Foetal development of endocrine organs in dog.

The canine adenohypophysis starts to be identifiable from 25 day of pregnancy. ACTH-immunoreactive cells migrate until day 38 after which the number of ACTH-producing cells increases but their distribution does not change. The STH- and LH-producing cells first appear on day 38 of pregnancy. The primordium of the adrenal glands appears as a slender structure on day 27 and forms the definitive cortical structure on day 35. The histological pattern of the foetal adrenal cortex differs from the post-natal structure in so far as the three cortical zones (definitive zone, transitional zone and foetal zone) extend from the outside towards the inside of gland. The mass of foetal and neonatal adrenals is more than 10 times larger than the adult adrenals relative to body weight. The cortisol concentration in the amnion is slightly lower than in the allantois but the foetal serum cortisol concentration is significantly higher than in the maternal and foetal fluid compartments. The thyroxine concentrations in the allantois and amnion fluids exceed the foetal serum concentrations except in the ninth week of pregnancy, but thyroxine levels in foetal fluids and serum are below the physiological levels of adult animals. The exocrine and endocrine functions of the pancreas develop and act in parallel. Pancreatic cells are first detected at 30 days when the branched structure is clearly detectable immunohistochemically, and at that time, insulin-positive ß-cells and α-cells are visible as well. The foetal serum glucose concentration exceeds the healthy adult range, but the glucose concentration in the allantois and amnion fluid remains below the physiological blood glucose concentration of mature dogs. The insulin concentration in the allantois fluid greatly exceeds the foetal serum and amnion insulin concentrations.

Changes in the concentrations of four maternal steroids during embryonic development in the threespined stickleback (Gasterosteus aculeatus).

Embryonic exposure to steroids often leads to long-term phenotypic effects. It has been hypothesized that mothers may be able to create a steroid environment that adjusts the phenotypes of offspring to current environmental conditions. Complicating this hypothesis is the potential for developing embryos to modulate their early endocrine environment. This study utilized the threespined stickleback (Gasterosteus aculeatus) to characterize the early endocrine environment within eggs by measuring four steroids (progesterone, testosterone, estradiol, and cortisol) of maternal origin. We then examined how the concentrations of these four steroids changed over the first 12 days post fertilization (dpf). Progesterone, testosterone, estradiol, and cortisol of maternal origin could be detected within unfertilized eggs and levels of all four steroids declined in the first 3 days following fertilization. While levels of progesterone, testosterone, and estradiol remained low after the initial decline, levels of cortisol rose again by 8 dpf. These results demonstrate that G. aculeatus embryos begin development in the presence of a number of maternal steroids but levels begin to change quickly following fertilization. This suggests that embryonic processes change the early endocrine environment and hence influence the ability of maternal steroids to affect development. With these findings, G. aculeatus becomes an intriguing system in which to study how selection may act on both maternal and embryonic processes to shape the evolutionary consequence of steroid-mediated maternal effects.

Developmental toxicity and endocrine disruption of naphthenic acids on the early life stage of zebrafish (Danio rerio).

Oil sands process-affected water (OSPW) has been reported to exhibit adverse effects on the environment and wildlife. Although the compounds responsible are unknown, naphthenic acids (NAs) have been considered to be implicated. The current study was designed to investigate whether NAs might cause developmental toxicity and endocrine disruption on the early life stage of zebrafish (Danio rerio). The success of embryo hatch was inhibited by 2.5 mg l(-1) oil sands NAs (OS-NAs) exposure, and both OSPW NAs and commercial NAs (C-NAs) exposure resulted in a variety of developmental lesions in the fish larvae, such as yolk sac edema, pericardial edema and spinal malformation. The transcription of genes involved cytochrome P450 aromatase (CYP19a and CYP19b), estrogen receptors (ERα, ERß1 and ERß2), and vitellogenin (VTG) was analyzed to evaluate the endocrine disrupting effects of NAs. Significant up-regulated gene expressions of CYP19b, ERα and VTG were observed in both OS-NAs and C-NAs groups, which indicated the deleteriously estrogenic potential of NAs. These results confirmed that NAs derived from crude petroleum could negatively impact the development and endocrine function of zebrafish, and be primarily responsible for the toxicity of OSPW.

