Expression of insulin-like growth factor-I gene in normal and diabetic rat eye

The expression of insulin-like growth factor I (IGF-I) gene was studied in both normal and diabetic rat eyes via in situ hybridization. The results revealed the regional expression of IGF-I gene in the rat eyes. The expression of IGF-I mRNA in the internal nuclear layer and ganglion cell layer is strong, in choroid, retinal pigment epithelium and external nuclear layer is moderate, in sclera is weak in degree and no such mRNA is detected in the cornea. The abundance of IGF-I mRNA in diabetic eye tissues is significantly (P < 0.05 or P < or = 0.01) higher than that in normal eye. These findings suggest that (1) functionally, the eye ball be considered to be an "IGF-I paracrine-autocrine system", and (2) the high expression of IGF-I indicate its initiation of diabetic retinopathy and its promotion of the progression of the lesion.     

PA26, a novel target of the p53 tumor suppressor and member of the GADD family of DNA damage and growth arrest inducible genes

We report the cystathionine-beta synthase (CBS) gene expression pattern during early human embryogenesis (3 to 6 weeks post conception) by in situ hybridization and in fetal and adult tissue by Northern Blot analysis. Probes were chosen to recognize either the common sequence to all known CBS mRNAs or the sequences of two different major exons 1 issued of we have previously identified. We demonstrate by in situ hybridization that CBS is continuously expressed from the earliest stages studied (22 days post conception) during embryogenesis in the tissues of developing embryos which will after birth present clinical abnormalities in homocystinuria patients. It is expressed at an especially high level in the neural and cardiac systems until the liver primordium appears. In embryonic central nervous system, the whole neural tube and primary brain vesicles are labeled. Secondary brain vesicles labeling are dependent on the neuroepithelium differentiation. The ventricular layer of the rhombencephalon, cranial nerve nuclei and then after cerebellar cortex derived from rhombencephalon ventricular layer are strongly labeled. Thalamus and other derivatives of the diencephalon plate, the neuroblastic layer of the retina, lens and dorsal root ganglia are labeled. After 35 days post conception, CBS mRNAs was detected in endocardial cells and in cells derived from the neural crest of the heart and in particular developing mesodermic regions such as the primitive hepatocytes of the liver, mesonephros vesicles, various endocrine glands and developing bones. We could not detect tissue specificity of different probes at this embryonic stage. Northern blot analysis consistently detected mRNA species in fetal 25 weeks post conception brain, liver and kidney. The common cDNA probe revealed the 2.5 and 3.7 kb mRNA species from brain, liver and kidney. The exon 1b probe detected only the 2.5 kb mRNA and the exon 1c probe the 3.7 kb mRNA in these three tissues. In adult tissue, the 1b probe detected only the 2.5 kb mRNA and the 1c probe only the 3.7 kb mRNA in the liver.     

Modulation of the chaperone heat shock cognate 70 by embryonic (pro)insulin correlates with prevention of apoptosis

Insights have emerged concerning insulin function during development, from the finding that apoptosis during chicken embryo neurulation is prevented by prepancreatic (pro)insulin. While characterizing the molecules involved in this survival effect of insulin, we found insulin-dependent regulation of the molecular chaperone heat shock cognate 70 kDa (Hsc70), whose cloning in chicken is reported here. This chaperone, generally considered constitutively expressed, showed regulation of its mRNA and protein levels in unstressed embryos during early development. More important, Hsc70 levels were found to depend on endogenous (pro)insulin, as shown by using antisense oligodeoxynucleotides against (pro)insulin mRNA in cultured neurulating embryos. Further, in the cultured embryos, apoptosis affected mainly cells with the lowest level of Hsc70, as shown by simultaneous Hsc70 immunostaining and terminal deoxynucleotidyltransferase-mediated UTP nick end labeling. These results argue in favor of Hsc70 involvement, modulated by embryonic (pro)insulin, in the prevention of apoptosis during early development and suggest a role for a molecular chaperone in normal embryogenesis.     

