadotropin-releasing hormone in the brain and pituitary of the self-fertilizing fish, Kryptolebias marmoratus

The localization of gonadotropin-releasing hormone (GnRH) in the brain and pituitary of the self-fertilizing mangrove killifish Kryptolebias marmoratus was examined by immunohistochemistry and in situ hybridization to understand its neuroendocrine system. The genome assembly of K. marmoratus did not have any sequence encoding GnRH1, but sequences encoding GnRH2 (chicken GnRH-II) and GnRH3 (salmon GnRH) were found. Therefore, GnRH1 was identified by in silico cloning. The deduced amino acid sequence of the K. marmoratus GnRH1 (mature peptide) was identical to that of the medaka GnRH. GnRH1 neurons were detected in the ventral part of the preoptic nucleus by immunohistochemistry and in situ hybridization, and GnRH1-immunoreactive (ir) fibers were observed throughout the brain. GnRH1-ir fibers were in close contact with luteinizing hormone (LH)-ir cells in the pituitary using double immunohistochemistry. GnRH2 neurons were detected in the midbrain tegmentum by immunohistochemistry and in situ hybridization. Although GnRH2-ir fibers were observed throughout the brain, they were not detected in the pituitary. GnRH3 neurons were detected in the lateral part of the ventral telencephalic area by both methods. GnRH3-ir fibers were observed throughout the brain, and a few GnRH3-ir fibers were in close contact with LH-ir cells in the pituitary. These results indicate that GnRH1 and possibly GnRH3 are responsible for gonadal maturation through LH secretion and that all three forms of GnRH function as neurotransmitters or neuromodulators in the brain of K. marmoratus

THE ROLE OF DMRT GENES DURING NEUROGENESIS IN THE ZEBRAFISH FOREBRAIN

Ph.DDOCTOR OF PHILOSOPH

A Type IIb, but Not Type IIa, GnRH Receptor Mediates GnRH-Induced Release of Growth Hormone in the Ricefield Eel

Multiple gonadotropin-releasing hormone receptors (GnRHRs) are present in vertebrates, but their differential physiological relevances remain to be clarified. In the present study, we identified three GnRH ligands GnRH1 (pjGnRH), GnRH2 (cGnRH-II), and GnRH3 (sGnRH) from the brain, and two GnRH receptors GnRHR1 (GnRHR IIa) and GnRHR2 (GnRHR IIb) from the pituitary of the ricefield eel Monopterus albus. GnRH1 and GnRH3 but not GnRH2 immunoreactive neurons were detected in the pre-optic area, hypothalamus, and pituitary, suggesting that GnRH1 and GnRH3 may exert hypophysiotropic roles in ricefield eels. gnrhr1 mRNA was mainly detected in the pituitary, whereas gnrhr2 mRNA broadly in tissues of both females and males. In the pituitary, GnRHR1 and GnRHR2 immunoreactive cells were differentially distributed, with GnRHR1 immunoreactive cells mainly in peripheral areas of the adenohypophysis whereas GnRHR2 immunoreactive cells in the multicellular layers of adenohypophysis adjacent to the neurohypophysis. Dual-label fluorescent immunostaining showed that GnRHR2 but not GnRHR1 was localized to somatotropes, and all somatotropes are GnRHR2-positive cells and vice versa at all stages examined. GnRH1 and GnRH3 were shown to stimulate growth hormone (Gh) release from primary culture of pituitary cells, and to decrease Gh contents in the pituitary of ricefield eels 12 h post injection. GnRH1 and GnRH3 stimulated Gh release probably via PLC/IP3/PKC and Ca2+ pathways. These results, as a whole, suggested that GnRHs may bind to GnRHR2 but not GnRHR1 to trigger Gh release in ricefield eels, and provided novel information on differential roles of multiple GnRH receptors in vertebrates

Neural Crest and Placodal Cells Contributions to Cranial Sensory Development

The sensory system of vertebrates is incredibly complex. Many important components of the sensory system are located within the cranial region, including the sense organs and cranial sensory ganglia. Early in development two progenitor populations, the neural crest and the cranial placodes, arise at the neural plate border and throughout vertebrate development contribute to the developing vertebrate peripheral sensory system. The interactions and contributions of both of these cell populations to the development of the pituitary system, the eyes, the nose, the ears, and the cranial ganglia of the head and neck are vital for the appropriate development of an embryo’s nervous system. In this dissertation we explore the contributions of both the neural crest and placodal cells to the sensory system of the developing embryo. In Chapter 1 we review the origin of these two cell populations at the neural plate border and give an overview of the development of the various cranial peripheral sensory systems and their placode and neural crest contributions. In Chapter 2 we use replication incompetent avian retroviruses to lineage trace both the olfactory placode and the neural crest to their respective cellular contributions in the olfactory system. We confirm previous studies which showed that GnRH neurons of the nose receive contributions from both the olfactory placode and the neural crest and we show that both the olfactory placode and the neural crest contribute to the olfactory neurons of the olfactory epithelium. However, neural crest alone gives rise to the olfactory ensheathing cells which are critical for neuronal migration from the olfactory epithelium to the forebrain. We also show for the first time that the neural crest gives rise to the p63 positive horizontal basal stem cell population of the olfactory epithelium. In Chapter 3, along with collaborators from SUNY Buffalo, we show that multipotent and functional NC cells can be derived by induction with a growth factor cocktail containing FGF2 and IGF1 from cultures of human inter-follicular keratinocytes (KC) isolated from elderly donors. They also maintained their multipotency, as evidenced by their ability to differentiate into all NC-specific lineages including neurons, Schwann cells, melanocytes, and smooth muscle cells (SMC). Notably, upon implantation into chick embryos, adult NC cells behaved similar to their embryonic counterparts, migrated along stereotypical pathways, and contributed to multiple NC derivatives in ovo. These results suggest that KC-derived NC cells may provide an easily accessible, autologous source of stem cells that can be used for treatment of neurodegenerative diseases or as a model system for studying disease pathophysiology and drug development. Finally, in Chapter 4 we discuss future directions and experiments that I plan to pursue post-graduation. I propose to conduct a closer examination of the variants of GnRH neurons across developmental time in various representative taxa of cartilaginous fish and reptiles. Furthermore, I intend to identify and experimentally confirm a molecular regulatory region for GnRH2, the most highly conserved variant across vertebrates, within the chicken embryo. Once this regulatory region is identified, the sequence can also be used to probe the genomes of other non-model taxa. Finally, I would like to perform lineage analysis using DiI in a non-model system to probe the embryonic origins (neural crest vs. placode) of the GnRH neurons in more ancient taxa.</p

Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains

Fertility in mammals is controlled by hypothalamic neurons that secrete gonadotropin-releasing hormone (GnRH). These neurons differentiate in the olfactory placodes during embryogenesis and migrate from the nose to the hypothalamus before birth. Information regarding this process in humans is sparse. Here, we adapted new tissue-clearing and whole-mount immunohistochemical techniques to entire human embryos/fetuses to meticulously study this system during the first trimester of gestation in the largest series of human fetuses examined to date. Combining these cutting-edge techniques with conventional immunohistochemistry, we provide the first chronological and quantitative analysis of GnRH neuron origins, differentiation and migration, as well as a 3D atlas of their distribution in the fetal brain. We reveal not only that the number of GnRH-immunoreactive neurons in humans is significantly higher than previously thought, but that GnRH cells migrate into several extrahypothalamic brain regions in addition to the hypothalamus. Their presence in these areas raises the possibility that GnRH has non-reproductive roles, creating new avenues for research on GnRH functions in cognitive, behavioral and physiological processes

Stress-resilience differences related to emergence time in rainbow trout

In wild salmonid fish, individual behavioural traits have been suggested to be coupled with the timing of fry emergence form gravel spawning nests, in such a way that early emerging fish have shown to be more aggressive and to have a higher probability to become socially dominant than those fish emerging at a later stage. Besides aggression and dominance, other behavioural and metabolic traits such as boldness, metabolic rate or growth had also been coupled to emergence time. Altogether, early- and late-emerging fish have traits resembling those of proactive and reactive stress coping styles, respectively. Proactive fish are considered to be more resilient to stress. However, it is currently unclear if that coupling is maintained in farmed fish populations, which showed no consistent evidence of a clear relation between emergence time and stress coping style. In this study, fish were hatched and larvae were fractionated according their emergence time (Early fraction: first 20 % of fish to emerge; Intermediate fraction: mid 20 %; Late fraction: last 20 %). Several months later, the resilience against a mild stressor (30 min of high stocking density), along with the stress habituation ability was investigated in 5 g fish from the different fractions. Results showed that fish from different fractions di played a similar neuroendocrine response to a novel stressor. Interestingly, the capacity of habituation to stress was however better in the fish from the early emergence fraction, which showed no cortisol response to the stressor after being exposed daily for 15 days to another mild acute stressor (1.5 min of air exposure). These results demonstrate that at least some behavioural differences related to emergence time exist in a domesticated trout population. The aquaculture related implications of these stress resilience differences are currently under study

視床下部GnRH ニューロンを中心とした真骨魚類生殖中枢制御機構に関する神経内分泌学的研究

学位の種別:課程博士University of Tokyo(東京大学

Light Dependent Regulation of Sleep/Wake States by Prokineticin 2 in Larval Zebrafish

Sleep is an evolutionarily conserved behavior and essential to survival. The classic two process model of sleep regulation proposes that sleep results from the interaction between circadian and homeostatic processes, but the details remain elusive. Most sleep research is performed using nocturnal rodents, and diurnal vertebrates are under-represented. It is unclear whether circadian regulatory mechanisms of sleep in nocturnal animals can be directly translated into diurnal animals. In this thesis, I first briefly describe sleep behavior and the two process model of sleep regulation, focusing on the circadian process, and then discuss the advantages of using larval zebrafish as a model to study sleep behavior in diurnal vertebrates. In Chapter 2, I characterize the role of Prokineticin 2, a proposed circadian output factor in nocturnal animals, in sleep/wake regulation in larval zebrafish. I show that, similar to nocturnal rodents, Prok2 is both necessary for daytime sleep/wake behavior and sufficient to modulate sleep/wake states in a light dependent manner. However, unlike nocturnal rodents and similar to humans, Prok2 is not required for maintaining circadian rhythmicity in larval zebrafish after removing external light cue. This result demonstrates the potential functional difference of circadian output factors in different chronotypes, and establishes larval zebrafish as an alternative model for studying circadian regulation of sleep and possibly other behaviors in humans. In Chapter 3, I describe the adaptation and development of TRP channels to manipulate neuronal activity in larval zebrafish, in an effort to expand the existing repertoire of genetic tools for studying behavior in zebrafish. I show that three TRP channels, TRPV1, TRPM8 and TRPA1, can inducibly activate specific populations of neurons in larval zebrafish by using their appropriate agonists. At high agonist concentrations, TRPV1, can rapidly induce cell ablation. Adaptation of TRP channels for use in larval zebrafish expands the variety of behavioral experiments and combinatorial manipulation of neuronal activity that can be performed in zebrafish. In summary, this work deepens our understanding of sleep regulation, establishes larval zebrafish as an appropriate model for studying circadian regulation of sleep in diurnal vertebrates, and presents novel genetic tools for studying behavior in larval zebrafish