Social Environment Ameliorates Behavioral and Immune Impairments in Tyrosine Hydroxylase Haploinsufficient Female Mice.

The social environment can influence the functional capacity of nervous and immune systems, and consequently the state of health, especially in aged individuals. Adult female tyrosine hydroxylase haploinsufficient (TH-HZ) mice exhibit behavioral impairments, premature immunosenescence and oxidative- inflammatory stress. All these deteriorations are associated with a lower lifespan than wild type (WT) counterparts. The aim was to analyze whether the cohabitation with WT animals could revert or at least ameliorate the deterioration in the nervous and immune systems that female TH-HZ mice show at adult age. Female TH-HZ and WT mice at age of 3-4 weeks were divided into following groups: control TH-HZ (5 TH-HZ mice in the cage; TH-HZ100%), control WT (5 WT mice in the cage; WT100%), TH-HZ > 50% and WT < 50% (5 TH-HZ with 2 WT mice in each cage) as well as TH-HZ < 50% and WT > 50% (2 TH-HZ and 5 WT mice in each cage). At the age of 37-38 weeks, all mice were submitted to a battery of behavioral tests, evaluating sensorimotor abilities, exploratory capacities and anxiety-like behaviors. Subsequently, peritoneal leukocytes were extracted and several immune functions as well as oxidative and inflammatory stress parameters were analyzed. The results showed that the TH-HZ < 50% group had improved behavioral responses, especially anxiety-like behaviors, and the immunosenescence and oxidative stress of their peritoneal leukocytes were ameliorated. However, WT mice that cohabited with TH-HZ mice presented higher anxiety-like behaviors and deterioration in immune functions and in their inflammatory stress parameters. Thus, this social environment is capable of ameliorating the impairments associated with a haploinsufficiency of the th gene. Graphical Abstract.

Premature aging in behavior and immune functions in tyrosine hydroxylase haploinsufficient female mice. A longitudinal study.

Aging is accompanied by impairment in the nervous, immune, and endocrine systems as well as in neuroimmunoendocrine communication. In this context, there is an age-related alteration of the physiological response to acute stress, which is modulated by catecholamine (CA), final products of the sympathetic-adreno-medullary axis. The involvement of CA in essential functions of the nervous system is consistent with the neuropsychological deficits found in mice with haploinsufficiency (hemizygous; HZ) of tyrosine hydroxylase (TH) enzyme (TH-HZ). However, other possible alterations in regulatory systems have not been studied in these animals. The aim of the present work was to analyze whether adult TH-HZ female mice presented the impairment of behavioral traits and immunological responses that occurs with aging and whether they had affected their mean lifespan. ICR-CD1 female TH-HZ and wild type (WT) mice were used in a longitudinal study. Behavioral tests were performed on adult and old mice in order to evaluate their sensorimotor abilities and exploratory capacity, as well as anxiety-like behaviors. At the ages of 2 ±â€¯1, 4 ±â€¯1, 9 ±â€¯1, 13 ±â€¯1 and 20 ±â€¯1 months, peritoneal leukocytes were extracted and several immune functions were assessed (phagocytic capacity, Natural Killer (NK) cytotoxicity, and lymphoproliferative response to lipopolysaccharide (LPS) and concanavalin A (ConA)). In addition, several oxidative stress parameters (catalase, glutathione reductase and glutathione peroxidase activities, and reduced glutathione (GSH) concentrations as antioxidant compounds as well as xanthine oxidase activity, oxidized glutathione (GSSG) concentrations, and GSSG/GSH ratio as oxidants) were analyzed. As inflammatory stress parameters TNF-alpha and IL-10 concentrations, and TNF-alpha/IL-10 ratios as inflammatory/anti-inflammatory markers, were measured. Animals were maintained in standard conditions until their natural death. The results indicate that adult TH-HZ mice presented worse sensorimotor abilities and exploratory capacity than their WT littermates as well as greater anxiety-like behaviors. With regards to the immune system, adult TH-HZ animals exhibited lower values of phagocytic capacity, NK cytotoxicity, and lymphoproliferative response to LPS and ConA than WT mice. Moreover, immune cells of TH-HZ mice showed higher oxidative and inflammatory stress than those of WT animals. Although these differences between TH-HZ and WT, in general, decreased with aging, this premature immunosenescence and impairment of behavior of TH-HZ mice was accompanied by a shorter mean lifespan in comparison to WT counterparts. In conclusion, haploinsufficiency of th gene in female mice appears to provoke premature aging of the regulatory systems affecting mean lifespan.

