We have previously observed that an axon-sparing injury to the developing striatum induced by the excitotoxin quinolinate results in a decrease in dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the adult. This decrease occurs in the absence of direct injury to the SNpc. As the striatum is a major target for the SNpc dopaminergic system, we have hypothesized that a decrease in the size of the striatal target during development may result in an induced regressive event in the SNpc, similar to what has been described for many developing neural systems with peripheral targets. We have examined by morphologic and biochemical means the time course and character of cell death in SN following a unilateral striatal lesion with quinolinate in immature rats. The striatal lesion is associated with an induced cell death event in the ipsilateral SN, observed first in SNpc and then in SN pars reticulata. The morphologic characteristics of the dying cells were typical of apoptosis. Immunostaining for tyrosine hydroxylase indicated that some of the apoptotic cells in the SNpc were dopaminergic. We conclude that developmental striatal excitotoxic injury is associated with induced apoptotic cell death in SN.

pMHC25, a recombinant plasmid containing myosin heavy chain (MHC) cDNA sequences from differentiated myotubes of the L6E9 rat cell line, has been shown to hybridize to all sarcomeric MHC mRNAs so far tested but not to nonsarcomeric MHC mRNAs. In addition, pMHC25 hybridizes to multiple restriction endonuclease-digested fragments of rat genomic DNA corresponding to different MHC genomic sequences. Thus, the MHC gene represented by pMHC25 is a member of a sarcomeric MHC multigene family that has regions of sequence homology shared among its members. This sarcomeric MHC multigene family has been estimated to be composed of a minimum ofseven genes, some ofwhich are polymorphic in the rat. We have also determined that pMHC25 hybridizes to MHC gene sequences in genomic DNA of all species that have striated muscle, ranging from nematodes to man. Sarcomeric MHC genes, therefore, have been horizontally and vertically conserved in evolution. Additionally, we have used the pMHC25 plasmid to demonstrate that MHC genes do not undergo rearrangement or amplification during muscle cell differentiation.Myosin heavy chain (MHC), one of the major contractile proteins, is found in all muscle and nonmuscle cells (1-4). Immunological studies, peptide mapping, amino acid sequence analysis, as well as cDNA and genomic DNA cloning have demonstrated that MHC exists in several forms (5-13). The expression of some of these forms is tissue specific and developmentally regulated (5,6,12,14,15) MATERIALS AND METHODSCells and Tissue Culture. The rat myogenic L6E9 cell line (18), a subclone of the L6 line (19), was maintained as growing undifferentiated myoblasts and induced to differentiate according to described protocols (18).RNA Isolation. Total cytoplasmic RNA was isolated from growing L6E9 myoblasts, differentiated L6E9 myotubes, and rat primary fibroblasts as described (20). Total RNA was isolated from rat skeletal, cardiac, uterine, and fetal muscle tissues by using the hot phenol procedure (21).Electrophoresis of RNA on Agarose Gels, Transfer to Nitrocellulose Paper, and Hybridization with Plasmid DNA. Total RNA was fractionated by size on 1% agarose/3% (vol/vol) formaldehyde/20 mM 4-morpholinepropanesulfonic acid, pH 7.4/1 mM EDTA gels. Transfer to nitrocellulose filters was done as described (22). Hybridization was performed at 40°C for 16 hr in a mixture containing 50% (vol/vol) formamide, 750 mM NaCl, 75 mM sodium citrate, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% NaDodSO4, sonicated denatured calf thymus DNA at 250 ,ug/ml, 25 mM sodium phosphate buffer (pH 6.5), 10% dextran sulfate, and 8 X 105 cpm/ml ofplasmid [32P]DNA labeled by nick-translation (23) to a specific activity of greater than 1 X 108 cpm/,ug. The filters were washed in several changes of 15 mM NaCl/1.5 mM sodium citrate/0.2% NaDodSO4 at 55°C.Electrophoresis of Genomic DNA, Transfer to Nitrocellulose Paper, and Hybridization with Plasmid DNA. High molecular weight DNA from different sources was prepared essentially as described ...

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The concentration of the peptide mitogen epidermal growth factor (EGF) is hormonally and developmentally regulated in the granular convoluted tubule cells of the mouse submandibular gland. Using a labeled EGF nucleic acid probe, we have demonstrated that submandibular gland EGF mRNA concentrations increase during postnatal development of the gland and after the administration of testosterone or thyroid hormone. Recently, it was reported that EGF mRNA is present in kidney as well as a number of other mouse tissues. A comparison of EGF gene regulation in submandibular gland and kidney revealed that kidney EGF mRNA levels also increase during the postnatal period. Opposite sex differences were observed, with submandibular gland levels being about 16-fold higher in the male than in the female and kidney levels being 2- to 4-fold higher in the female than in the male. Renal EGF mRNA concentrations are less responsive to hormones than those in the submandibular gland. Renal EGF was localized immunocytochemically to the cells of distal convoluted tubules.

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