TTF-1, a homeodomain gene required for diencephalic morphogenesis, is postnatally expressed in the neuroendocrine brain in a developmentally regulated and cell-specific fashion.

TTF-1 is a member of the Nkx family of homeodomain genes required for morphogenesis of the hypothalamus. Whether TTF-1, or other Nkx genes, contributes to regulating differentiated hypothalamic functions is not known. We now report that postnatal hypothalamic TTF-1 expression is developmentally regulated and associated with the neuroendocrine process of female sexual development. Lesions of the hypothalamus that cause sexual precocity transiently activate neuronal TTF-1 expression near the lesion site. In intact animals, hypothalamic TTF-1 mRNA content also increases transiently, preceding the initiation of puberty. Postnatal expression of the TTF-1 gene was limited to subsets of hypothalamic neurons, including LHRH neurons, which control sexual maturation, and preproenkephalinergic neurons of the lateroventromedial nucleus of the basal hypothalamus, which restrain sexual maturation and facilitate reproductive behavior. TTF-1 mRNA was also detected in astrocytes of the median eminence and ependymal/subependymal cells of the third ventricle, where it colocalized with erbB-2, a receptor involved in facilitating sexual development. TTF-1 binds to and transactivates the erbB-2 and LHRH promoters, but represses transcription of the preproenkephalin gene. The singular increase in hypothalamic TTF-1 gene expression that precedes the initiation of puberty, its highly specific pattern of cellular expression, and its transcriptional actions on genes directly involved in neuroendocrine reproductive regulation suggest that TTF-1 may represent one of the controlling factors that set in motion early events underlying the central activation of mammalian puberty.

Direct effects of nerve growth factor on thecal cells from antral ovarian follicles.

TrkA, the nerve growth factor (NGF) tyrosine kinase receptor, is expressed not only in the nervous system, but also in nonneural cells, including discrete cellular subsets of the endocrine and immune system. In the rat ovary, trkA receptor abundance increases strikingly in thecal-interstitial cells during the hours preceding the first ovulation. Blockade of either trkA transducing capacity or NGF biological activity inhibited ovulation, suggesting a role for NGF in the ovulatory process of this species. To identify some of the processes that may be affected by trkA activation in the thecal compartment, we used purified thecal cells/thecal fibroblasts from bovine ovaries (heretofore referred to as thecal cells). Ribonuclease protection assays employing bovine-specific cRNA probes demonstrated the presence of the messenger RNAs (mRNAs) encoding NGF and its receptors, p75 NTR and trkA, in the thecal compartment of small, medium, and large antral follicles and showed that trkA mRNA is also expressed in granulosa cells. In situ hybridization and immunohistochemical examination of intact ovaries confirmed these cellular sites of NGF and trkA synthesis. TrkA mRNA, but not NGF mRNA, was lost within 48 h of placing thecal cells in culture. Thus, to study trkA-mediated actions of NGF on these cells we transiently expressed the receptor by transfection with a vector containing a full-length rat trkA complementary DNA under transcriptional control of the cytomegalovirus promoter. Because ovulation is preceded by an LH-dependent increase in androgen and progesterone production, the ability of NGF to modify the release of these steroids was determined in freshly plated cells still containing endogenous trkA receptors and in cells undergoing luteinization in culture that were transiently transfected with the trkA-encoding plasmid. NGF stimulated both androgen and progesterone release in freshly plated thecal cells, but not in luteinizing cells provided with trkA receptors. As ovulation in rodents requires an increased formation of PGE2 and has been shown to be antedated by proliferation of thecal fibroblasts, we determined the ability of NGF to affect these parameters in trkA-transfected thecal cells. The neurotrophin rapidly stimulated PGE2 release and amplified the early steroidal response to hCG in trkA-expressing cells, but not in cells lacking the receptor. Likewise, NGF stimulated [3H]thymidine incorporation into trkA-containing cells, but not into cells that had lost the receptor in culture. Induction of ovulation in immature rats by gonadotropin treatment verified that an increased cell proliferation in the thecal compartment, determined by the incorporation of bromodeoxyuridine into cell nuclei, occurs 4-5 h before ovulation in this species. These results suggest that the contribution of NGF to the ovulatory process includes a stimulatory effect of the neurotrophin on steroidogenesis, PGE2 formation, and proliferative activity of thecal compartment cells.