La docencia del área de Toxicología de la Universidad de La Laguna / The teaching of Toxicology at the University of La Laguna

La docencia de Toxicología en la Universidad de La Laguna (ULL) se integra en diversas Licenciaturas y Grados tales como Farmacia, Ciencia y Tecnología de los Alimentos (CTA), Medicina, Náutica y Transporte Marítimo y el Máster Oficial de Seguridad y Calidad de los Alimentos. En la Licenciatura de Farmacia en la ULL, el Área de Toxicología imparte la asignatura troncal Toxicología de 7 créditos y dos asignaturas optativas, Drogodependencias y Toxicología Clínica y Laboral, de 4,5 créditos cada una. En el Grado en Farmacia, se imparte la asignatura obligatoria de Toxicología (9 ECTS) y la asignatura optativa de Drogodependencias (6 ECTS). Asimismo, se imparte un total de 11 créditos en la Licenciatura de CTA quedando ésta extinguida en el curso académico 2013/2014. En la Licenciatura de Medicina, se imparte docencia de Toxicología en las asignaturas de Medicina Legal y Toxicología (asignado 1 crédito) y Toxicología clínica (4,5 créditos) mientras que en el Grado de Medicina se integra en las asignaturas Farmacología, anestesia y tratamiento del dolor (9 ECTS) y Aspectos éticos, aspectos legales y aspectos humanísticos de la Medicina (6 ECTS). En ellas, la Toxicología tiene asignado 1 y 0,5 ECTS, respectivamente. La Licenciatura de Náutica y Transporte Marítimo, la docencia de Toxicología se engloba en una asignatura obligatoria mientras que en el Grado en Ingeniería Radioelectrónica se incluye en una asignatura optativa (3 ECTS). En la docencia de Postgrado, la Toxicología se imparte en el Máster Oficial en Seguridad y Calidad de los Alimentos desde dos módulos con 6 ECTS cada uno de ellos (AU)

The Toxicology Department at the University of La Laguna (ULL) offers courses included in the curricula of several degrees such as Pharmacy, Food Science and Technology, Medicine, Marine and Maritime Transport, and Food Safety and Quality Master program. In the Bachelor of Pharmacy at ULL that will end in 2014/2015, the Toxicology Department offers the mandatory subject "Toxicology", with 7 credits, as well as two optional subjects ("Clinical and Professional Toxicology", and "Drug Addiction"), with 4.5 credits each. In the Degree in Pharmacy, the "Toxicology" course is a compulsory subject worth 9 credits in the European Credit Transfer System (ECTS) and the optional "Drug Addiction" subject remains, and will be worth 6 ECTS. The Toxicology Department also gives a total of 11 credits in the Food Science and Technology Bachelor that will end in the academic course 2013/2014. In the Bachelor of Medicine, Toxicology is taught in two courses: "Pharmacology, anesthesia and pain management" (9 ECTS, of which 1 ECTS is for toxicology) and "Ethical, legal and humanistic aspects of medicine" (6 ECTS, of which 0.5 ECTS is for toxicology). In the Bachelor of Nautical and Maritime Transport, Toxicology is included in a mandatory subject while in the Bachelor of Radio Electronics Engineering, Toxicology is an optional subject (3 ECTS). As for postgraduate teaching, Toxicology is taught in the Master program in Food Safety and Quality, in two modules of 6 ECTS each (AU)

Differentiation of mangoes (Magnifera indica L.) conventional and organically cultivated according to their mineral content by using support vector machines.

