Ptf1a/Rbpj complex inhibits ganglion cell fate and drives the specification of all horizontal cell subtypes in the chick retina.

During development, progenitor cells of the retina give rise to six principal classes of neurons and the Müller glial cells found within the adult retina. The pancreas transcription factor 1 subunit a (Ptf1a) encodes a basic-helix-loop-helix transcription factor necessary for the specification of horizontal cells and the majority of amacrine cell subtypes in the mouse retina. The Ptf1a-regulated genes and the regulation of Ptf1a activity by transcription cofactors during retinogenesis have been poorly investigated. Using a retrovirus-mediated gene transfer approach, we reported that Ptf1a was sufficient to promote the fates of amacrine and horizontal cells from retinal progenitors and inhibit retinal ganglion cell and photoreceptor differentiation in the chick retina. Both GABAergic H1 and non-GABAergic H3 horizontal cells were induced following the forced expression of Ptf1a. We describe Ptf1a as a strong, negative regulator of Atoh7 expression. Furthermore, the Rbpj-interacting domains of Ptf1a protein were required for its effects on cell fate specification. Together, these data provide a novel insight into the molecular basis of Ptf1a activity on early cell specification in the chick retina.

A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX.

cDNA and genomic clones encoding alpha 7, a novel neuronal nicotinic acetylcholine receptor (nAChR) alpha subunit, were isolated and sequenced. The mature alpha 7 protein (479 residues) has moderate homology with all other alpha and non-alpha nAChR subunits and probably assumes the same transmembrane topology. alpha 7 transcripts transiently accumulate in the developing optic tectum between E5 and E16. They are present in both the deep and the superficial layers of E12 tectum. In Xenopus oocytes, the alpha 7 protein assembles into a homo-oligomeric channel responding to acetylcholine and nicotine. The alpha 7 channel desensitizes very rapidly, rectifies strongly above -20 mV, and is blocked by alpha-bungarotoxin. A bacterial fusion protein encompassing residues 124-239 of alpha 7 binds labeled alpha-bungarotoxin. We conclude that alpha-bungarotoxin binding proteins in the vertebrate nervous system can function as nAChRs.

Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida.

The reproductive development of alligators from a contaminated and a control lake in central Florida was examined. Lake Apopka is adjacent to an EPA Superfund site, listed due to an extensive spill of dicofol and DDT or its metabolites. These compounds can act as estrogens. Contaminants in the lake also have been derived from extensive agricultural activities around the lake that continue today and a sewage treatment facility associated with the city of Winter Garden, Florida. We examined the hypothesis that an estrogenic contaminant has caused the current failure in recruitment of alligators on Lake Apopka. Supporting data include the following: At 6 months of age, female alligators from Lake Apopka had plasma estradiol-17 beta concentrations almost two times greater than normal females from the control lake, Lake Woodruff. The Apopka females exhibited abnormal ovarian morphology with large numbers of polyovular follicles and polynuclear oocytes. Male juvenile alligators had significantly depressed plasma testosterone concentrations comparable to levels observed in normal Lake Woodruff females but more than three times lower than normal Lake Woodruff males. Additionally, males from Lake Apopka had poorly organized testes and abnormally small phalli. The differences between lakes and sexes in plasma hormone concentrations of juvenile alligators remain even after stimulation with luteinizing hormone. Our data suggest that the gonads of juveniles from Lake Apopka have been permanently modified in ovo, so that normal steroidogenesis is not possible, and thus normal sexual maturation is unlikely.

On the transcriptional regulation of neuronal nAChR genes.

The promoters driving transcription of the neuronal nicotinic genes alpha 7 and beta 3 have been characterized in the chicken. Although their regulatory modalities are thoroughly different, they nevertheless lead to co-expression in the same neurons.

Indomethacin influences arginine vasotocin-induced parturition and oviposition in lizards (Sceloporus jarrovi and Sceloporus undulatus ).

