The genes encoding mammalian subtilisin-like endoproteases furin, PC1, and PC2 have been isolated and are implicated in endoproteolytic cleavage of precursor molecules, which is a key step in posttranslational maturation of proproteins and neuropeptide precursors. Following endoproteolytic cleavage, the carboxyl-terminal basic amino acid residues are removed by carboxypeptidase E (CPE). We have examined the expression of these genes during rat development by in situ hybridization and compared their expression patterns to those of potential substrates. In the primitive streak stage of embryogenesis (e7) furin is expressed in both endoderm and mesoderm. This overall expression pattern is maintained until e10, when a distinctly higher level of furin expression is observed in the heart and liver primordia. In mid- and late gestational stages furin is broadly expressed in the peripheral tissues, and, therefore, may contribute to the proteolytic processing of numerous fetal proproteins, such as the precursors for natriuretic factors in heart and IGF-II throughout the embryo. In contrast, the expressions of PC1 and PC2 are initiated much later (e13) and are mainly confined to the developing nervous system, but with distinct spatial distributions. At midgestational ages, PC1 mRNA is mainly expressed in the hypothalamus and peripheral ganglia, while PC2 is expressed not only in these tissues but also in the thalamus, midbrain, pons, medulla oblongata, cortical plate, and spinal cord. Besides neuropeptide precursor processing in the nervous system, PC1 and PC2 may also be involved in the proteolytic processing in additional regions as evidenced by the finding that both PC1 and PC2 mRNAs are expressed in the embryonic pituitary and pancreas. CPE mRNA is expressed in both neural tissues and some non-neural tissues. In the developing nervous system, the expression of CPE encompasses all the regions where PC1 and PC2 are expressed and in fact includes most brain regions as neurogenesis proceeds. CPE mRNA is also expressed in some peripheral tissues, such as the embryonic heart and cartilage primordia, and in some cases its expression overlaps with furin expression. Thus, CPE may functionally collaborate during development with the subtilisin family of endoproteases in the completion of proteolytic processing of neuropeptide precursors in the nervous system and proproteins in the peripheral tissues. In the pituitary, the endoproteolytic processing of polyfunctional precursor proopiomelanocortin (POMC) occurs in a developmentally regulated manner. We have shown that while PC2 mRNA is predominantly expressed in the intermediate lobe in the adult, we observed an increased expression of PC2 mRNA in developing rat anterior lobe, peaking at early postnatal stages.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND:The European Centre for the Validation of Alternative Methods (ECVAM) designed the Embryonic Stem Cell Test (EST) as a tool for classifying developmentally toxic compounds. An in vitro tool to assess developmental toxicity would be of great value to the pharmaceutical industry to help with toxicity-associated attrition. METHODS: ECVAM's EST protocol was used, but employing a different mouse embryonic stem cell (ESC) line and an alternative differentiation medium. A subset of the compounds used to validate the EST assay along with a number of in-house pharmaceutical compounds plus marketed pharmaceutical compounds were used to assess the EST performance with receptor-mediated compounds. RESULTS: Our results with ECVAM compounds mirrored ECVAM's. Compounds that were developmentally toxic in vivo were classified by the EST as moderate risk. Overall, the accuracy was 75% with the current set of data and the predictivity of low-, moderate-, and high-risk compounds was 90, 71, and 60% while the precision was 59, 86, and 100%, respectively. Interestingly, a number of the non-developmentally toxic compounds had values for the 3T3 IC 50 values, which were lower than the ESC IC 50 and ID 50, a situation not taken into account by ECVAM when designing the EST algorithm. CONCLUSIONS: The assay as currently constructed has a significant falsepositive rate (B40%), but a very low false-negative rate (B7%). Additional moderate-and high-risk compounds need to be assessed to increase confidence, accuracy, and understanding in the EST's predictivity. Birth Defects Res (Part B) 83: 104-111, 2008.

Insulin-like growth factors (IGFs), when isolated from serum or tissue fluids, are usually found as part of a protein complex which also contains one of several IGF binding proteins (IGFBPs). Although some IGFBPs have been shown to alter interactions of IGFs with their receptors in vitro and can modify the responses of cultured cells to exogenous IGFs, the in vivo functions of IGFBPs remain unclear. This study examines expression of a recently described IGFBP gene, IGFBP-5, in the rat embryo and fetus and in selected adult tissues. Embryonic IGFBP-5 messenger RNA (mRNA) can be detected as early as embryonic day 10.5 and has an mRNA expression pattern distinct from the previously characterized pattern of IGFBP-2 mRNA expression. Major sites of IGFBP-5 expression during early postimplantation stages of development include the notochord, the floor plate, regions of the surface ectoderm, muscle precursor cells, and specific axial regions of neuroepithelium. Later in development IGFBP-5 mRNA is found in several regions of the central nervous system, including the proliferative zone of the external granule layer of the cerebellum and the mitral neurons of the olfactory bulb, as well as in muscle precursor populations of the developing limb, and in most cells of the anterior pituitary. In addition, only a subset of pituicytes in the adult posterior pituitary express IGFBP-5, which provides the first evidence that this cell population is biochemically heterogeneous. Taken together, these data suggest functions for IGFBP-5 during development of several organ systems.

No abstract

As background for an antibody-based therapeutic program against the IGF receptor, we undertook a review of available information on the early pregnancy-specific regulation and localization of IGFs, IGF-binding proteins (BPs), IGFBP-specific proteases, and the type 1 IGF receptor relative to placental maintenance, function of placental nutrient transporters, placental cellular differentiation/turnover/apoptosis, and critical hormone signaling needed to maintain pregnancy. Possible adverse outcomes of altered IGF signaling include prenatal loss, fetal growth retardation, and maldevelopment are also discussed. It appears that the IGF axes in both the conceptus and mother are important for normal embryo-fetal growth. Thus, all molecules (i.e., both small and large) that disrupt the IGF axis could be expected to have some degree of fetal consequences.