Mangoes of uniform genetics (Lippens variety) cultivated in the Gomera Island (Canary Islands) by conventional and organic farming were used to analyze the mineral content in order to differentiate crops cultivated in the same geographic area by the cultivation practices. Farming differences as well as soil differences may be reflected in the mineral content of the mangoes cultivated in these extensions. Concentration metal profiles consisting of the content of Ca, Co, Cu, Fe, K, Mg, Mn, Na, Ni and Zn in mangoes were obtained by using atomic absorption spectrometry (AAS). Pattern recognition classification procedures were applied for discriminating purposes. Linear discriminant analysis (LDA) allows to a classification performance of about 73% and support vector machines (SVM) found up to a 93% of prediction ability. The classification success when applying support vector machines techniques is due to their ability for modeling non-linear class boundaries.

Trihalometanos en aguas de consumo humano / Trihalomethanes in water supply

Los trihalometanos (THMs) son subproductos de desinfección que resultan de la cloración de las aguas. Los THMs engloban a sustancias como el cloroformo (CHCl ), el 3 bromodiclorometano (CHBrCl ), el clorodibromometano (CHBr Cl) 2 2 y el tribromometano (CHBr ). La Agencia Internacional para la 3 Investigación del Cáncer ha clasificado al cloroformo y al bromodiclorometano en el grupo 2B que incluye a las sustancias posiblemente carcinogénicas en humanos. Debido a su toxicidad y a que el agua de consumo humano constituye la principal vía de exposición del hombre a estos compuestos, su presencia en las aguas de consumo humano está limitada por la Directiva Europea 98/83/CE a 100 ìg/L. En esta revisión se destacan los aspectos más relevantes sobre su toxicidad, reglamentación, determinación, concentración en aguas, variaciones estacionales y eliminación (AU)

Trihalomethanes (THMs) are disinfection byproducts resulting from chlorination of water. THMs comprise substances such as chloroform (CHCl ), 3 bromodichloromethane (CHBrCl ), chlorodibromomethane 2 (CHBr Cl) and tribromomethane (CHBr ). The International Agency 2 3 for Research on Cancer has classified chloroform and bromodichloromethane in Group 2B, which includes potentially carcinogenic substances in humans. Because of its toxicity and that drinking water is the main human exposure to these compounds, their presence in drinking water is limited by the European Directive 98/83/EC to 100 mg/L. This review highlights the most relevant aspects of its toxicity, regulation, determination, concentration in water, and removing seasonal variations (AU)

Proinsulin in development: New roles for an ancient prohormone.

In postnatal organisms, insulin is well known as an essential anabolic hormone responsible for maintaining glucose homeostasis. Its biosynthesis by the pancreatic beta cell has been considered a model of tissue-specific gene expression. However, proinsulin mRNA and protein have been found in embryonic stages before the formation of the pancreatic primordium, and later, in extrapancreatic tissues including the nervous system. Phylogenetic studies have also confirmed that production of insulin-like peptides antecedes the morphogenesis of a pancreas, and that these peptides contribute to normal development. In recent years, other roles for insulin distinct from its metabolic function have emerged also in vertebrates. During embryonic development, insulin acts as a survival factor and is involved in early morphogenesis. These findings are consistent with the observation that, at these stages, the proinsulin gene product remains as the precursor form, proinsulin. Independent of its low metabolic activity, proinsulin stimulates proliferation in developing neuroretina, as well as cell survival and cardiogenesis in early embryos. Insulin/proinsulin levels are finely regulated during development, since an excess of the protein interferes with correct morphogenesis and is deleterious for the embryo. This fine-tuned regulation is achieved by the expression of alternative embryonic proinsulin transcripts that have diminished translational activity.

Balance of pro-apoptotic transforming growth factor-beta and anti-apoptotic insulin effects in the control of cell death in the postnatal mouse retina.