Porcine somatotropin (pST) increases IGF-I mRNA abundance in liver and subcutaneous adipose tissue but not in skeletal muscle of growing pigs

The present study was undertaken to determine the effects of exogenous porcine somatotropin (pST) on IGF-I gene expression in liver, skeletal muscle (longissimus dorsi), and s.c. adipose tissue of growing pigs. Twenty prepubertal gilts (approximately 60 kg BW) were allotted to four treatment groups (n = 5) and treated with either 0, 35, 70, or 140 micrograms/kg BW of recombinantly derived pST by daily i.m. injection for 7 d. Serum concentrations of IGF-I were determined by RIA and IGF-I mRNA levels were determined by direct counting of individual samples on slot blots. Administration of pST increased IGF-I concentration in serum. This was accompanied by significant increases (P < .05) in IGF-I mRNA abundance in liver and s.c. adipose tissue; the effects were maximal at the lowest dose of pST. Insulin-like growth factor I mRNA levels were increased 2.5- and 4.5-fold, respectively. Levels of IGF-I mRNA were very low in longissimus muscle and were unaffected by administration of pST. When expressed as picograms of mRNA/10 micrograms of total RNA, IGF-I mRNA levels were highest in s.c. adipose tissue. Levels of IGF-I mRNA were 1.9-fold higher in s.c. adipose tissue than in liver of control animals, and pST administration increased this difference to 3.2-fold. Our results suggest that 1) the effects of pST administered by daily i.m. injection on IGF-I gene expression in pigs are tissue-specific and 2) the stimulatory effects of pST administered in this manner on muscle growth in pigs are not associated with increased expression of the IGF-I gene in skeletal muscle.     

Early ontogeny of catecholaminergic cell lineage in brain and peripheral neurons monitored by tyrosine hydroxylase-lacZ transgene

As the first and rate limiting enzyme in the biosynthetic pathway for catecholamine (CA) neurotransmitters, tyrosine hydroxylase (TH) is a specific phenotypic marker for CA cells in the central and peripheral nervous systems of adult animals. During embryogenesis, TH expression appears permanently within cells destined to be CA-secreting during adult life, and transiently in several cell types that will not express TH in adulthood. In this study, we examined the early ontogeny of TH expression in transgenic mouse embryos by following the expression of a lacZ reporter, driven by the tissue-specific promoter of the rat TH gene. The lacZ reporter product, beta-galactosidase (beta-gal), visualized by X-gal staining, first became apparent in primordia of sensory ganglia serving the glossopharyngeal (IX) and vagal (X) cranial nerves at embryonic day (E)9.0. Between E9.5 and E10.5, beta-gal expression extended to the remaining cranial sensory ganglia serving the trigeminal (V) and facial (VII) nerves, dorsal root ganglia, ventrolateral neural tube and sympathetic ganglion primordia. During that same period, the first beta-gal expression in the embryonic brain also appeared within distinct regions, such as the ventral prosencephalon, the ventral and dorsolateral mesencephalon and the rostral and caudal rhombencephalon. The level of beta-gal expression in all these tissues decreased at E13.5, but a distinct adult pattern of beta-gal expression started to emerge in the substantia nigra and ventral tegmental area in the central nervous system and the adrenal medulla in the periphery. Our findings indicate that the proximal 9.0 kb of the 5' promoter region of the rat TH gene encodes sufficient information to direct development of the appropriate catecholaminergic lineage cells in the central and most peripheral nervous systems during embryogenesis.     

Expression of galanin and a galanin receptor in several sensory systems and bone anlage of rat embryos

Using in situ hybridization and immunohistochemistry the expression of, respectively, prepro-galanin (pre-pro-GAL) mRNA and GAL receptor-1 mRNA, as well as GAL-like and GAL message-associated peptide-like immunoreactivities, were studied in rats from embryonic day 14 (E14) to postnatal day 1. GAL expression was observed already at E14 in trigeminal and dorsal root ganglion neurons and at E15 in the sensory epithelia in developing ear, eye, and nose, as well as at E19 during bone formation. Also, GAL receptor-1 mRNA was expressed in the sensory ganglia of embryos but appeared later than the ligand. These findings suggest that GAL and/or GAL message-associated peptide may have a developmental role in several sensory systems and during bone formation.     

Direct evidence that FK506 inhibition of FcepsilonRI-mediated exocytosis from RBL mast cells involves calcineurin

The development of human endocrine pancreas has been the subject of many immunohistochemical studies but very little is known at the molecular level. We have determined the patterns of gene expression of glucagon, somatostatin and pancreatic polypeptide during fetal life (16-41 weeks of gestation) using the dot-blot hybridization method. In spite of some dispersion in the mRNA levels, no progressive increase or decrease during this period of fetal life could be found, as previously observed for insulin. In keeping with these molecular data, no increase in immunostaining of the four hormones was observed, but a dispersion of endocrine cells within the exocrine tissue was noticed at 20 weeks of gestation followed by a clear differentiation of the Langerhans islets at 31 weeks. Interestingly, the mRNA levels of the four hormones were always higher in the fetal pancreas than in the adult pancreas.