Intraovarian excess of nerve growth factor increases androgen secretion and disrupts estrous cyclicity in the rat.

A single injection of estradiol valerate induces a form of cystic ovary resembling some aspects of the human polycystic ovarian syndrome. Preceding the development of follicular cysts, there is an increase in intraovarian synthesis of nerve growth factor (NGF) and the low affinity NGF receptor (p75 NGFR). Selective blockade of NGF actions and p75 NGFR synthesis in the ovary restored estrous cyclicity and ovulatory capacity in estradiol valerate-treated rats, suggesting that an increase in NGF-dependent, p75 NGFR-mediated actions within the ovary contributes to the development of cystic ovarian disease. We have tested this hypothesis by grafting NGF-producing neural progenitor cells into the ovary of juvenile rats that have been induced to ovulate precociously by a single injection of PMSG. The NGF-producing cells, detected by their content of immunoreactive p75 NGFR material, were found scattered throughout the ovary with some of them infiltrating the granulosa cell compartment of large, precystic follicles. Ovarian NGF content was 2-fold higher than in the ovary of rats receiving control cells. Estrous cyclicity was disrupted, with the animals showing prolonged periods of persistent estrus, and an almost continuous background of vaginal cornified cells at other phases of the estrous cycle. Morphometric analysis revealed that the presence of NGF-producing cells neither reduced the total number of corpora lutea per ovary nor significantly increased the formation of follicular cysts. However, the ovaries receiving these cells showed an increased incidence of precystic, type III follicles, accompanied by a reduced number of healthy antral follicles, and an increased size of both healthy and atretic follicles. These changes in follicular dynamics were accompanied by a selective increase in serum androstenedione levels. The results show that an abnormally elevated production of NGF within the ovary suffices to initiate several of the structural and functional alterations associated with the development of follicular cysts in the rat ovary.

Neuregulins signaling via a glial erbB-2-erbB-4 receptor complex contribute to the neuroendocrine control of mammalian sexual development.

Activation of erbB-1 receptors by glial TGFalpha has been shown to be a component of the developmental program by which the neuroendocrine brain controls mammalian sexual development. The participation of other members of the erbB family may be required, however, for full signaling capacity. Here, we show that activation of astrocytic erbB-2/erbB-4 receptors plays a significant role in the process by which the hypothalamus controls the advent of mammalian sexual maturation. Hypothalamic astrocytes express both the erbB-2 and erbB-4 genes, but no erbB-3, and respond to neuregulins (NRGs) by releasing prostaglandin E(2) (PGE(2)), which acts on neurosecretory neurons to stimulate secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual development. The actions of TGFalpha and NRGs in glia are synergistic and involve recruitment of erbB-2 as a coreceptor, via erbB-1 and erbB-4, respectively. Hypothalamic expression of both erbB-2 and erbB-4 increases first in a gonad-independent manner before the onset of puberty, and then, at the time of puberty, in a sex steroid-dependent manner. Disruption of erbB-2 synthesis in hypothalamic astrocytes by treatment with an antisense oligodeoxynucleotide inhibited the astrocytic response to NRGs and, to a lesser extent, that to TGFalpha and blocked the erbB-dependent, glia-mediated, stimulation of LHRH release. Intracerebral administration of the oligodeoxynucleotide to developing animals delayed the initiation of puberty. Thus, activation of the erbB-2-erbB-4 receptor complex appears to be a critical component of the signaling process by which astrocytes facilitate the acquisition of female reproductive capacity in mammals.

The Oct-2 POU domain gene in the neuroendocrine brain: a transcriptional regulator of mammalian puberty.

