Elevated levels of inducible heat shock 70 proteins in human brain.

Differential expression of heat shock genes can modulate protein folding and stress-related cell death. There have been no comparisons of their levels of expression in animals and humans. Levels of expression of heat shock 70 genes in human brain were compared to levels in non-stressed and heat-stressed brain of rat. Levels of hsp70 proteins in human brain were 43-fold higher than in non-stressed rat brain and 14-fold higher than highest induced levels in brains of heat-shocked rats. Levels of constitutively synthesized hsc70 proteins were approximately 1.5-fold higher in human than in rat. Higher levels of hsp70 proteins in human brain may serve to protect brain cells against stress-related death or dysfunction throughout the lifespan.

Lesioning of the inferior olive using a ventral surgical approach. Characterization of temporal and spatial astrocytic responses at the lesion site and in cerebellum.

Activated astrocytes, intrinsic components of both local and remote (axonal target regions) central nervous system injury responses, are now recognized as active metabolic and regulatory mediators in many neurological disorders. To further define these responses, we devised a new ventral surgical approach to unilaterally lesion the inferior olivary nuclear complex, which has a single predominant remote target, the cerebellum. Activated astrocyte number, volume, and density, as well as the total volume of brainstem involved in the astrocytic response, all peaked at postlesion day (pld) 4, returning toward, but not to, unoperated control values at pld 24 (p < 0.05). In contrast, the peak astrocyte response in the cerebellum was delayed, being greatest at pld 6 (p < 0.05 compared to control or pld 2). These responses were associated with increases in overexpression of S100 beta, an astrocyte-derived neurite growth factor, and with an increase in cerebellar steady-state levels of a neuronal injury response protein, the beta-amyloid precursor protein (beta-APP). This is similar to correlated increases in these two proteins that are found in epilepsy and Alzheimer disease. Our studies defining remote astrocytic and neuronal responses may be important for understanding glial-neuronal mechanisms underlying the spread of neuropathological changes in conditions such as Alzheimer disease.

Differential expression of heat shock 70 proteins in primary cultures from rat cerebellum.

While a number of studies have described the heat shock response in established cell lines and in primary cultures of cells derived from the nervous system, there has been no systematic analysis comparing expression and localization of the inducible heat shock 70 (hsp70) proteins and the constitutively synthesized members of the family (hsc70) in neurons and glia. In the present communication, we utilized specific probes to compare the expression of hsp70 and hsc70 mRNAs and proteins in two types of primary cultures, astroglial and neuro-astroglial, from postnatal rat cerebellum. Conditions were adjusted to maintain physiological numbers of microglia in both types of culture, and cultures were analyzed at a number of different time points following a precisely defined heat shock. The northern, in situ hybridization and immunohistochemical analyses resulted in a number of novel observations concerning the nature of the heat shock response in these neuronal and glial cells. In postnatal day 4-5 cultures, hsp70 mRNA levels were elevated for at least 10 h in both types of culture, but in situ hybridization analysis showed no evidence for hsp70 mRNAs in neurons. Microglia were the only cell type in which hsp70 was detected in non-stressed cultures and this cell type contained the highest concentrations of hsp70 proteins in stressed cultures. Hsc70 mRNA levels were also increased after heat shock, but the increase was more transient. Hsc70 mRNAs and proteins were present in all cell types, again with the highest concentrations being present in microglia. Hsc70 mRNAs and proteins were localized in the cytoplasm at all time points examined, with hsc70 protein also being localized in nucleoli. Hsp70 mRNAs and proteins were diffusely localized over nuclei of astrocytes, as well as of most microglia. Hsp70, but not hsc70, was localized on chromosomes in glia once they had resumed cell division after heat shock, suggesting a role for hsp70 either in targeting damaged chromosomal proteins or in cell division. Some cytoplasmic hsp70 was observed in astrocytes of the mixed neuro-astroglial cultures and a delayed hsp70 immunoreactivity was observed in granule neurons in these cultures, suggesting either that translation of low levels of hsp70 mRNAs was more efficient in neurons, or that glial-neuronal translocation of hsp70 proteins had taken place. These results suggest that metabolism and functions of different heat shock protein family members may not always be identical and that care must be taken in extrapolation of results from one cell type to another.

