In situ hybridization: use of 35S-labeled probes on paraffin tissue sections.

The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with (35)S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided.

Timed appearance of lymphocytic choriomeningitis virus after gastric inoculation of mice.

Arenaviruses present an emerging health threat in agrarian areas of Africa and South America; however, the natural routes of arenaviral infections are not clearly understood. Our previous studies with lymphocytic choriomeningitis virus (LCMV), the prototype arenavirus, implicate oral and intragastric routes as natural routes of infection. Our studies raised many questions about the primary site of infection and the route of dissemination after gastric infection. In this report, we use in situ hybridization to detect LCMV in various organs at different time points (0 to 96 hours). After gastric inoculation, the gastric mucosa is the initial site of viral infection, followed by infection of the spleen and liver, then ileum and last, lung, kidney, brain, and esophagus. Furthermore, our observations suggest that virus is disseminated lymphatically rather than by a hematogenous route. Infectious center assays using mononuclear cells from stomach, blood, and spleen of mice infected by the gastric route confirmed active infection with LCMV and the presence of mononuclear cells producing infectious virus in these tissues. This is the first identification of gastric epithelia as a primary site of virus infection.

In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse.

We have developed an in vitro gene trap screen for novel murine genes that allows one to determine, prior to making chimeric or transgenic animals, if these genes are expressed in one or more specific embryonic tissues. Totipotent embryonic stem (ES) cells are infected with a retroviral gene trap construct encoding a selectable lacZ/neo fusion gene, which is expressed only if the gene trap inserts within an active transcription unit. G418-resistant ES cell clones are induced to differentiate in vitro, and neurons, glia, myocytes, and chondrocytes are screened for expression of beta-galactosidase (beta-gal). cDNAs of the gene trap transcripts are obtained by 5' rapid amplification of cDNA ends and are sequenced to determine if they represent novel genes. In situ hybridization analyses show that trapped genes are expressed in vivo within the cell types that express beta-gal in vitro. Gene traps and their wild-type alleles are characterized in terms of copy number, alternate splicing of their transcripts, and the proportion of endogenous mRNA sequence that is replaced by lacZ/neo in the hybrid gene trap transcript. This approach, which we term "in vitro preselection," is more economical than standard in vivo gene trap screening because tissue-specific expression of probable knockout alleles is verified before transgenic animals are generated. These results also highlight the utility of ES cell differentiation in vitro as a method with which to study the molecular mechanisms regulating the specification and commitment of a variety of cell and tissue types.

Cardiomyopathy in transgenic myf5 mice.

To explore the compatibility of skeletal and cardiac programs of gene expression, transgenic mice that express a skeletal muscle myogenic regulator, bmyf5, in the heart were analyzed. These mice develop a severe cardiomyopathy and exhibit a significantly shorter life span than do their nontransgenic littermates. The transgene was expressed from day 7.5 post coitum forward, resulting in activation of skeletal muscle genes not normally seen in the myocardium. Cardiac pathology was not apparent at midgestation but was evident by day 2 of postnatal life, and by 42 days, hearts exhibited multifocal interstitial inflammation, fibrosis, cellular hypertrophy, and occasional myocyte degeneration. All four chambers of the heart were enlarged to varying degrees, with the atria demonstrating the most significant hypertrophy (>100% in 42-day-old mice). The transgene and several skeletal muscle-specific genes were expressed only in patchy areas of the heart in heterozygous mice. However, molecular markers of hypertrophy (such as alpha-skeletal actin and atrial myosin light chain- 1) were expressed with a wider distribution, suggesting that their induction was secondary to the expression of the transgene, In older (28-week-old) mice, lung weights were also significantly increased, consistent with congestive heart failure. The life span of bmyf5 mice was significantly shortened, with an average life span of 109 days, compared with at least a twofold longer life expectancy for nontransgenic littermates. Expression of the transgene was associated with an increase in Ca2+-stimulated myofibrillar ATPase in myofibrils obtained from the left ventricles of 42-day-old bmyf5 mice. Myocardial bmyf5 expression therefore induces a program of skeletal muscle gene expression that results in progressive cardiomyopathy that may be due to incompatibility of heart and skeletal muscle structural proteins.

