Pei-San Tsai

Fibroblast Growth Factor 8 Signaling through Fibroblast Growth Factor Receptor 1 Is Required for the Emergence of Gonadotropin-Releasing Hormone Neurons

GnRH neurons are essential for the onset and maintenance of reproduction. Mutations in both fibroblast growth factor receptor (Fgfr1) and Fgf8 have been shown to cause Kallmann syndrome, a disease characterized by hypogonadotropic hypogonadism and anosmia, indicating that FGF signaling is indispensable for the formation of a functional GnRH system. Presently it is unclear which stage of GnRH neuronal development is most impacted by FGF signaling deficiency. GnRH neurons express both FGFR1 and -3; thus, it is also unclear whether FGFR1 or FGFR3 contributes directly to GnRH system development. In this study, we examined the developing GnRH system in mice deficient in FGF8, FGFR1, or FGFR3 to elucidate the individual contribution of these FGF signaling components. Our results show that the early emergence of GnRH neurons from the embryonic olfactory placode requires FGF8 signaling, which is mediated through FGFR1, not FGFR3. These data provide compelling evidence that the developing GnRH system is exquisitely sensitive to reduced levels of FGF signaling. Furthermore, Kallmann syndrome stemming from FGF signaling deficiency may be due primarily to defects in early GnRH neuronal development prior to their migration into the forebrain.

Novel FGF8 mutations associated with recessive holoprosencephaly, craniofacial defects, and hypothalamo-pituitary dysfunction

CONTEXT Fibroblast growth factor (FGF) 8 is important for GnRH neuronal development with human mutations resulting in Kallmann syndrome. Murine data suggest a role for Fgf8 in hypothalamo-pituitary development; however, its role in the etiology of wider hypothalamo-pituitary dysfunction in humans is unknown. OBJECTIVE The objective of this study was to screen for FGF8 mutations in patients with septo-optic dysplasia (n = 374) or holoprosencephaly (HPE)/midline clefts (n = 47). METHODS FGF8 was analyzed by PCR and direct sequencing. Ethnically matched controls were then screened for mutated alleles (n = 480-686). Localization of Fgf8/FGF8 expression was analyzed by in situ hybridization in developing murine and human embryos. Finally, Fgf8 hypomorphic mice (Fgf8(loxPNeo/-)) were analyzed for the presence of forebrain and hypothalamo-pituitary defects. RESULTS A homozygous p.R189H mutation was identified in a female patient of consanguineous parentage with semilobar HPE, diabetes insipidus, and TSH and ACTH insufficiency. Second, a heterozygous p.Q216E mutation was identified in a female patient with an absent corpus callosum, hypoplastic optic nerves, and Moebius syndrome. FGF8 was expressed in the ventral diencephalon and anterior commissural plate but not in Rathkes pouch, strongly suggesting early onset hypothalamic and corpus callosal defects in these patients. This was consolidated by significantly reduced vasopressin and oxytocin staining neurons in the hypothalamus of Fgf8 hypomorphic mice compared with controls along with variable hypothalamo-pituitary defects and HPE. CONCLUSION We implicate FGF8 in the etiology of recessive HPE and potentially septo-optic dysplasia/Moebius syndrome for the first time to our knowledge. Furthermore, FGF8 is important for the development of the ventral diencephalon, hypothalamus, and pituitary.

The Emergence and Loss of Gonadotropin-Releasing Hormone in Protostomes: Orthology, Phylogeny, Structure, and Function

Abstract Gonadotropin-releasing hormone (GNRH) is a neuropeptide critical for reproductive activation and maintenance in vertebrates. The recent elucidation of molluscan GNRH-like sequences led to several important questions regarding the evolution of the GNRH family. For instance, are molluscan and chordate GNRHs true orthologs? Has GNRH been retained in most protostomian lineages? What was the function of the ancestral GNRH? The goal of this review is to provide a critical analysis of GNRH evolution based on data available from the known forms of protostomian GNRH. Judging from the orthology between chordate and protostomian GNRH receptors, conservation of several structural motifs on the GNRH peptide, and exon/intron arrangement conserved between protostomian and chordate GNRH genomic sequences, we conclude that chordate and protostomian GNRHs likely share a common ancestor. Based on our analysis of phylogenetic distribution, we also hypothesize that GNRH may have been lost in the ecdysozoan lineage but preserved in lophotrochozoans. Lastly, we propose that the ancestral function of GNRH is to serve as a general neural regulator, and its considerable specialization in reproduction seen in chordates is a consequence of neofunctionalization following gene duplication..