The effect of the prostaglandin blocker indomethacin on arginine vasotocin-induced birth was examined. Gravid female, oviparous (Sceloporus undulatus ) and viviparous (Sceloporus jarrovi ) lizards were pretreated with saline or indomethacin, a potent blocker of PG synthesis. Pretreatment was followed by an intraperitoneal injection of AVT. Pretreatment with indomethacin significantly delayed the onset of AVT-induced oviposition in S. undulatus , whereas it had no effect on latency to birth in S. jarrovi . Female S. jarrovi treated with indomethacin, however, gave birth to only part of the total litter, whereas control females gave birth to complete litters. In viviparous females, an interaction of embryonic age with pretreatment was evident; females having more developed embryos decreased birth latency significantly and increased the percentage of parturition when compared with females that had embryos at earlier stages of development. Our data suggest that although exogenous AVT can stimulate oviposition or parturition, these events occur more rapidly and completely when prostaglandin synthesis is not inhibited.

The relation between polyoma T-antigen and increased 5S RNA synthesis in cell-free extracts from polyoma-infected mouse kidney cell cultures.

In polyoma-infected mouse kidney cell cultures 5S RNA synthesis began to increase around 16 h, i.e. 7-9 h after the onset of polyoma T-antigen synthesis. The rate of polyoma-induced 5S RNA synthesis reached a maximum plateau around 25 h when it was 1.8-2.0 times higher than in mock-infected parallel cultures. Stimulation of 5S RNA synthesis in vivo thus coincided in time with the increase in total cellular RNA and protein. Cell-free extracts (S100) prepared at 15 h from mock-(S100-M) or polyoma-infected (S100-Py) mouse kidney cell cultures were indistinguishable with respect to protein concentration and 5S RNA synthesis, using a cloned somatic Xenopus borealis 5S gene as template. S100-Py extracted 25 h after infection contained 30% more protein and synthesized 1.5-2.0 times more 5S RNA than S100-M. Complete removal of the polyoma T-antigens from S100-Py by 3 cycles of immunoprecipitation with hamster anti-T serum remained without effect on stimulated 5S RNA synthesis. However, a linear relationship between 5S RNA synthesis and protein concentration of S100-M and S100-Py was observed.

Expression of neuronal nicotinic acetylcholine receptor genes in the developing chick visual system.

Expression of the neuronal non-alpha nicotinic acetylcholine receptor (n alpha nAChR) gene is transiently stimulated in the chick optic tectum between embryonic days 7 and 16 with a peak value reached around embryonic day 12. This stimulation takes place at the time when optic nerve axons are invading this region of the brain and proceeds along a rostral to caudal gradient. Transcripts of the n alpha nAChR gene are localized in the superficial layers of the tectum at the time when cells in these layers are forming synapses with retina axons. The transient expression of n alpha nAChR gene does not take place in the optic tectum of 'eyeless' embryos. The results of our study suggest that the neuronal n alpha nAChR gene may play a role in neurogenesis of retino-tectal connections.

Central monoamines in free-ranging lizards: differences associated with social roles and territoriality.

During the breeding season different social classes of field-active lizards, Sceloporus jarrovi, exhibit regionally specific changes in central monoaminergic activation. Changes in serotonergic content and turnover between lizards from different social classes are seen in forebrain structures (telencephalon and diencephalon) and reflect events associated with reproductive behaviors, stress and aggression. Males without territories (satellite males) exhibit higher forebrain serotonin (5-HT) system activation compared to territorial males and adult females. This serotonergic activation includes increased 5-hydroxyindoleacetic acid (5-HIAA) and 5-HIAA/5-HT ratio, suggesting increased release and catabolism. Satellite males also exhibit higher 5-HIAA/5-HT ratios (serotonergic turnover) compared to territorial males following agonistic interactions. Territorial males, immediately following aggressive defense of territories against intruder males, exhibit increased 5-hydroxytryptophan (5-HTP) levels, higher 5-HIAA levels and 5-HIAA/5-HT ratio, higher epinephrine levels, greater MHPG/NE, more DOPAC and larger DOPAC/DA ratio compared to territorial males that did not have an aggressive encounter. These differences suggest activation of 5-HT, norepinephrine (NE), and dopamine (DA) systems by the synthesis and release of more 5-HT and the release of more NE and DA during aggressive defense of territory. The highest activity of serotonergic system is exhibited by satellite males compared to territorial males, perhaps reflecting stress in subordinate animals from social and ecological sources.