Transforming growth factor (TGF)-beta and insulin display opposite effects in regulating programmed cell death during vertebrate retina development; the former induces apoptosis while the latter prevents it. In the present study we investigated coordinated actions of TGF-beta and insulin in an organotypic culture system of early postnatal mouse retina. Addition of exogenous TGF-beta resulted in a significant increase in cell death whereas exogenous insulin attenuated apoptosis and was capable of blocking TGF-beta-induced apoptosis. This effect appeared to be modulated via insulin-induced transcriptional down-regulation of TGF-beta receptor II levels. The analysis of downstream signalling molecules also revealed opposite effects of both factors; insulin provided survival signalling by increasing the level of anti-apoptotic Bcl-2 protein expression and phosphorylation and down-regulating caspase 3 activity whereas pro-apoptotic TGF-beta signalling reduced Bcl-2 mRNA levels and Bcl-2 phosphorylation and induced the expression of TGF-induced immediate-early gene (TIEG), a Krüppel-like zinc-finger transcription factor, mimicking TGF-beta activity.

Functional inactivation of the IGF-I and insulin receptors in skeletal muscle causes type 2 diabetes.

Peripheral insulin resistance and impaired insulin action are the primary characteristics of type 2 diabetes. The first observable defect in this major disorder occurs in muscle, where glucose disposal in response to insulin is impaired. We have developed a transgenic mouse with a dominant-negative insulin-like growth factor-I receptor (KR-IGF-IR) specifically targeted to the skeletal muscle. Expression of KR-IGF-IR resulted in the formation of hybrid receptors between the mutant and the endogenous IGF-I and insulin receptors, thereby abrogating the normal function of these receptors and leading to insulin resistance. Pancreatic beta-cell dysfunction developed at a relative early age, resulting in diabetes. These mice provide an excellent model to study the molecular mechanisms underlying the development of human type 2 diabetes.

Mice transgenically overexpressing sulfonylurea receptor 1 in forebrain resist seizure induction and excitotoxic neuron death.

The ability of the sulfonylurea receptor (SUR) 1 to suppress seizures and excitotoxic neuron damage was assessed in mice transgenically overexpressing this receptor. Fertilized eggs from FVB mice were injected with a construct containing SUR cDNA and a calcium-calmodulin kinase IIalpha promoter. The resulting mice showed normal gross anatomy, brain morphology and histology, and locomotor and cognitive behavior. However, they overexpressed the SUR1 transgene, yielding a 9- to 12-fold increase in the density of [(3)H]glibenclamide binding to the cortex, hippocampus, and striatum. These mice resisted kainic acid-induced seizures, showing a 36% decrease in average maximum seizure intensity and a 75% survival rate at a dose that killed 53% of the wild-type mice. Kainic acid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons or expression of heat shock protein 70, whereas wild-type mice lost 68-79% of pyramidal neurons in the CA1-3 subfields and expressed high levels of heat shock protein 70 after kainate administration. These results indicate that the transgenic overexpression of SUR1 alone in forebrain structures significantly protects mice from seizures and neuronal damage without interfering with locomotor or cognitive function.

Characterization of the mouse sulfonylurea receptor 1 promoter and its regulation.

The ATP-sensitive potassium channels (K+ATP channels) are heteromultimeric structures formed by a member of the sulfonylurea receptor (SUR) family and a member of the inwardly rectifying potassium channel family (Kir6.x). The K+ATP channels play an essential role in nutrient-induced insulin secretion from the pancreatic beta-cell. We have cloned and characterized the promoter region of the mouse SUR1 gene, and have shown that it lacks CAAT and TATA boxes or an initiator element. Studies of transcription initiation in several tissues showed that there is a common SUR1 promoter in brain, heart, and pancreas and in the pancreatic beta-cell line, betaTC3. The SUR1 gene uses multiple transcription start sites with the major site located 54 base pairs 5'-upstream of the translation initiation site. Transient transfection experiments in pancreatic beta-cell lines showed that the proximal promoter fragment -84/+54 is sufficient for significant transcriptional activity. The proximal promoter region contains multiple SP1-binding sites, and cotransfection experiments of the SUR1 promoter-luciferase vector with SP1 expression vector in Drosophila SL2 cells demonstrated a stimulatory effect of SP1 on SUR1 transcriptional activity. The mobility shift assays confirmed the interaction of the SP1 transcription factor with the proximal promoter region of the SUR1 gene. Together, these results indicate that SP1 may mediate transcription initiation of the SUR1 gene. In addition, we have described the coordinate regulation of the gene expression of both K+ATP channel subunits by glucocorticoids. SUR1 and Kir6.2 mRNA levels are down-regulated by approximately 40-50% in response to glucocorticoid treatment. Interestingly, the extent of the inhibitory effect as well as the kinetics and sensitivity are very similar for both mRNAs. Studies of mRNA turnover demonstrate that glucocorticoids most likely decrease the transcriptional activity of both SUR1 and Kir6.2 genes since glucocorticoids failed to affect the stability of each mRNA. Likewise, the reduction in mRNA levels was correlated with a decrease in SUR1 and Kir6.2 protein levels.