POU homeodomain genes are transcriptional regulators that control development of the mammalian forebrain. Although they are mostly active during embryonic life, some of them remain expressed in the postnatal hypothalamus, suggesting their involvement in regulating differentiated functions of the neuroendocrine brain. We show here that Oct-2, a POU domain gene originally described in cells of the immune system, is one of the controlling components of the cell-cell signaling process underlying the hypothalamic regulation of female puberty. Lesions of the anterior hypothalamus cause sexual precocity and recapitulate some of the events leading to the normal initiation of puberty. Prominent among these events is an increased astrocytic expression of the gene encoding transforming growth factor-alpha (TGF alpha), a tropic polypeptide involved in the stimulatory control of LHRH secretion. The present study shows that such lesions result in the rapid and selective increase in Oct-2 transcripts in TGF alpha-containing astrocytes surrounding the lesion site. In both lesion-induced and normal puberty, there is a preferential increase in hypothalamic expression of the Oct-2a and Oct-2c alternatively spliced messenger RNA forms of the Oct-2 gene, with an increase in 2a messenger RNA levels preceding that in 2c and antedating the peripubertal activation of gonadal steroid secretion. Both Oct-2a and 2c trans-activate the TGF alpha gene via recognition motifs contained in the TGF alpha gene promoter. Inhibition of Oct-2 synthesis reduces TGF alpha expression in astroglial cells and delays the initiation of puberty. These results suggest that the Oct-2 gene is one of the upstream components of the glia to neuron signaling process that controls the onset of female puberty in mammals.

Progress toward positional cloning of ovine neuronal ceroid lipofuscinosis, a model of the human late-infantile variant CLN6.

The neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage diseases with severe neurodegenerative pathology. An ovine model (OCL) has well-defined parallels with the human disease at the biochemical and pathological levels. The gene for OCL is located in the chromosomal region OAR7q13-15. This region is syntenic with HAS15q21-23, suggesting that OCL and CLN6 represent mutations in orthologous genes. New microsatellite markers have been developed enabling further refinement of the OCL critical region.

Ovine neuronal ceroid lipofuscinosis: a large animal model syntenic with the human neuronal ceroid lipofuscinosis variant CLN6.

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited degenerative neurological diseases affecting children. A number of non-allelic variants have been identified within the human population and the genes for some of these have recently been identified. The underlying mechanism for the neuropathology remains an enigma; however, pioneering studies with the naturally occurring ovine model (OCL) have led to the proposal that these diseases represent lesions in specific hydrophobic protein degradation pathways. In this study, we show linkage between OCL and microsatellite markers on OAR 7q13-15. Using interspecies chromosome painting we establish that OAR 7q13-15 is syntenic with human chromosome 15q21-23, the region which was recently defined as the location of a newly identified late infantile variant (CLN6). We propose that our ovine model represents a mutation in the gene orthologous to that mutated in the human late infantile variant CLN6. The ovine linkage flock, consisting of 56 families, represents a powerful resource for positional cloning of this NCL gene. The availability of such a large animal model will have important implications for experimentation in downstream corrective therapies.

Genetic variation within the ovine cystic fibrosis transmembrane conductance regulator gene.

We report here the results of a preliminary screening programme to identify natural mutations in the ovine cystic fibrosis transmembrane conductance regulator (CFTR) gene. Nine regions of the ovine CFTR gene were screened, corresponding to human CFTR gene exons 4, 6b, 7, 9, 10, 11, 12, 17b and 20. DNA samples from up to 2000 individual sheep were examined by single-stranded conformation polymorphism (SSCP) of each exon. In addition to the mutation (R297Q) reported previously, we have found several interesting variants, including intronic DNA variants and exonic polymorphisms.

Use of the Indian muntjac idiogram to align conserved chromosomal segments in sheep and human genomes by chromosome painting.

We have hybridized all 28 chromosome-specific painting probes from the domestic sheep (Ovis aries, 2n = 54) onto metaphase chromosomes of the Indian muntjac deer (Muntiacus muntjak vaginalis, 2n = 6,7) and identified 35 conserved chromosomal segments. Results from this study show that most of the sheep acrocentric chromosomes hybridized to single regions in the Indian muntjac genome. This conserved hybridization pattern supports the concept that the large Indian muntjac chromosomes were derived from multiple tandem fusions from an ancestral deer species. Using previously reported fluorescence in situ hybridization data in which human chromosomes were hybridized onto the Indian muntjac genome, we were able to align chromosomal segments of the sheep and human genomes. Using this three-species genome alignment approach, we have identified a minimum of 42 conserved chromosomal segments between sheep and human genomes including 7 new regions not previously reported.