Activity of the beta 3 nicotinic receptor promoter is a marker of neuron fate determination during retina development.

We have previously shown that transcription of the beta 3 nicotinic receptor gene within the chick CNS is regulated by a promoter 143 base pairs (bp) in length. Here, we demonstrate that in the developing visual system this promoter is active in a subset of retinal cells, the majority of which are ganglion cells. Because the beta 3 promoter is activated very early during retina development, it can provide a marker of ganglion cell induction and differentiation. Transfection of neuroretina explants enabled us to detect activity of the beta 3 promoter in premigratory cells localized on the ventricular side of the retina. Double-labeling experiments showed that activation of the beta 3 promoter takes place before the last S-phase, suggesting that this particular phenotypic trait is determined when precursor cells are still proliferating. The beta 3 phenotype is induced in about one-tenth of the total pool of retinal progenitor cells and is stable upon changing the cellular environment. Our study suggests that at the very early stages of retina neurogenesis, some lineage restrictions have already occurred in the population of retinal progenitor cells.

Sequential stimulation of cellular RNA synthesis in polyoma-infected mouse kidney cell cultures.

Lytic infection with polyoma virus leads in Go-arrested primary mouse kidney cell cultures to a mitotic host response. In the present work we focused our attention on cellular RNA synthesis shortly after onset of polyoma T-antigen synthesis. Onset of polyoma-induced stimulation of 45S pre-rRNA synthesis was determined by hybridization of total cellular RNA with a plasmid (pMrSalB) containing the 5'-end of the mouse ribosomal gene and of the other cellular RNA species by standard biochemical analysis of cellular fractions. The results showed that polyoma-induced stimulation of cellular hnRNA (hnRNP) synthesis, the earliest presently known host cell reaction, preceded onset of stimulated 45S pre-rRNA synthesis and that the latter was paralleled by polyoma-induced stimulation of 5S RNA, tRNA and overall protein synthesis. The polyoma-induced mitotic response is similar to that triggered by simian virus 40 and by certain nonviral mitogens.

Polyoma-induced stimulation of nucleoplasmic transcription is paralleled by development of resistance against actinomycin D.

Polyoma virus induced in quiescent, Go-arrested mouse kidney cells a lytic infection. Synthesis of the polyoma T-antigens began 7-8 h after infection and was followed by a mitotic reaction of the host cell comprising stimulated synthesis and accumulation of cellular (mainly ribosomal) RNA and protein and duplication of the host cell chromatin (S-phase). In the present work we focused attention on nucleoplasmic transcription, i.e. synthesis of hnRNA, 5S RNA and tRNA. To inhibit selectively nucleolar transcription we used low concentrations of actinomycin D (act. D). Synthesis of 45S precursor- ribosomal RNA in mock- and polyoma-infected mouse kidney cells was completely blocked by 0.05 micrograms/ml act.D within 2 h. In mock-infected cells also nucleoplasmic transcription was rather sensitive against 0.05 micrograms/ml act.D. Polyoma- induced stimulation of nucleoplasmic transcription began around 12 h and was paralleled by the development of resistance against act.D. Resistance of nucleoplasmic transcription in virus-infected cells was thus similar to that observed by others in uninfected, proliferating mammalian cells. The possible biological implications of these results are discussed.

Simian virus 40 and polyoma virus stimulate overall cellular RNA and protein synthesis.

In lytic infection with simian virus 40 and polyoma virus of monkey and mouse cells in tissue culture, synthesis of the viral tumor (T) antigens (T antigens) is rapidly followed by a mitogenic response of the host cell. The latter begins with virus-induced stimulation of overall cellular RNA and protein synthesis, leading to a substantial increase in cytoplasmic and nuclear RNA and protein. Stimulation begins within 1 hr after onset of T-antigen synthesis and also occurs if virus-induced DNA synthesis is blocked by metabolic inhibitors. The broad spectrum of biological and molecular effects induced by simian virus 40 and polyoma virus is, at least phenotypically, reminescent of the pleiotropic impact exerted on target cells by nonviral mitogens and by certain growth-promoting steroid and polypeptide hormones.