Heat shock proteins in retinal neurogenesis: identification of the PM1 antigen as the chick Hsc70 and its expression in comparison to that of other chaperones.

While the role of heat shock proteins under experimental stress conditions is clearly characterized, their expression in unstressed cells and tissues and their functions in normal cell physiology, besides their chaperone action, remain largely undetermined. We report here the identification in chicken of the antigen recognized by the monoclonal antibody PM1 [Hernández-Sánchez et al. (1994) Eur. J. Neurosci., 6,1801-1810] as the noninducible chaperone heat-shock cognate 70 (Hsc70). Its identity was determined by partial peptide sequencing, immuno-crossreactivity and two-dimensional gel-electrophoresis. In addition, we examined its expression during chick embryo retinal neurogenesis. The early widespread Hsc70 immunostaining corresponding to most, if not all, of the neuroepithelial cells becomes restricted to a subpopulation of these cells in the peripheral retina as development proceeds. On the other hand, retinal ganglion cells, differentiating in the opposite central-to-peripheral gradient, retained Hsc70 immunostaining. Other molecular chaperones, the heat-shock proteins Hsp40, Hsp60 and Hsp90, did not seem to compensate the loss of Hsc70. They also showed decreasing immunostaining patterns as neurogenesis proceeds, although distinctive from that of Hsc70, whereas Hsp70 was not detected in the embryonic retina. This precise cellular and developmental regulation of Hsc70, a generally considered constitutive molecular chaperone, in unstressed embryos, together with the expression of other chaperones, provides new tools and a further insight on neural precursor heterogeneity, and suggests possible specific cellular roles of chaperone function during vertebrate neurogenesis.

Differential regulation of insulin-like growth factor-I (IGF-I) receptor gene expression by IGF-I and basic fibroblastic growth factor.

Insulin-like growth factor-I receptor (IGF-IR) gene expression is regulated by various stimuli, including hormones, growth factors, and nutritional status. We have investigated the molecular mechanism by which two growth factors, insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) regulate IGF-IR gene expression. bFGF increases the endogenous IGF-IR mRNA levels and IGF-IR promoter activity. This effect is mediated by a region of the IGF-IR promoter located between nucleotides -476 and -188 in the 5'-flanking region. In contrast, IGF-I decreases the IGF-IR mRNA levels. IGF-I down-regulates IGF-IR transcriptional activity as deduced from experiments in which the levels of pre-mRNA and mRNA were measured. IGF-I reduced pre-mRNA and mRNA levels in parallel, while the mRNA stability was found to be unchanged by IGF-I treatment. While these results strongly suggest an effect of IGF-I on IGF-IR transcriptional activity, no specific IGF-I response element was demonstrated in the 5'-untranslated region or 5'-flanking region studied. Thus, bFGF and IGF-I have differential effects on IGF-IR gene transcription, with the IGF-I response region as yet unidentified.

Developmental and tissue-specific sulfonylurea receptor gene expression.