Isolation of chromosome-specific paints from high-resolution flow karyotypes of the sheep (Ovis aries).

High-resolution bivariate flow karyotypes were obtained using fibroblast cell lines from a sheep with a normal karyotype (2n = 54), from sheep carrying Robertsonian translocation chromosomes and from sheep-hamster somatic cell hybrids. By taking advantage of the presence of chromosome polymorphisms, translocation chromosomes and sheep-hamster somatic cell hybrids, all sheep chromosomes were isolated by flow sorting. Chromosome-specific paints were generated from each sorted peak using degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The sheep chromosome present in each peak was identified by chromosome-specific microsatellite analysis of the DOP-PCR products and fluorescence in situ hybridization (FISH) onto DAPI-banded sheep metaphase chromosomes. The chromosome-specific DNA obtained in this study can be used for the production of genomic libraries and as a resource for mapping randomly cloned DNA sequences that will greatly aid the construction of genetic and physical maps in the sheep. The chromosome-specific paints will facilitate chromosome identification and contribute to the study of karyotype evolution in the sheep and related species.

Targeting transforming growth factor alpha expression to discrete loci of the neuroendocrine brain induces female sexual precocity.

Precocious puberty of cerebral origin is a poorly understood disorder of human sexual development, brought about by the premature activation of those neurons that produce luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual maturation. An increased production of transforming growth factor alpha (TGF alpha) in the hypothalamus has been implicated in the mechanism underlying both normal and precocious puberty. We have now used two gene delivery systems to target TGF alpha overexpression near LHRH neurons in immature female rats. Fibroblasts infected with a retroviral construct in which expression of the human TGF alpha gene is constitutively driven by the phosphoglycerate kinase promoter, or transfected with a plasmid in which TGF alpha expression is controlled by an inducible metallothionein promoter, were transplanted into several regions of the hypothalamus. When the cells were in contact with LHRH nerve terminals or in the vicinity of LHRH perikarya, sexual maturation was accelerated. These results suggest that precocious puberty of cerebral origin may result from a focal disorder of TGF alpha production within the confines of the LHRH neuron microenvironment.

Involvement of nerve growth factor in the ovulatory cascade: trkA receptor activation inhibits gap junctional communication between thecal cells.

Activation of trkA, the nerve growth factor (NGF) tyrosine kinase receptor, has been recently implicated in the process of mammalian ovulation. During the hour preceding follicular rupture, a marked increase in trkA and NGF gene expression occurs in thecal-interstitial cells of the ovary. Immunoneutralization of NGF actions or pharmacological blockade of trkA transducing activity inhibits ovulation, suggesting that activation of the NGF-trkA complex in nonneural cells of the periovulatory follicle is a physiological component of the ovulatory cascade. As thecal cells of Graafian follicles are functionally coupled by gap junctions, and the ovulatory rupture requires dissociation of thecal cell-cell communication, we sought to determine whether NGF affects the integrity of this communication. We now report that NGF-induced activation of trkA receptors in isolated ovarian thecal cells disrupts cell to cell communication by affecting the functional integrity of gap junctions. Bovine thecal cells expressing trkA receptors, but not cells lacking the receptors, respond to NGF with a reduction in the transfer of calcein, a fluorescent dye that passes through gap junctions. This effect was associated with a rapid (10-30 min) increase in serine phosphorylation of connexin-43, the main protein constituent of gap junctions in the ovary. The reduction in dye transfer was not observed when the cells were exposed to epidermal growth factor or other neurotrophins, including neurotrophin 3, neurotrophin 4, and brain-derived neurotrophic factor. Thus, cell-specific activation of trkA receptors in periovulatory follicles may provide one of the signals involved in inducing the cellular dissociation of the follicular wall that precedes ovulatory rupture.

A linkage map of the ovine X chromosome.