Specification of neurotransmitter receptor identity in developing retina: the chick ATH5 promoter integrates the positive and negative effects of several bHLH proteins.

Genetic studies in Drosophila and in vertebrates have implicated basic helix-loop-helix (bHLH) transcription factors in neural determination and differentiation. In this report, we analyze the role that several bHLH proteins play in the transcriptional control of differentiation in chick retina. Our experimental system exploits the properties of the promoter for the beta 3 subunit of the neuronal acetylcholine receptors, important components of various phenotypes in the CNS of vertebrates. The beta 3 subunit contributes to define ganglion cell identity in retina and its promoter, whose activation is an early marker of ganglion cell differentiation, is under the specific control of the chick atonal homolog ATH5. Functional analysis of the ATH5 promoter indicates that interactions between ATH5 and several other bHLH transcription factors underlie the patterning of the early retinal neuroepithelium and form a regulatory cascade leading to transcription of the gene for beta 3. ATH5 appears to coordinate the transcriptional pathways that control pan-neuronal properties with those that regulate the subtype-specific features of retinal neurons.

Neuronal specificity of the alpha 7 nicotinic acetylcholine receptor promoter develops during morphogenesis of the central nervous system.

A transient transfection assay has been developed to analyse promoter activity in neuronal cells freshly dissociated from the chick central nervous system. The assay enabled us to identify cis-acting regulatory elements within the 5'-flanking region of the alpha 7 nicotinic acetylcholine receptor gene. In differentiated retina, regulatory elements direct reporter gene expression to a small subset of neurons which has been identified as ganglion cells, i.e. to the population of neurons in which alpha 7 transcripts were localized by in situ hybridization. However, these promoter elements exhibit ubiquitous activity in undifferentiated neural cells and in mesodermal stem cells. Our study supports the idea that alpha 7 regulatory elements acquire their neuronal specificity in the course of embryogenesis.

NeuroM, a neural helix-loop-helix transcription factor, defines a new transition stage in neurogenesis.

Genes encoding transcription factors of the helix-loop-helix family are essential for the development of the nervous system in Drosophila and vertebrates. Screens of an embryonic chick neural cDNA library have yielded NeuroM, a novel neural-specific helix-loop-helix transcription factor related to the Drosophila proneural gene atonal. The NeuroM protein most closely resembles the vertebrate NeuroD and Nex1/MATH2 factors, and is capable of transactivating an E-box promoter in vivo. In situ hybridization studies have been conducted, in conjunction with pulse-labeling of S-phase nuclei, to compare NeuroM to NeuroD expression in the developing nervous system. In spinal cord and optic tectum, NeuroM expression precedes that of NeuroD. It is transient and restricted to cells lining the ventricular zone that have ceased proliferating but have not yet begun to migrate into the outer layers. In retina, NeuroM is also transiently expressed in cells as they withdraw from the mitotic cycle, but persists in horizontal and bipolar neurons until full differentiation, assuming an expression pattern exactly complementary to NeuroD. In the peripheral nervous system, NeuroM expression closely follows cell proliferation, suggesting that it intervenes at a similar developmental juncture in all parts of the nervous system. We propose that availability of the NeuroM helix-loop-helix factor defines a new stage in neurogenesis, at the transition between undifferentiated, premigratory and differentiating, migratory neural precursors.

Functional properties of the neuronal nicotinic acetylcholine receptor beta3 promoter in the developing central nervous system.

Within the chick central nervous system, expression of the beta3 nicotinic acetylcholine receptor gene is restricted to a subset of retinal neurons, the majority of which are ganglion cells. Transient transfection in retinal neurons and in neural and non-neural cells from other regions of the chick embryo allowed the identification of the cis-regulatory domain of the beta3 gene. Within this domain, a 75-base pair fragment located immediately upstream of the transcription start site suffices to reproduce the neuron-specific expression pattern of beta3. This fragment encompasses an E-box and a CAAT box, both of which are shown to be key positive regulatory elements of the beta3 promoter. Co-transfection experiments into retinal, telencephalic, and tectal neurons with plasmid reporters of beta3 promoter activity and a number of vectors expressing different neuronal (ASH-1, NeuroM, NeuroD, CTF-4) and non-neuronal (MyoD) basic helix-loop-helix transcription factors indicate that the cis-regulatory domain of beta3 has the remarkable property of discriminating accurately between related members of the basic helix-loop-helix protein family. The sequence located immediately 3' of the E-box participates in this selection, and the E-box acts in concert with the nearby CAAT box.