New findings in pancreatic and intestinal endocrine development to advance regenerative medicine.

PURPOSE OF REVIEW: We highlight some of the major recent advances that have been made towards understanding the mechanisms that control endocrine differentiation and cell identity in the pancreas and intestine. RECENT FINDINGS: Notch signaling plays a complex role in the fate choice between endocrine, duct, and acinar lineages in the developing pancreas. New approaches to dissecting the role of mesenchymal cells in the developing endocrine pancreas reveal inhibitory signals from the endothelium. Epigenetic mechanisms represent another layer of control over pancreatic development and ß cell identity. Further details on the transcriptional control of enteroendocrine cell development have emerged and revealed a surprising role for FoxO1 in restraining insulin expression in the gut. Incremental progress is being made in the field of directed differentiation of embryonic stem cells to pancreatic ß cells and the first reported differentiation of human embryonic stem cells into intestinal organoids containing enteroendocrine cells represents a major breakthrough. SUMMARY: Greater knowledge of the fundamental processes controlling endocrine development in the pancreas and intestine has the potential to advance the field of regenerative medicine by providing a pathway to successfully create cell types of clinical interest.

Eliminating SF-1 (NR5A1) sumoylation in vivo results in ectopic hedgehog signaling and disruption of endocrine development.

Sumoylation is generally considered a repressive mark for many transcription factors. However, the in vivo importance of sumoylation for any given substrate remains unclear and is questionable because the extent of sumoylation appears exceedingly low for most substrates. Here, we permanently eliminated SF-1/NR5A1 sumoylation in mice (Sf-1(K119R, K194R, or 2KR)) and found that Sf-1(2KR/2KR) mice failed to phenocopy a simple gain of SF-1 function or show elevated levels of well-established SF-1 target genes. Instead, mutant mice exhibited marked endocrine abnormalities and changes in cell fate that reflected an inappropriate activation of hedgehog signaling and other potential SUMO-sensitive targets. Furthermore, unsumoylatable SF-1 mutants activated Shh and exhibited preferential recruitment to Shh genomic elements in cells. We conclude that the sumoylation cycle greatly expands the functional capacity of transcription factors such as SF-1 and is leveraged during development to achieve cell-type-specific gene expression in multicellular organisms.

Expression of ezrin in human embryonic, fetal, and normal adult tissues.

Ezrin, which cross-links the cytoskeleton and plasma membrane, was involved in a wide variety of cellular processes. Here, to investigate the distribution of ezrin, tissue microarray technology was employed to perform immunohistochemical experiments on human embryos, fetuses at 4 to 22 weeks' gestation, and adult tissue specimens. Results showed that ezrin was widely expressed in the gastrointestinal tract throughout the human developmental stages studied. At 6 to 8 weeks' gestation, ezrin was found in epithelial cells, and this staining pattern was particularly pronounced in the brush border of mature absorptive cells lining the villus in later developmental stages and adult tissues. Throughout neural development, ezrin was only expressed in the neural tube at 4 weeks' gestation. Ezrin was also detected in the cortex and medulla of the adrenal gland at 8 to 12 weeks' gestation, whereas its immunoreactivity was increased from the zona glomerulosa through the zona reticularis and was essentially undetectable in the adrenal medulla of adult tissues. Significant expression of ezrin was seen throughout development in the kidney, spleen, lymph nodes, and cells of stratified squamous epithelia. However, ezrin was undetectable in lung, liver, heart, and blood vessels. These results demonstrated that the expression pattern of ezrin was highly time specific and tissue specific.

The origin of neuroendocrine tumors and the neural crest saga.