A genetic linkage map of the ovine X chromosome containing type I and type II markers has been constructed. The map contains 7 known gene markers and 14 microsatellite markers with a recombination length of 141.9 cM. Segregation of polymorphic markers was observed in a three-generation pedigree containing 480 animals. The maximum number of informative meioses was 912. Additional information was obtained for some markers by following segregation in the AgResearch International Mapping Flock, consisting of nine three-generation full-sib pedigrees. A pseudoautosomal region containing two markers has been identified at one end of the linkage map. Comparisons with mouse and human X chromosomes confirms the observation of Ohno (1973) that the gene content of the mammalian X chromosome is retained. In particular, the conserved grouping of the genes PHKA1, ATP7A, and XIST observed in both the human and the mouse X chromosome appears to be conserved in the sheep X chromosome, and XIST has been mapped to near the center of the chromosome. This study provides the first reported genetic linkage map combining both type I and type II markers for any ruminant X chromosome.

An ovine CFTR variant as a putative cystic fibrosis causing mutation.

This report describes a DNA variant in the ovine cystic fibrosis transmembrane conductance regulator (CFTR) gene that has been previously reported as a putative cystic fibrosis causing mutation in humans. The variant is a guanine to adenine base change at position 1019 of the ovine CFTR cDNA, corresponding to an arginine (R) to glutamine (Q) amino acid substitution at position 297 in the predicted CFTR polypeptide. The equivalent R297Q mutation in exon 7 of the human CFTR gene has been reported in a CF patient. This is the first putative cystic fibrosis mutation to be detected in another animal species.

Basic fibroblast growth factor regulates the conversion of pro-luteinizing hormone-releasing hormone (Pro-LHRH) to LHRH in immortalized hypothalamic neurons.

Growth factors are commonly associated with the regulation of cellular proliferation and differentiation. In established cells, growth factors can also serve as trophic agents. Immortalized LHRH neurons contain basic fibroblast growth factor (bFGF) receptors. Although these receptors are coupled to activation of protein kinase C, and phorbol esters are strong activators of protein kinase C-stimulated LHRH release, bFGF did not influence LHRH secretion from these cells. To clarify this discrepancy, the effects of bFGF and phorbol ester on pro-LHRH biosynthesis, protein processing, and secretion were examined in GT1-7 cells. Phorbol ester stimulated LHRH secretion, whereas bFGF either had no effect or stimulated LHRH release depending upon the antiserum used. Pro-LHRH levels in lysate and medium were depressed by phorbol esters; concentrations in bFGF-treated cells were somewhat lower than those in unstimulated controls. HPLC analyses revealed that both agents enhanced the release of LHRH intermediate products into the medium. C-Terminally extended forms of LHRH, especially LHRH-[Gly11], were prominent in medium from bFGF-stimulated neurons. Levels of LHRH were depressed relative to those in the control or phorbol ester groups. These data indicate that phorbol esters control the biosynthesis, secretion, and, to some extent, processing of pro-LHRH. The effects of bFGF are novel because this factor regulates processing of the prohormone so that LHRH-intermediate products are predominantly secreted instead of LHRH. By enhancing the secretion of these intermediates over that of LHRH, bFGF can control the biological activity of the decapeptide and regulate LHRH neuronal function.

Neural and glial-mediated effects of growth factors acting via tyrosine kinase receptors on luteinizing hormone-releasing hormone neurons.

