Catalina Hernández-Sánchez

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 IGFs role, local IGFBPs expression can contribute to the fine modulation.

Evolution of the Insulin Receptor Family and Receptor Isoform Expression in Vertebrates

The molecular phylogeny of the vertebrate insulin receptor (IR) family was reconstructed under maximum likelihood (ML) to establish homologous relationships among its members. A sister group relationship between the orphan insulin-related receptor (IRR) and the insulin-like growth factor 1 receptor (IGF1R) to the exclusion of the IR obtained maximal bootstrap support. Although both IR and IGF1R were identified in all vertebrates, IRR could not be found in any teleost fish. The ancestral character states at each position of the receptor molecule were inferred for IR, IRR + IGF1R, and all 3 paralogous groups based on the recovered phylogeny using ML in order to determine those residues that could be important for the specific function of IR. For 18 residues, ancestral character state of IR was significantly distinct (probability >0.95) with respect to the corresponding inferred ancestral character states both of IRR + IGF1R and of all 3 vertebrate paralogs. Most of these IR distinct (shared derived) residues were located on the extracellular portion of the receptor (because this portion is larger and the rate of generation of IR shared derived sites is uniform along the receptor), suggesting that functional diversification during the evolutionary history of the family was largely generated modifying ligand affinity rather than signal transduction at the tyrosine kinase domain. In addition, 2 residues at positions 436 and 1095 of the human IR sequence were identified as radical cluster-specific sites in IRR + IGF1R. Both Ir and Irr have an extra exon (namely exon 11) with respect to Igf1r. We used the molecular phylogeny to infer the evolution of this additional exon. The Irr exon 11 can be traced back to amphibians, whereas we show that presence and alternative splicing of Ir exon 11 seems to be restricted exclusively to mammals. The highly divergent sequence of both exons and the reconstructed phylogeny of the vertebrate IR family strongly indicate that both exons were acquired independently by each paralog.

Upstream AUGs in embryonic proinsulin mRNA control its low translation level

Proinsulin is expressed prior to development of the pancreas and promotes cell survival. Here we study the mechanism affecting the translation efficiency of a specific embryonic proinsulin mRNA. This transcript shares the coding region with the pancreatic form, but presents a 32 nt extended leader region. Translation of proinsulin is markedly reduced by the presence of two upstream AUGs within the 5′ extension of the embryonic mRNA. This attenuation is lost when the two upstream AUGs are mutated to AAG, leading to translational efficiency similar to that of the pancreatic mRNA. The upstream AUGs are recognized as initiator codons, because expression of upstream ORF is detectable from the embryonic transcript, but not from the mutated or the pancreatic mRNAs. Strict regulation of proinsulin biosynthesis appears to be necessary, since exogenous proinsulin added to embryos in ovo decreased apoptosis and generated abnormal developmental traits. A novel mechanism for low level proinsulin expression thus relies on upstream AUGs within a specific form of embryonic proinsulin mRNA, emphasizing its importance as a tightly regulated developmental signal.

Developmental regulation of a proinsulin messenger RNA generated by intron retention

Proinsulin gene expression regulation and function during early embryonic development differ remarkably from those found in postnatal organisms. The embryonic proinsulin protein content decreased from gastrulation to neurulation in contrast with the overall proinsulin messenger RNA increase. This is due to increasing levels of a proinsulin mRNA variant generated by intron 1 retention in the 5′ untranslated region. Inclusion of intron 1 inhibited proinsulin translation almost completely without affecting nuclear export or cytoplasmic decay. The novel proinsulin mRNA isoform expression was developmentally regulated and tissue specific. The proportion of intron retention increased from gastrulation to organogenesis, was highest in the heart tube and presomitic region, and could not be detected in the pancreas. Notably, proinsulin addition induced cardiac marker gene expression in the early embryonic stages when the translationally active transcript was expressed. We propose that regulated unproductive splicing and translation is a mechanism that regulates proinsulin expression in accordance with specific requirements in developing vertebrates.

A Comparative Study of Age-Related Hearing Loss in Wild Type and Insulin-Like Growth Factor I Deficient Mice

Insulin-like growth factor-I (IGF-I) belongs to the family of insulin-related peptides that fulfils a key role during the late development of the nervous system. Human IGF1 mutations cause profound deafness, poor growth and mental retardation. Accordingly, Igf1−/− null mice are dwarfs that have low survival rates, cochlear alterations and severe sensorineural deafness. Presbycusis (age-related hearing loss) is a common disorder associated with aging that causes social and cognitive problems. Aging is also associated with a decrease in circulating IGF-I levels and this reduction has been related to cognitive and brain alterations, although there is no information as yet regarding the relationship between presbycusis and IGF-I biodisponibility. Here we present a longitudinal study of wild type Igf1+/+ and null Igf1−/− mice from 2 to 12 months of age comparing the temporal progression of several parameters: hearing, brain morphology, cochlear cytoarchitecture, insulin-related factors and IGF gene expression and IGF-I serum levels. Complementary invasive and non-invasive techniques were used, including auditory brainstem-evoked response (ABR) recordings and in vivo MRI brain imaging. Igf1−/− null mice presented profound deafness at all the ages studied, without any obvious worsening of hearing parameters with aging. Igf1+/+ wild type mice suffered significant age-related hearing loss, their auditory thresholds and peak I latencies augmenting as they aged, in parallel with a decrease in the circulating levels of IGF-I. Accordingly, there was an age-related spiral ganglion degeneration in wild type mice that was not evident in the Igf1 null mice. However, the Igf1−/− null mice in turn developed a prematurely aged stria vascularis reminiscent of the diabetic strial phenotype. Our data indicate that IGF-I is required for the correct development and maintenance of hearing, supporting the idea that IGF-I-based therapies could contribute to prevent or ameliorate age-related hearing loss.