Expression of a Dominant Negative FGF Receptor in Developing GNRH1 Neurons Disrupts Axon Outgrowth and Targeting to the Median Eminence

Abstract During development, neurons that synthesize and release gonadotropin-releasing hormone (GNRH1) extend their axons to the median eminence (ME) to establish neurosecretory contacts necessary for hormone secretion. Signals that coordinate this process are not known, but could involve the activation of fibroblast growth factor receptors (FGFRs) expressed on developing GNRH1 neurons. Using both whole-animal and cell culture approaches, this study examines the direct role of FGFR signaling in the extension and guidance of GNRH1 axons to the ME. In vivo retrograde labeling with fluorogold (FG) first showed a significant reduction in the projections of GNRH1 axons to the circumventricular organs (including the ME) in transgenic mice expressing a dominant negative FGF receptor (dnFGFR) in GNRH1 neurons. Using a primary GNRH1 neuronal culture system, we examined if compromised axon extension and directional growth led to the reduced axon targeting efficiency seen in vivo. Primary cultures of GNRH1 neurons were established from Embryonic Day 15.5 embryos, an age when GNRH1 neurons are actively targeting the ME. Cultured GNRH1 neurons expressing dnFGFR (dnFGFR neurons) exhibited attenuated activation of signaling pathways and reduced neurite outgrowth in response to FGF2. Further, dnFGFR neurons failed to preferentially target neurites toward cocultured ME explant and FGF2-coated beads, suggesting a defect in axon pathfinding. Together, these findings describe a direct role of FGFR signaling in the elongation and guidance of GNRH1 axons to the ME.

Localization of gonadotropin-releasing hormone in the central nervous system and a peripheral chemosensory organ of Aplysia californica

Gonadotropin-releasing hormone (GnRH) is a neurohormone crucial for the regulation of reproductive and neural functions in vertebrates. Recent discoveries of GnRH immunoreactivity (IR) in a number of invertebrates raised the possibility that GnRH may be an ancient molecule that had arisen before the emergence of Phylum Chordata. We previously demonstrated the presence of a GnRH IR similar to the mammalian (m) and tunicate I (tI) forms of GnRH in the hemolymph and ovotestis of an opisthobranch mollusk, Aplysia californica; however, the presence of GnRH in the central nervous system (CNS) of A. californica could not be detected with the available antisera against various forms of chordate GnRH. In the present study, we performed immunohistochemistry (IHC) to localize the presence of GnRH in the CNS and a peripheral chemosensory organ, the osphradium, of A. californica. A newly generated antiserum against tI-GnRH revealed the strong expression of GnRH IR in neurons of all CNS ganglia. A notable asymmetry in immunostaining was detected in the left and right abdominal hemiganglia. The CNS is rich in tI-GnRH immunoreactive neurons but lacks mGnRH IR, whereas the osphradium contains abundant mGnRH immunoreactive neurons but lacks tI-GnRH IR. The extract of CNS failed to stimulate the release of LH from mouse pituitary, demonstrating that the A. californica GnRH IR is structurally different from what is required to bind and activate mammalian GnRH receptor. Together, these results indicate the presence of at least two distinct GnRH systems in A. californica. The presence of GnRH in the osphradium is consistent with the long-standing anatomical relationship between GnRH and the chemosensory system observed in vertebrates.

Differential distribution of chicken-I and chicken-II GnRH in the turtle brain.

Chicken-I and chicken-II gonadotropin-releasing hormone (cI-GnRH and cII-GnRH) were shown to be differentially distributed in the brain of a turtle, Trachemys scripta, by HPLC and specific radioimmunoassays. The cI-GnRH was most concentrated in the median eminence (ME), while cII-GnRH was most concentrated in the caudal brain regions, especially medulla and cerebellum. The ratio of cI- to cII-GnRH in the ME of adults was 8:1. Age- and sex-related differences in GnRH concentrations were observed exclusively in the ME: adult females had significantly higher cI-GnRH than younger females and adult males; adult females also had significantly higher cII-GnRH than hatching females. Their differential distribution and sex- and age-related differences suggest that the two peptides may have distinct physiological roles; cI-GnRH is likely the form responsible for stimulating gonadotropin release.