In this essay, the role of the neural crest in the development of the vertebrate embryo is briefly described. The techniques used to document the neural crest origin of various cell types and the tumors arising from them are discussed, with emphasis on Le Douarin's quail-chick chimera model. The current dogma on the origin of the cells of the diffuse endocrine system is presented, and some personal conjectures based on the microscopic appearances of various types of normal, vestigial and neoplastic human tissues are offered to the reader as 'food for thought.'

Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease.

Fibroblast growth factors (FGFs) are a family of structurally related polypeptides that are essential for embryonic development and that function postnatally as homoeostatic factors, in the response to injury, in the regulation of electrical excitability of cells and as hormones that regulate metabolism. In humans, FGF signalling is involved in developmental, neoplastic, metabolic and neurological diseases. Fgfs have been identified in metazoans but not in unicellular organisms. In vertebrates, FGFs can be classified as having intracrine, paracrine and endocrine functions. Paracrine and endocrine FGFs act via cell-surface FGF receptors (FGFRs); while, intracrine FGFs act independent of FGFRs. The evolutionary history of the Fgf family indicates that an intracrine Fgf is the likely ancestor of the Fgf family. During metazoan evolution, the Fgf family expanded in two phases, after the separation of protostomes and deuterostomes and in the evolution of early vertebrates. These expansions enabled FGFs to acquire diverse actions and functions.

Review: the role of neural crest cells in the endocrine system.

The neural crest is a pluripotent population of cells that arises at the junction of the neural tube and the dorsal ectoderm. These highly migratory cells form diverse derivatives including neurons and glia of the sensory, sympathetic, and enteric nervous systems, melanocytes, and the bones, cartilage, and connective tissues of the face. The neural crest has long been associated with the endocrine system, although not always correctly. According to current understanding, neural crest cells give rise to the chromaffin cells of the adrenal medulla, chief cells of the extra-adrenal paraganglia, and thyroid C cells. The endocrine tumors that correspond to these cell types are pheochromocytomas, extra-adrenal paragangliomas, and medullary thyroid carcinomas. Although controversies concerning embryological origin appear to have mostly been resolved, questions persist concerning the pathobiology of each tumor type and its basis in neural crest embryology. Here we present a brief history of the work on neural crest development, both in general and in application to the endocrine system. In particular, we present findings related to the plasticity and pluripotency of neural crest cells as well as a discussion of several different neural crest tumors in the endocrine system.

The chicken embryo as a model for developmental endocrinology: development of the thyrotropic, corticotropic, and somatotropic axes.

The ease of in vivo experimental manipulation is one of the main factors that have made the chicken embryo an important animal model in developmental research, including developmental endocrinology. This review focuses on the development of the thyrotropic, corticotropic and somatotropic axes in the chicken, emphasizing the central role of the pituitary gland in these endocrine systems. Functional maturation of the endocrine axes entails the cellular differentiation and acquisition of cell function and responsiveness of the different glands involved, as well as the establishment of top-down and bottom-up anatomical and functional communication between the control levels. Extensive cross-talk between the above-mentioned axes accounts for the marked endocrine changes observed during the last third of embryonic development. In a final paragraph we shortly discuss how genomic resources and new transgenesis techniques can increase the power of the chicken embryo model in developmental endocrinology research.

Notch signaling in development and cancer.

Notch is an evolutionarily conserved local cell signaling mechanism that participates in a variety of cellular processes: cell fate specification, differentiation, proliferation, apoptosis, adhesion, epithelial-mesenchymal transition, migration, and angiogenesis. These processes can be subverted in Notch-mediated pathological situations. In the first part of this review, we will discuss the role of Notch in vertebrate central nervous system development, somitogenesis, cardiovascular and endocrine development, with attention to the mechanisms by which Notch regulates cell fate specification and patterning in these tissues. In the second part, we will review the molecular aspects of Notch-mediated neoplasias, where Notch can act as an oncogene or as a tumor suppressor. From all these studies, it becomes evident that the outcome of Notch signaling is strictly context-dependent and differences in the strength, timing, cell type, and context of the signal may affect the final outcome. It is essential to understand how Notch integrates inputs from other signaling pathways and how specificity is achieved, because this knowledge may be relevant for future therapeutic applications.

Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types.

All pancreatic endocrine cells, producing glucagon, insulin, somatostatin, or PP, differentiate from Pdx1+ progenitors that transiently express Neurogenin3. To understand whether the competence of pancreatic progenitors changes over time, we generated transgenic mice expressing a tamoxifen-inducible Ngn3 fusion protein under the control of the pdx1 promoter and backcrossed the transgene into the ngn3(-/-) background, devoid of endogenous endocrine cells. Early activation of Ngn3-ER(TM) almost exclusively induced glucagon+ cells, while depleting the pool of pancreas progenitors. As from E11.5, Pdx1+ progenitors became competent to differentiate into insulin+ and PP+ cells. Somatostatin+ cells were generated from E14.5, while the competence to make glucagon+ cells was dramatically decreased. Hence, pancreas progenitors, similar to retinal or cortical progenitors, go through competence states that each allow the generation of a subset of cell types. We further show that the progenitors acquire competence to generate late-born cells in a mechanism that is intrinsic to the epithelium.

An amylase/Cre transgene marks the whole endoderm but the primordia of liver and ventral pancreas.

Mice bearing a Cre-encoding transgene driven by a compound [SV40 small t antigen/mousealpha-amylase-2] promoter expressed the recombinase at early developmental stages broadly in the embryonic endoderm before the pancreas and lungs begin to outgrow, but not in other germ layers, as determined indirectly by beta-galactosidase and YFP reporter activity, indicating that the transgene is in fact an endodermic marker. Interestingly, the liver and ventral pancreas were excluded from this expression pattern, denoting that the chimerical alpha-amylase-2 promoter was not active in the anterior leading edge of the endoderm (the presumptive region from which liver and ventral pancreas form). These transgenics thus confirm, among other findings, that dorsal and ventral pancreatic primordia have different intrinsic transcriptional capabilities. In conclusion, we have generated a new transgenic mouse that should be useful to target endoderm at early stages, without affecting the liver or ventral pancreas before embryonic day E12.5.

The proneural gene ascl1a is required for endocrine differentiation and cell survival in the zebrafish adenohypophysis.

Mammalian basic helix-loop-helix proteins of the achaete-scute family are proneural factors that, in addition to the central nervous system, are required for the differentiation of peripheral neurons and sensory cells, derivatives of the neural crest and placodal ectoderm. Here, in identifying the molecular nature of the pia mutation, we investigate the role of the zebrafish achaete-scute homologue ascl1a during development of the adenohypophysis, an endocrine derivative of the placodal ectoderm. Similar to mutants deficient in Fgf3 signaling from the adjacent ventral diencepahalon, pia mutants display failure of endocrine differentiation of all adenohypophyseal cell types. Shortly after the failed first phase of cell differentiation, the adenohypophysis of pia mutants displays a transient phase of cell death, which affects most, but not all adenohypophyseal cells. Surviving cells form a smaller pituitary rudiment, lack expression of specific adenohypophyseal marker genes (pit1, neurod), while expressing others (lim3, pitx3), and display an ultrastructure reminiscent of precursor cells. During normal development, ascl1a is expressed in the adenohypophysis and the adjacent diencephalon, the source of Fgf3 signals. However, chimera analyses show that ascl1a is required cell-autonomously in adenohypophyseal cells themselves. In fgf3 mutants, adenohypophyseal expression of ascl1a is absent, while implantation of Fgf3-soaked beads into pia mutants enhances ascl1a, but fails to rescue pit1 expression. Together, this suggests that Ascl1a might act downstream of diencephalic Fgf3 signaling to mediate some of the effects of Fgf3 on the developing adenohypophysis.

Members of the Plag gene family are expressed in complementary and overlapping regions in the developing murine nervous system.