We have studied the developmental regulation of mouse sulfonylurea receptor (SUR) gene expression throughout several embryonic stages as well as in the adult mouse. To this end we used a 229-bp mouse complementary DNA corresponding to the 3'-end of the SUR gene for in situ hybridization and solution hybridization/ribonuclease protection assays. We found that the SUR gene was expressed as early as embryonic day 12 in the developing pancreas, heart, and central nervous system. These tissues maintained significant levels of SUR messenger RNA (mRNA) throughout development. In addition, SUR mRNA was detected in the submandibular gland, anterior duodenum, dorsal root ganglia, lens, retina, and vibrissae by late developmental stages. SUR mRNA is widely distributed in adult mouse tissues, with the exception of the liver. In the adult pancreas, the SUR gene was expressed exclusively in endocrine tissue. Although significant levels of SUR mRNA were broadly seen throughout the brain, neurons of the cerebellum, hippocampus, and thalamus had especially high levels of SUR mRNA. These findings support the idea that the SUR has important functions in many other tissues in addition to the islets of the pancreas.

The role of the tyrosine kinase domain of the insulin-like growth factor-I receptor in intracellular signaling, cellular proliferation, and tumorigenesis.

Insulin and insulin-like growth factor (IGF-I) receptors are heterotetrameric proteins consisting of two alpha-and two beta-subunits and members of the transmembrane tyrosine kinase receptors. Specific ligand binding to the receptor triggers a cascade of intracellular events, which begins with autophosphorylation of several tyrosine residues of the beta-subunit of the receptor. The triple cluster in the tyrosine kinase domain of the beta-subunit is the earliest and major autophosphorylation site. Previous studies have shown that substitutions of these three tyrosines by phenylalanines of both insulin and IGF-I receptors practically abolish any activation of cellular signaling pathways. We have studied the effect of double tyrosine mutations on IGF-I induced receptor autophosphorylation, activation of Shc and IRS-1 pathways, and cell proliferation and tumorigenicity. Substitution of tyrosines 1131/1135 blocks any detectable autophosphorylation, whereas substitution of tyrosines 1131/1136 or 1135/1136 only reduces autophosphorylation levels in some clones by approximately 50%. Nevertheless, all the cells expressing IGF-I receptors with double tyrosine substitutions demonstrated markedly reduced signaling through Shc and IRS-1 pathways. In addition, they were unable to respond to IGF-I-stimulated cell growth in culture, and tumor formation in nude mice was abrogated. These data suggest that the presence of tyrosine 1131 or 1135 essential for receptor autophosphorylation, whereas the presence of each of these tyrosines is necessary for a fully functional receptor.

Autocrine/paracrine role of insulin-related growth factors in neurogenesis: local expression and effects on cell proliferation and differentiation in retina.

Early neurogenesis progresses by an initial massive proliferation of neuroepithelial cells followed by a sequential differentiation of the various mature neural cell types. The regulation of these processes by growth factors is poorly understood. We intend to understand, in a well-defined biological system, the embryonic chicken retina, the role of the insulin-related growth factors in neurogenesis. We demonstrate the local presence of signaling elements together with a biological response to the factors. Neuroretina at days 6-8 of embryonic development (E6-E8) expressed proinsulin/insulin and insulin-like growth factor I (IGF-I) mRNAs as well as insulin receptor and IGF type I receptor mRNAs. In parallel with this in vivo gene expression, E5 cultured neuroretinas synthesized and released to the medium a metabolically radiolabeled immunoprecipitable insulin-related peptide. Furthermore, insulin-related immunoreactive material with a HPLC mobility close to that of proinsulin was found in the E6-E8 vitreous humor. Exogenous chicken IGF-I, human insulin, and human proinsulin added to E6 cultured neuroretinas showed relatively close potencies stimulating proliferation, as determined by [methyl-3H]thymidine incorporation, with a plateau reached at 10(-8) M. These factors also stimulated neuronal differentiation, indicated by the expression of the neuron-specific antigen G4. Thus, insulin-related growth factors, interestingly including proinsulin, are present in the developing chicken retina and appear to play an autocrine/paracrine stimulatory role in the progression of neurogenesis.

The regulation of IGF-I receptor gene expression.