It is becoming increasingly evident that the secretory activity of LHRH neurons is regulated not only by transsynaptic inputs but also by trophic molecules of glial and neuronal origin. The present experiments were undertaken to gain insights into the potential cell-cell mechanisms by which basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF alpha), two growth factors produced in the hypothalamus, may affect LHRH neuronal function. Northern blot analysis showed that the LHRH-producing cell line GT1-7 contains the messenger RNA (mRNA) encoding the type 1 fibroblast growth factor receptor (FGFR-1) but not that encoding the epidermal growth factor (EGF) receptors, which mediates the biological actions of both TGF alpha and EGF. Ligand-induced receptor phosphorylation experiments demonstrated that GT1-7 cells possess biologically active FGFR-1s but not EGF receptors. Exposure of the cells to bFGF resulted not only in FGFR-1 tyrosine phosphorylation, but also in tyrosine phosphorylation of phospholipase C gamma, one of the initial enzymes in the intracellular signaling cascade initiated by FGFR activation. GT1-7 cells proliferated in response to this activation. Despite the presence of biologically active receptors, bFGF did not significantly stimulate release of the mature LHRH decapeptide. Instead, bFGF increased the steady-state levels of the mRNA encoding the LHRH precursor processing endoprotease PC2, with a time course comparable to that of phorbol esters, suggesting that, as shown in the companion paper, the actions of the growth factor on LHRH neurons involve facilitation of the initial step in LHRH prohormone processing. The increase in PC2 gene expression was not accompanied by changes in LHRH mRNA levels. Unlike these direct actions of bFGF on GT-1 cells, TGF alpha appears to act indirectly via astroglial intermediacy. Exposure of GT1-7 cells to TGF alpha or EGF failed to affect several parameters of cellular activity including LHRH release, LHRH and PC2 mRNA levels, and cell proliferation. In contrast, astrocyte culture medium conditioned by treatment with TGF alpha led to sustained stimulation of LHRH release with no changes in LHRH gene expression and a transient increase in PC2 mRNA levels. Although no definitive evidence for the presence of FGFR-1 in normal LHRH neurons could be obtained by either double immunohistochemistry or double in situ hybridization procedures, fetal LHRH neurons in primary culture responded to bFGF with neurite outgrowth. Thus, normal LHRH neurons may have an FGFR-1 content too low for detection by regular histochemical procedures, and/or detectable expression of the receptor may be confined to a much earlier developmental stage. The mitogenic effect of bFGF on GT1-7 cells supports this possibility and suggests a role for FGF in the cell proliferation events that precede acquisition of the LHRH neuronal phenotype. It appears that once this phenotype is established, bFGF may promote the differentiation of LHRH neurons. The results also suggest that the secretory capacity of LHRH neurons develops under a dual trophic influence, one on peptide processing exerted directly by bFGF on early neurons, and another on LHRH release, exerted by TGF alpha via the intermediacy of astroglial cells.

A role for trkA nerve growth factor receptors in mammalian ovulation.

Several members of the neurotrophin (NT) family, including nerve growth factor (NGF), NT-3, and NT-4/5, are expressed in the mammalian ovary. As their respective receptor tyrosine kinases are also found in the gland, the possibility exists that NTs act directly on the gonads to exert effects unrelated to their support of the ovarian innervation. We now report that trkA, the NGF receptor tyrosine kinase, is involved in the acute activational process that leads to the first ovulation. The trkA gene becomes transiently expressed in periovulatory follicules at the time of the first preovulatory surge of gonadotropins at puberty; the increase in trkA messenger RNA (mRNA) content is dramatic ( > 100-fold), but transient (approximately 9 h). No such changes in trkB or trkC mRNA were observed; the abundance of these mRNAs, which encode the receptor tyrosine kinase for NT-4/5 and brain-derived neurotrophic factor, and NT-3, respectively, remained at very low levels throughout puberty. In vivo and in vitro experiments demonstrated that the activation of trkA gene expression is brought about by the proestrous discharge of LH. The increase in trkA mRNA levels is mainly localised to cells of the follicular wall and interstitial tissue of the ovary. NGF mRNA abundance also increases at proestrus, with peak values detected about 5 h before ovulation; as in the case of trkA mRNA, NGF mRNA was found in thecal-interstitial cells. Both trkA and NGF protein, detected by immunohistochemistry, were localized to this same ovarian compartment. Interleukin-1 beta (IL-1 beta), a putative mediator of LH action, enhances both trkA and NGF gene expression in ovarian cells, an effect prevented by IL-1ra, a natural IL-1 beta receptor antagonist. Il-1 beta also stimulates PGE2 release, and this effect was inhibited by both NGF antibodies and a trk receptor blocker, NGF antibodies administered in vivo attenuated the increase in ovarian PGE2 synthesis that antedates ovulation. Immunoneutralization of NGF action or pharmacological blockade of trk tyrosine kinase activity targeted to one ovary resulted in the ipsilateral inhibition of ovulation. The remarkably narrow time frame of trkA gene activation at the completion of follicular growth suggests that NGF acting as a neuroendocrinotrophic factor in a developmentally restricted manner contributes to the acute cytodifferentiation process that leads to the first ovulation in mammals.