In the developing nervous system, cell fate specification and proliferation are tightly coupled events, ensuring the coordinated generation of the appropriate numbers and correct types of neuronal and glial cells. While it has become clear that tumor suppressor genes and oncogenes are key regulators of cell division in tumor cells, their role in normal cellular and developmental processes is less well understood. Here we present a comparative analysis of the expression profiles of the three members of the pleiomorphic adenoma gene (Plag) family, which encode zinc finger transcription factors previously characterized as tumor suppressors (Zac1) or oncogenes (Plag1, Plag-l2). We focused our analysis on the developing nervous system of mouse where we found that the Plag genes were expressed in both unique and overlapping patterns in the central and peripheral nervous systems, and in olfactory and neuroendocrine lineages. Based on their patterns of expression, we suggest that members of the Plag gene family might control cell fate and proliferation decisions in the developing nervous system and propose that deciphering these functions will help to explain why their inappropriate inactivation/activation leads to tumor formation.

Macrophages in the murine pancreas and their involvement in fetal endocrine development in vitro.

Macrophages are a heterogeneous population of cells that belong to the mononuclear phagocyte system. They play an important role in tissue homeostasis and remodeling and are also potent immune regulators. Pancreatic macrophages are critically involved in the development and pathogenesis of autoimmune diabetes. To elucidate the ontogeny of pancreatic macrophages, we characterized in this study the macrophages present in the adult and developing fetal pancreas of normal mice. We additionally examined the presence of local macrophage precursors and the involvement of macrophages in the growth of endocrine tissue in the fetal pancreas. We identified two phenotypically distinct macrophage subsets in the adult pancreas. The majority of macrophages was CD45(+)ER-MP23(+)MOMA-1(+). Under noninflammatory conditions, only a minority ( approximately 5%) of the pancreatic macrophages additionally expressed the macrophage marker F4/80. In contrast, in the fetal pancreas, phenotypically, mature macrophages were identified exclusively by their expression of F4/80 and lacked detectable staining with ER-MP23 and MOMA-1 antibodies. In fetal pancreas organ cultures, we could show that macrophages develop from pre-existing precursors, which are present in the fetal pancreas at embryonic age 12.5. Moreover, the number of macrophages increased significantly when macrophage-colony stimulating factor was added to these cultures. It is important that this increase of F4/80-positive cells was paralleled by an increase in the number of insulin-producing cells, suggesting that macrophages support the growth of these endocrine cells.

Hedgehog signaling regulates expansion of pancreatic epithelial cells.

Embryonic Hedgehog signaling is essential for proper tissue morphogenesis and organ formation along the developing gastrointestinal tract. Hedgehog ligands are expressed throughout the endodermal epithelium at early embryonic stages but excluded from the region that will form the pancreas. Ectopic activation of Hedgehog signaling at the onset of pancreas development has been shown to inhibit organ morphogenesis. In contrast, Hedgehog signaling components are found within pancreatic tissue during subsequent stages of development as well as in the mature organ, indicating that a certain level of pathway activation is required for normal organ development and function. Here, we ectopically activate the Hedgehog pathway midway through pancreas development via expression of either Sonic (Shh) or Indian Hedgehog (Ihh) under control of the human Pax4-promoter. Similar pancreatic defects are observed in both Pax4-Shh and Pax4-Ihh transgenic lines, suggesting that regulation of the overall level of Hedgehog activity is critical for proper pancreas development. We also show that Hedgehog signaling controls mesenchymal vs. epithelial tissue differentiation and that pathway activation impairs formation of epithelial progenitors. Thus, tight control of Hedgehog pathway activity throughout embryonic development ensures proper pancreas organogenesis.

Relationship between ethinylestradiol-mediated changes in endocrine function and reproductive impairment in Japanese medaka (Oryzias latipes).