Balance of pro‐apoptotic transforming growth factor‐β and anti‐apoptotic insulin effects in the control of cell death in the postnatal mouse retina

Transforming growth factor (TGF)‐β 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‐β and insulin in an organotypic culture system of early postnatal mouse retina. Addition of exogenous TGF‐β resulted in a significant increase in cell death whereas exogenous insulin attenuated apoptosis and was capable of blocking TGF‐β‐induced apoptosis. This effect appeared to be modulated via insulin‐induced transcriptional down‐regulation of TGF‐β 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‐β 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‐β activity.

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 non‐inducible chaperone heat‐shock cognate 70 (Hsc70). Its identity was determined by partial peptide sequencing, immuno‐crossreactivity & 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 & 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 & developmental regulation of Hsc70, a generally considered constitutive molecular chaperone, in unstressed embryos, together with the expression of other chaperones, provides new tools & a further insight on neural precursor heterogeneity & suggests possible specific cellular roles of chaperone function during vertebrate neurogenesis.

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 × 103Mr 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.

Tyrosine hydroxylase is expressed during early heart development and is required for cardiac chamber formation

AIMS Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in catecholamine biosynthesis. Whereas the neuroendocrine roles of cathecolamines postnatally are well known, the presence and function of TH in organogenesis is unclear. The aim of this study was to define the expression of TH during cardiac development and to unravel the role it may play in heart formation. METHODS AND RESULTS We studied TH expression in chick embryos by whole mount in situ hybridization and by quantitative reverse transcription-polymerase chain reaction and analysed TH activity by high-performance liquid chromatography. We used gain- and loss-of-function models to characterize the role of TH in early cardiogenesis. We found that TH expression was enriched in the cardiac field of gastrulating chick embryos. By stage 8, TH mRNA was restricted to the splanchnic mesoderm of both endocardial tubes and was subsequently expressed predominantly in the myocardial layer of the atrial segment. Overexpression of TH led to increased atrial myosin heavy chain (AMHC1) and T-box 5 gene (Tbx5) expression in the ventricular region and induced bradyarrhythmia. Similarly, addition of l-3,4-dihydroxyphenylalanine (l-DOPA) or dopamine induced ectopic expression of cardiac transcription factors (cNkx2.5, Tbx5) and AMHC1 as well as sarcomere formation. Conversely, blockage of dopamine biosynthesis and loss of TH activity decreased AMHC1 and Tbx5 expression, whereas exposure to retinoic acid (RA) induced TH expression in parallel to that of AMHC1 and Tbx5. Concordantly, inhibition of endogenous RA synthesis decreased TH expression as well as that of AMHC1 and Tbx5. CONCLUSION TH is expressed in a dynamic pattern during the primitive heart tube formation. TH induces cardiac differentiation in vivo and it is a key regulator of the heart patterning, conferring atriogenic identity.

Non-neural tyrosine hydroxylase, via modulation of endocrine pancreatic precursors, is required for normal development of beta cells in the mouse pancreas

Aims/hypothesisApart from transcription factors, little is known about the molecules that modulate the proliferation and differentiation of pancreatic endocrine cells. The early expression of tyrosine hydroxylase (TH) in a subset of glucagon+ cells led us to investigate whether catecholamines have a role in beta cell development.MethodsWe studied the immunohistochemical characteristics of TH-expressing cells in wild-type (Th+/+) mice during early pancreas development, and analysed the endocrine pancreas phenotype of TH-deficient (Th−/−) mice. We also studied the effect of dopamine addition and TH-inhibition on insulin-producing cells in explant cultures.ResultsIn the mouse pancreas at embryonic day (E)12.5–E13.5, the ∼10% of early glucagon+ cells that co-expressed TH rarely proliferated and did not express the precursor marker neurogenin 3 at E13.5. The number of insulin+ cells in the Th−/− embryonic pancreas was decreased as compared with wild-type embryos at E13.5. While no changes in pancreatic and duodenal homeobox 1 (PDX1)+-progenitor cell number were observed between groups at E12.5, the number of neurogenin 3 and NK2 homeobox 2 (NKX2.2)-expressing cells was reduced in Th−/− embryonic pancreas, an effect that occurred in parallel with increased expression of the transcriptional repressor Hes1. The potential role of dopamine as a pro-beta cell stimulus was tested by treating pancreas explants with this catecholamine, which resulted in an increase in total insulin content and insulin+ cells relative to control explants.Conclusions/interpretationA non-neural catecholaminergic pathway appears to modulate the pancreatic endocrine precursor and insulin producing cell neogenesis. This finding may have important implications for approaches seeking to promote the generation of beta cells to treat diabetes.