ABSTRACT
The non-inducible chaperone heat shock cognate 70 kDa (Hsc70) is regulated during development. We now characterize its dynamic expression pattern from gastrulation to early organogenesis. Throughout this developmental period, hsc70 transcripts were largely restricted to neuroectoderm- and mesoderm-derived structures. In stage 10 embryos, Hsc70 protein was expressed in the neural tube with increasing rostrocaudal and decreasing dorsoventral gradients, and in some somite cells. This highly regulated expression of Hsc70 is likely to reflect specific developmental functions, besides its well-characterized role in protein folding.
Subject(s)
Carrier Proteins/genetics , HSP70 Heat-Shock Proteins , Molecular Chaperones/genetics , Animals , Chick Embryo , Gene Expression Regulation, Developmental , HSC70 Heat-Shock Proteins , In Situ Hybridization , Nervous System/embryology , Nervous System/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
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.
Subject(s)
Apoptosis/physiology , Carrier Proteins/physiology , Heat-Shock Proteins , Molecular Chaperones/physiology , Proinsulin/physiology , Amino Acid Sequence , Animals , Apoptosis/genetics , Base Sequence , Carrier Proteins/genetics , Chick Embryo , Cloning, Molecular , Cysteine Endopeptidases/physiology , DNA Primers/genetics , DNA, Complementary/genetics , Endoplasmic Reticulum Chaperone BiP , Gene Expression Regulation, Developmental , HSC70 Heat-Shock Proteins , HSP70 Heat-Shock Proteins/genetics , Humans , Molecular Chaperones/genetics , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
We recently demonstrated that thyroid hormone is an important regulator of mitochondrial gene expression during brain development. To gain further insights into the consequences of this regulation, we have performed functional and structural analysis of brain mitochondria from control and hypothyroid neonatal rats. Flow cytometric analysis showed a significant decrease in the mitochondrial transmembrane potential in hypothyroid animals compared with controls, which was reversed after 48 h, but not after 2 h, of thyroid hormone administration, suggesting that the functional alterations observed are the consequence of changes in mitochondrial gene expression. In addition, band shift studies showed a protein bound to the rat mitochondrial promoter differentially regulated by thyroid state. Electron microscopic analysis of cerebral cortex, striatum, and hippocampus revealed marked differences in the morphology of neuronal mitochondria from control and hypothyroid neonates. Hypothyroid mitochondria presented a decrease in the area of the inner membrane plus cristae in all areas studied, except for the hippocampal CA1 neurons and nonneuronal cell types. The observations reported here provide a basis for the known biochemical action of thyroid hormone on brain development.
Subject(s)
Animals, Newborn , Brain/growth & development , Hypothyroidism/pathology , Membrane Potentials , Mitochondria/ultrastructure , Neurons/ultrastructure , Animals , Brain/ultrastructure , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/genetics , Female , Flow Cytometry , Hypothyroidism/physiopathology , Intracellular Membranes/physiology , Microscopy, Electron , Promoter Regions, Genetic , Rats , Rats, Wistar , Triiodothyronine/pharmacologyABSTRACT
The effect of thyroid hormone and retinoic acid on the expression of CCAAT/enhancer binding proteins (C/EBPs) alpha and beta was investigated in rat liver during development. Congenital hypothyroidism caused a significant decrease in both C/EBP alpha and C/EBP beta gene expression at early stages of postnatal development. This effect was tissue specific since thyroid hormone had no effect on the level of C/EBP mRNAs in brown fat. Injection of 15-, and 30-day-old hypothyroid animals with thyroid hormone resulted in a slow recovery of the hepatic levels of both C/EBP alpha and beta mRNA levels. Retinoic acid was a very rapid and potent stimulator compared to thyroid hormone in hypothyroid animals. C/EBP alpha and beta protein levels were markedly diminished in hypothyroid neonates and the kinetics of induction of these proteins by thyroid hormone was faster than the one observed for the corresponding transcripts. The discrepancies observed between mRNA and protein levels suggest a translational or post-translational regulation of these genes as the major point of thyroid hormone action on these genes.
Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation/drug effects , Liver/drug effects , Nuclear Proteins/genetics , Tretinoin/pharmacology , Triiodothyronine/pharmacology , Animals , Animals, Newborn , CCAAT-Enhancer-Binding Proteins , Liver/growth & development , Liver/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RatsABSTRACT
Thyroid hormone (T3) plays a critical role in the development of the central nervous system and its deficiency during the early neonatal period results in severe brain damage. However the mechanisms involved and the genes specifically regulated by T3 during brain development are largely unknown. By using a subtractive hybridization technique we have isolated a number of cDNAs that represented mitochondrial genes (12S and 16S rRNAs and cytochrome c oxidase subunit III). The steady state level of all three RNAs was reduced in hypothyroid animals during the postnatal period and T3 administration restored control levels. During fetal life the level of 16S rRNA was decreased in the brain of hypothyroid animals, suggesting a prenatal effect of thyroid hormone on brain development. Since T3 does not affect the amount of mitochondrial DNA, the results suggest that the effect of T3 is at transcriptional and/or postranscriptional level. In addition, the transcript levels for two nuclear-encoded mitochondrial cytochrome c oxidase subunits: subunits IV and VIc were also decreased in the brains of hypothyroid animals. Hypothyroidism-induced changes in mitochondrial RNAs were followed by a concomitant 40% decrease in cytochrome c oxidase activity. This study shows that T3 is an important regulator of mitochondrial function in the neonatal brain and, more importantly, provides a molecular basis for the specific action of this hormone in the developing brain.