Many biochemical endpoints currently are used to describe endocrine function in fish; however, the sensitivity of these parameters as biomarkers of impaired reproduction or sexual development is not well understood. In the present study, adult Japanese medaka (Oryzias latipes) were assessed for reproductive output and endocrine function, including circulating steroid concentrations, ex vivo steroidogenesis from the gonads, aromatase activity, hepatic estrogen receptor (ER), and plasma vitellogenin (VTG) after exposure to 0, 0.2, 5, 500, and 2,000 ng/L of 17alpha-ethinylestradiol (EE) for 14 d. The EE altered these biochemical responses at various sites along the hypothalamus-pituitary-gonadal axis at concentrations as low as 0.2 ng/L, but it only depressed reproductive function at concentrations of 500 ng/L or greater. Offspring also had reduced ability to hatch at 500 ng/L of EE, but this concentration did not produce any other observed changes in development or sexual phenotype. The reproductive parameters correlated well with VTG, ER, and gonadosomatic index (GSI) in both sexes of adult medaka, which could be indicative of the ER-mediated mode of action for EE. Vitellogenin and ER were elevated at higher concentrations of EE in both sexes, whereas GSI was decreased. Overall, most biochemical endpoints were more sensitive than reproduction or development to exposure, indicating that reproductive function may be relatively protected.

Development of pancreatic islets in pancreatic polypeptide-overexpressing mice.

Recently we produced pancreatic polypeptide transgenic (PPTG) mice and found that PP was overexpressed in pancreatic islets. The present study examines development of four islet hormones in PPTG mice at embryonic days (ED) 15, 17, and 19, and in adult animals. Adult PPTG mice showed massive aggregation of PP-positive cells and glucagon-positive cells seen at the central area of the islets. Confocal laser microscopic study showed that three islet hormones (insulin, glucagon and PP) were completely overlapped in islets of PPTG mice. Overlapping of somatostatin/glucagon and somatostatin/PP were also increased at the peripheral area of the islets in adult PPTG mice compared to wild-type mice. In prenatal development of pancreatic islets of PPTG mice, somatostatin/glucagon overlapping cells appeared at ED 15, two days earlier than in wild-type mice. Differentiation of these somatostatin/glucagon double-positive cells into single-positive cells was disturbed in the PPTG mice during perinatal to postnatal periods. Differentiation of glucagon/insulin-double positive cells into single-positive cells was disturbed remarkably in postnatal development of the islets of PPTG mice. The present results suggest that early and overexpression of PP may engender the early appearance of somatostatin producing cells; however, that may disturb differentiation of multihormonal immature endocrine cells into single hormonal mature endocrine cells.

The complex between urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-I) independently predicts response to first-line endocrine therapy in advanced breast cancer.

It has been shown that urokinase-type plasminogen activator (uPA) and its main inhibitor (PAI-I) have predictive value for therapy success in advanced breast cancer. Levels of the complex between uPA and PAI-I, formed when both molecules are in their active form, might have superior predictive power. Here, we investigate the association between levels of uPA:PAI-I complex and rate of response to first-line systemic therapy for advanced breast cancer. Tumor tissues of 170 patients with advanced breast cancer were analyzed for uPA:PAI-I complex concentrations using a quantitative enzyme-linked immunosorbent assay. The patients received either endocrine therapy (n=96) or chemotherapy (n=74) as first-line treatment after diagnosis of advanced disease. Of the endocrine treated patients, those with high levels of uPA:PAI-I complex showed a shorter progression-free survival (PFS) compared to patients with lower uPA:PAI-I complex levels (P=0.035). Furthermore, in the multivariate regression analysis a significant lower rate of response to first-line endocrine therapy was found in patients with high uPA:PAI-I complex levels compared to patients with low uPA:PAI-I complex levels (odds ratio (OR)=0.27, 95% CI, 0.09-0.59, P=0.018), in addition to the predictive impact of the steroid hormone receptor (ER/PgR) status (OR=2.68, 95% CI, 1.08-6.63, P=0.033). Complex levels did not predict efficacy of chemotherapy in patients with advanced breast cancer. The results show that the plasminogen activation system affects the response to endocrine therapy independent of steroid hormone receptor status and may be of help to further refine the indication for this treatment in individual patients. Further studies are warranted to explain this underlying resistance to endocrine therapy when uPA:PAI-I levels are high.