Characterization of the nicotinic acetylcholine receptor beta 3 gene. Its regulation within the avian nervous system is effected by a promoter 143 base pairs in length.

Genomic and cDNA clones encoding the chicken neuronal nicotinic acetylcholine receptor beta 3 subunit were isolated and sequenced. The beta 3 gene consists of six protein-encoding exons and the deduced protein has the structural features found in all other members of the neuronal nicotinic acetylcholine receptor subunit family. Although they are undetectable in most brain compartments, beta 3 mRNAs are relatively abundant in the developing retina and in the trigeminal ganglion. In situ hybridization and immunohistochemical analysis demonstrated that in retina, beta 3 transcripts and protein are confined to subpopulations of cells in the inner nuclear and ganglion cell layers. Beta 3 is expressed in the proximal and distal regions of the developing trigeminal ganglion, i.e. in both placode- and neural crest-derived neurons. Transient transfection assays in cells freshly dissociated from selected regions of the central nervous system at different developmental stages allowed the identification of genetic elements involved in the neuronal-selective expression of the beta 3 gene. A promoter fragment 143 base pairs in length and containing TATA, CAAT, and other consensus sequences is sufficient to restrict reporter gene expression to a subpopulation of retinal neurons. This promoter is totally inactive upon transfection into neuronal and non-neuronal cells from other regions of the central nervous system.

Plasma estradiol-17 beta, progesterone, prostaglandin F, and prostaglandin E2 concentrations during natural oviposition in the loggerhead turtle (Caretta caretta).

Changes in plasma concentrations of steroids and prostaglandins (PGs) during natural nesting and oviposition in the loggerhead turtle were studied. Blood samples were obtained during nine distinct behavioral stages of oviposition. Emerging females had no detectable prostaglandin F (PGF) or prostaglandin E2 (PGE2) whereas plasma estradiol-17 beta averaged 255 pg/ml and mean plasma progesterone was 395 pg/ml. Plasma steroid concentrations did not vary significantly during nesting. In contrast, plasma PGF and PGE2 exhibited significant elevations during nest digging about 15 min after emergence. A further significant increase in plasma PGs was observed 10 min later during early oviposition. Plasma PGE2 peaked during mid oviposition whereas maximal plasma PGF levels occurred during nest covering although mean values were not significantly different than those observed during oviposition. Both PGs showed an abrupt decline (within 10 min) during body pit covering to concentrations similar to those observed during nest construction. Our data suggest that PGs have an active role during oviposition and nesting in the loggerhead turtle and are consistent with hypotheses that PGF2 alpha stimulates uterine contractions promoting egg expulsion while PGE2 may be more important in promoting cervical relaxation.

The neuronal alpha6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells.

We examine some of the biological and physiological properties of the avian alpha6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, alpha6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as alpha3, beta2 and beta4. In contrast, alpha6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the alpha6 subunit has long failed the critical test of functional reconstitution. Here we use patch-clamp techniques and confocal laser microscopy to measure ACh-activated currents and nicotine-elicited Ca2+ transients in human BOSC 23 cells transfected with chick alpha6 in combination with other chick nAChR neuronal subunits. Heterologously expressed alpha6 and beta4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 microM). Likewise, ACh elicits measurable currents in cells transfected with alpha6 and beta2. Hill analysis of the dose-response curves in cells transfected with alpha3, beta4 and alpha6 cDNAs, suggests the assembly of functional alpha3beta4alpha6 receptor, with an apparent affinity for ACh threefold lower than alpha3beta4. Our results indicate that alpha6-containing nAChRs assemble in heterologous expression systems and are probably present in retinal cells.