The insulin-like growth factor I (IGF-I) receptor mediates most of the biological effects of IGF-I and -II. Despite its structural similarity to the insulin receptor, the IGF-I receptor is mainly involved in the transduction of growth and differentiation types of signals. The IGF-I receptor gene is constitutively expressed by most cells in the organism as well as in culture, consistent with the role of the IGFs as survival factors. In addition, the expression of the IGF-I receptor gene is modulated by a number of physiological and pathological factors, including developmental stage, nutritional status, hormones, growth disorders and malignancy. The regulatory region of the IGF-I receptor gene has been characterized and shown to display a high level of basal promoter activity. Transcription factor Sp1 is a strong activator of IGF-I receptor gene expression, whereas tumor suppressor WT1 represses its activity. The biological implications of these findings in both normal development and disease are described in this review.

Insulin and insulin-like growth factor system components gene expression in the chicken retina from early neurogenesis until late development and their effect on neuroepithelial cells.

To better understand the role of insulin-related growth factors in neural development, we have characterized by in situ hybridization in chicken embryonic retina the patterns of gene expression for insulin, insulin-like growth factor I (IGF-I), their respective receptors and the IGF binding protein 5 (IGFBP5) from early stages (E6) until late stages (E18)--an analysis not performed yet in any species. In addition, we studied the effect of insulin and IGF-I on cultured neuroepithelial cells. Insulin receptor mRNA and IGF-I receptor mRNA were both present and showed a similar, widespread pattern throughout retina development. Insulin mRNA could be detected only by reverse transcription coupled to polymerase chain reaction. IGF-I mRNA was concentrated in the ciliary processes and extraocular muscles early in development (embryonic day 6; E6) and in maturing retinal ganglion cells subsequently (E9-15). IGFBP5 mRNA was preferentially localized in the more differentiated central retinal zone and was maximally concentrated in the inner nuclear and ganglion cell layers at E9. These findings suggest a near constitutive expression of insulin receptor and IGF-I receptor genes, while IGF-I and IGFBP5 showed a highly focal spatiotemporal regulation of gene expression. Insulin and IGF-I, already at 10(-8) M, increased the proportion of PM1-positive neuroepithelial cells found in E5 retinal cultures without affecting significantly the total number of proliferating cells. Together, these data support the finding that, during early neurogenesis in chicken retina, insulin and IGF-I have a specific paracrine/autocrine action. This action, as well as possible effects elicited subsequently, may be dictated by restricted-local synthesis of the ligands and limited access to the factors contained in the vitreous humour. In the case of IGF's role, local IGFBPs expression can contribute to the fine modulation.

Heterogeneity among neuroepithelial cells in the chick retina revealed by immunostaining with monoclonal antibody PM1.

Neuroepithelial cells appear as a homogeneous population of cells in the cell cycle that seem to behave as pluripotent neural precursors. The study of the intrinsic heterogeneity and subtle developmental changes among neuroepithelial cells has been hindered by the lack of specific markers. To address that study, a panel of monoclonal antibodies was produced against early developing chick retina. The monoclonal antibody precursor marker 1 (PM1) labels most, if not all, of the early neuroepithelial cells in embryonic day 4 retinal sections. This pattern is transient since the labelling becomes restricted to the peripheral retina as development proceeds and eventually disappears from the neuroepithelial cells. However, apparently in parallel, the differentiating retinal ganglion cells become PM1-positive. The expression of the PM1 antigen, a 73 x 10(3) M(r) protein, as shown by western blotting, also decreases with development. In addition, a chick retina dissociated-cell culture system, where retinal neuroepithelial cells actively proliferate and undergo differentiation under defined conditions, in combination with monoclonal antibody PM1, allowed us to characterize and quantify the proliferating and differentiating neuroepithelial cells. Interestingly, the fraction of total neuroepithelial cells that are stained with PM1 sharply decreases as retinal development proceeds, in correlation with the staining pattern in sections from matched stages. These data thus reveal that the pluripotent neural precursors in the chick retina already represent an intrinsically heterogeneous population, and that this population changes with development.