Genetic and physical mapping of the ovine cystic fibrosis gene.

Two yeast artificial chromosomes (YACs 48B6 and 88F7) that span the ovine cystic fibrosis transmembrane conductance regulator (CFTR) gene locus have been isolated. These YAC clones have been physically assigned to the interval 4q23-->q25 on sheep chromosome 4, using fluorescence in situ hybridization. A sequence polymorphism in exon 7 of the CFTR gene was identified in Merino sheep and present at a low frequency. This polymorphism segregated in several flocks. Linkage analysis confirmed the location of CFTR on sheep chromosome 4. The most likely location is within a 13.7-cM region close to markers MAF70 and TGLA116.

Sheep linkage mapping: RFLP markers for comparative mapping studies.

Restriction fragment length polymorphisms (RFLPs) detected using cDNA probes for conserved genes provide an important set of markers that anchor or link syntenic groups in a range of divergent mammalian species. DNA probes from sheep, cattle, pig, human and mouse were screened against sheep DNA samples and 24 new RFLP markers for sheep were identified. Among the loci tested, 22 had a homologue that has been mapped in humans. An RFLP for fibronectin (FN1) was linked to alpha-inhibin (INHA) at a distance of 5cM. The FN1 locus has been assigned to sheep chromosome 2q41-q44 and linkage between FN1 and INHA assigns INHA to the same chromosome in sheep. In addition to the new loci reported here, 28 RFLPs have been published previously by this group and these are collated together with RFLPs published from other laboratories. RFLPs have been reported for 86 loci in sheep. Fifty-four loci have been mapped to 16 different chromosomes.

Neurotrophins and the neuroendocrine brain: different neurotrophins sustain anatomically and functionally segregated subsets of hypothalamic dopaminergic neurons.

Hypothalamic neurons control a variety of important hormonal and behavioral functions. Little is known, however, about the neurotrophic factors that these neurons may require for survival and/or maintenance of their differentiated functions. We conducted experiments to examine this issue, utilizing a combination of immunohistochemical, in situ hybridization and cell culture approaches. We found that the low affinity receptor for nerve growth factor (p75 NGFR) is present in small subsets of hypothalamic peptidergic neurons identified as such by their content of galanin, luteinizing hormone-releasing hormone (LHRH) and vasointestinal peptide (VIP). More prominently, however, examination of hypothalamic dopaminergic (DA) neurons for the presence of p75 NGFR-like immunoreactivity revealed that the receptor was present on tyrosine hydroxylase (TH)-positive neurons of the zona incerta and periventricular region, but not on neuroendocrine DA neurons of the tuberoinfundibular region. In situ hybridization experiments using a p75 NGFR cRNA confirmed this distribution. Regardless of the presence or absence of p75 NGFR, neither DA group expresses trkA mRNA, indicating that these two major hypothalamic subsets of DNA neurons are NGF-insensitive. A substantial fraction of TH mRNA-positive cells in the zona incerta expresses trkB mRNA, which encodes the receptor for brain derived neurotrophic factor (BDNF); in turn BDNF supports the in vitro survival of hypothalamic TH neurons bearing p75-NGFR, suggesting that BDNF is trophic for DNA neurons of the zona incerta. In contrast, tuberoinfundibular DA neurons do not express trkB mRNA, but some have trkC mRNA, which encodes the receptor for neurotrophin-3 (NT-3). The in vitro survival of TH neurons devoid of p75-NGFR is supported by NT-3, implying that NT-3 may be trophic for a subset of tuberoinfundibular DA neurons. These results suggest that, in spite of expressing an identical neurotransmitter phenotype, anatomically and functionally segregated DA neurons of the neurodendocrine brain are sustained by different neurotrophic factors.