Abnormal hypothalamic oxytocin system in fibroblast growth factor 8-deficient mice

Oxytocin (OT) is a nonapeptide essential for maternal care. The development of the OT neuroendocrine system is a multi-step process dependent on the action of many transcription factors, but upstream signaling molecules regulating this process are still poorly understood. In this study, we examined if fibroblast growth factor 8 (FGF8), a signaling molecule critical for forebrain development, is essential for the proper formation of the OT system. Using immunohistochemistry, we showed a significant reduction in the number of neurons immunoreactive for the mature OT peptide in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) in the hypothalamus of homozygous (HOMO) FGF8 hypomorphic mice compared to wild-type mice. The number of neurons positive for oxyphysin prohormone in the SON but not the PVN was also significantly reduced in FGF8 HOMO hypomorphs. However, steady-state mRNA levels of the oxyphysin prohormone were not significantly different between FGF8 hypomorphs and WT mice. These data suggest that a global reduction in FGF8 signaling leads to an overall reduction of mature OT and oxyphysin prohormone levels that may have resulted from defects in multiple stages of the hormone-synthesis pathway. Since proper hormone synthesis is a hallmark of mature OT neurons, this study suggests that FGF8 signaling may contribute to the phenotypic maturation of a neuroendocrine system that originates within the diencephalon.

Gonadotropin-releasing hormone in protostomes: insights from functional studies on Aplysia californica.

Several protostomian molecules that structurally resemble chordate gonadotropin-releasing hormone (GnRH) have been identified through cloning, biochemical purification or data mining. These molecules share considerable sequence and structural similarities with chordate GnRH, leading to the current belief that protostomian and chordate forms of GnRH share a common ancestor. However, the physiological significance of these protostomian GnRH-like molecules remains poorly understood. This knowledge gap hampers our understanding of how GnRH has evolved functionally over time. This review provides a summary of our recent functional characterization of a GnRH-like molecule (ap-GnRH) in a gastropod mollusk, Aplysia californica, and presents preliminary proof for a cognate ap-GnRH receptor (ap-GnRHR). Our data reveal that ap-GnRH is a general neural regulator capable of exerting diverse central and motor effects, but plays little or no role in reproductive activation. This notion is supported by the abundance of a putative ap-GnRHR transcript in the central nervous system and the foot. Comparing these results to the available functional data from a cephalopod mollusk, Octopus vulgaris, we surmise that protostomian GnRH-like molecules are likely to assume a wide range of physiological roles, and reproductive activation is not an evolutionarily conserved role of these molecules. Future functional studies using suitable protostomian models are required to identify functional changes in protostomian GnRH-like molecules that accompany major taxa-level transitions.

Role of fibroblast growth factor signaling in gonadotropin-releasing hormone neuronal system development.

There is growing evidence demonstrating that fibroblast growth factor (FGF) signaling is important for the development of the gonadotropin-releasing hormone (GnRH) neuronal system. In humans, loss-of-function mutations in FGF receptor 1 (Fgfr1) and Fgf8 lead to hypogonadotropic hypogonadism (HH) with or without anosmia. Insights into how FGF signaling deficiency disrupts the GnRH system in humans are beginning to emerge from studies using transgenic mouse models. In this review, we summarize GnRH system defects in several lines of FGF signaling-deficient mice. We showed that FGF signaling is critically required for olfactory placode induction, differentiation, and GnRH neuronal fate specification and postnatal maintenance. Extrapolating from these transgenic mouse data, we suggest that idiopathic HH in patients harboring loss-of-function Fgfr1 and/or Fgf8 mutations is not merely a result of defective GnRH neuronal migration, but also insults accumulated in the GnRH system during fate specification and postnatal development.

Epidermal growth factor receptor levels in reproductive organs of female mice exposed neonatally to diethylstilbestrol.

Abstract Binding of epidermal growth factor (EGF) to membrane preparations of vagina, uterus, ovary, oviduct, and liver was examined in mice treated neonatally with diethylstilbestrol (DES) and compared with that in untreated mice. Binding in the vagina (12.5 ± 0.73 fmol/mg protein) was somewhat higher than in the uterus (8.0 ± 0.34 fmol/mg protein). Level of specific binding was of the order: liver (18.4 ± 1.09 and 16.0 ± 1.53 fmol/mg protein) > vagina (12.5 ± 0.73 and 8.2 ± 0.57 fmol/mg protein) > uterus (8.0 ± 0.34 and 6.8 ± 0.56 fmol/mg protein) > ovary (6.8 ± 0.36 and 8.0 ± 1.05 fmol/mg protein) > oviduct (2.1 ± 0.32 and 1.7 ± 0.05 fmol/mg protein) in control and neonatally DES-exposed mice, respectively. Thus, neonatal DES exposure significantly lowered the binding site level only in the vagina, without modifying the binding affinity (Kd = 5.4 × 10-9 M in controls vs 4.6 × 10-9 M in DES-exposed mice). Reduction of EGF receptor level in the vagina correlates with ovary-independent persistent proliferation and keratinization of the vagina induced by neonatal DES exposure. EGF receptors were immunohistochemically demonstrated in epithelial cells of vagina, uterus, and oviduct and in stromal cells in uterus and oviduct using a polyclonal antibody to human EGF receptor protein.