Enhanced expression of mouse c-ski accompanies terminal skeletal muscle differentiation in vivo and in vitro.

Overexpression of either v-ski, or the proto-oncogene, c-ski, in quail embryo fibroblasts induces the expression of myoD and myogenin, converting the cells to myoblasts capable of differentiating into skeletal myotubes. In transgenic mice, overexpression of ski also influences muscle development, but in this case it effects fully formed muscle, causing hypertrophy of fast skeletal muscle fibers. In attempts to determine whether endogenous mouse c-ski plays a role in either early muscle cell determination or late muscle cell differentiation, we analyzed mRNA expression during muscle development in mouse embryos and during in vitro terminal differentiation of skeletal myoblasts. To generate probes for these studies we cloned coding and 3' non-coding regions of mouse c-ski. In situ hybridization revealed low c-ski expression in somites, and only detected elevated levels of mRNA in skeletal muscle beginning at about 12.5 days of gestation. Northern analysis revealed a two-fold increase in c-ski mRNA during terminal differentiation of skeletal muscle cell lines in vitro. Our results suggest that c-ski plays a role in terminal differentiation of skeletal muscle cells not in the determination of cells to the myogenic lineage.

Expression of mef2 genes in the mouse central nervous system suggests a role in neuronal maturation.

Members of the myocyte enhancer factor 2 (MEF2) gene family are expressed in a dynamic pattern during development of the CNS of pre- and postnatal mice. The four MEF2 genes, Mef2A, -B, -C, -D, encode transcription factors belonging to the MADS (MCM1-agamous-deficiens-serum response factor) superfamily of DNA binding proteins. MEF2 factors have previously been shown to be positive regulators of gene expression in terminally differentiated muscle cells. To begin to determine the role of MEF2 factors in CNS development, we used in situ hybridization with gene-specific cRNA probes to define the expression patterns of each of the four Mef2 mRNAs in the developing and mature mouse CNS. Mef2C mRNA was first detected in a ventral portion of the telencephalon at 11.5 d postcoitum (p.c.). By 13.5 d p.c., each of the four Mef2 genes were expressed in overlapping yet distinct patterns in regions of the frontal cortex, midbrain, thalamus, hippocampus, and hindbrain. Temporal and spatial patterns of embryonic Mef2 gene expression appeared to follow gradients of neuron maturation and suggested that the onset of Mef2 gene expression coincides with withdrawal from the cell cycle and initiation of neuronal differentiation. This correlation is particularly striking for Purkinje cells in the cerebellum. Since the molecular mechanisms that regulate neuron differentiation are unknown, we propose that the MEF2 factors are likely to play an important role in this process.

Protooncogene c-ski is expressed in both proliferating and postmitotic neuronal populations.

The cellular protooncogene, c-ski, is expressed in all cells of the developing mouse at low but detectable levels. In situ hybridization and Northern blot analyses reveal that some cells and tissues express this gene at higher levels at certain stages of embryonic and postnatal development. RT-PCR results indicate that alternative splicing of exon 2, known to occur in chickens (Sutrave and Hughes [1989] Mol. Cell. Biol. 9:4046-4051; Grimes et al. [1993] Oncogene 8:2863-2868) does not occur in adult mouse tissues. In the embryo, neural crest cells express the c-ski gene during migration at 8.5 to 9.5 days post coitum (p.c.). Neural crest derivatives such as dorsal root ganglia and melanocytes stain positively with an antibody to the ski protein. At 9 days p.c., the entire neural tube has high levels of c-ski gene expression. By 12-13.5 days only the ependymal layer expresses c-ski above background levels. At 14-16 days p.c., c-ski mRNAs are detected at high levels in the cortical layers of the brain and in the olfactory bulb. In 2 week and 6 week postnatal brains, c-ski gene transcripts are also detected in the hippocampus and in the granule cell layer of the cerebellum. The allantois and placenta exhibit high levels of c-ski mRNAs. Neonatal lung tissue increases c-ski gene expression approximately two-fold compared to prenatal levels. These results suggest that ski plays a role in both the proliferation and differentiation of specific cell populations of the central and peripheral nervous systems and of other tissues.