Developmental characterization of thymosin beta 4 and beta 10 expression in enriched neuronal cultures from rat cerebella.

The beta 4 and beta 10 thymosins are G-actin binding proteins that exhibit complex patterns of expression during rat cerebellar development. Their expression in vivo is initially high in immature granule cells and diminishes as they migrate and differentiate, ceasing altogether by postnatal day 21. Thymosin beta 4 is present in a subset of glia throughout postnatal development, and its synthesis is also induced in maturing Bergmann glia. In contrast, thymosin beta 10 is only present at very low levels in a very small subpopulation of glia in the adult cerebellum. To study the factors differentially regulating expression of the beta-thymosins, we characterized their patterns of expression in primary cultures of rat cerebellum. Both beta-thymosins were initially expressed in granule cells, although expression, especially of thymosin beta 4, was truncated compared with the in vivo time course. As in vivo, thymosin beta 4 was synthesized at much higher levels in astrocytes and microglia in cultures from postnatal cerebellum than was thymosin beta 10. Unlike in vivo, the latter was expressed in glia cultured from fetal cerebellum. The similarities between the in vivo and in vitro expression of the beta-thymosins show that modulation of tissue culture conditions could be used to identify factors regulating beta-thymosin expression in vivo. The differences would identify regulatory mechanisms that are not evident from the in vivo studies alone.

Heat-shock 70 messenger RNA levels in human brain: correlation with agonal fever.

Systematic review of antemortem clinical information on randomly selected Alzheimer disease (AD) patients revealed that approximately 40% of the patients had a recorded fever of > or = 39.2 degrees C at or near death. Using isolation and quantitation techniques appropriate for analysis of human brain mRNAs, we found that low levels of inducible heat-shock protein 70 (hsp70) mRNAs were present in cerebellum of afebrile AD patients and that mRNA levels were usually lower in two brain regions affected in AD, i.e., hippocampus and temporal cortex. Levels of hsp70 mRNAs were increased three- to 33-fold in cerebellum of febrile patients compared with levels in patients whose recorded temperatures were < or = 37.5 degrees C. Levels of hsp70 mRNAs were also increased in hippocampus and cortex of these febrile patients, but to a lesser extent than cerebellum. Heat-shock cognate 70 (hsc70) mRNAs were present at highest levels in afebrile cerebellum and were also present in the other brain regions. In cerebellum of patients with the highest temperatures, hsc70 mRNAs were induced severalfold over basal levels. Although there was a low and variable induction of hsc70 mRNAs in temporal cortex of these patients, there was no evidence for any induction in hippocampus. Increased heat-shock 70 mRNA levels did not correlate with hypoxia, coma, hypertension, hypoglycemia, seizures, or medication. These results indicate that a specific agonal stress, namely fever, can increase the levels of heat shock 70 mRNAs in AD brain; however, there is no evidence to suggest that affected regions of AD brain have higher overall levels of these mRNAs. Failure to obtain adequate agonal state information could result in inaccurately identifying short-term stress-related changes in postmortem brain as neuropathology characteristic of a chronic disease state.

Cell size and the heat-shock response in rat brain.

The expression of mRNAs encoding two members of the heat-shock protein 70 family, the constitutively-expressed heat-shock cognate (hsc70) mRNA and the strictly heat-inducible (hsp70) mRNA, was quantitated in cerebellar and hippocampal cells of rats 3 h after amphetamine-induced or heat-induced hyperthermia. Intracellular heat-shock mRNA levels in specific cell types were compared with those of total polyadenylic acid [poly(A)] mRNA or 18S rRNA in the same cell type. Levels of poly(A) mRNAs, 18S rRNAs, and hsc70 mRNAs were highest in large neurons and lowest in glia. hsp70 mRNAs were also present at highest levels in large neurons, suggesting that hsp70 mRNAs accumulated as rapidly in these cell types as they did in small neurons and glia. However, compared with levels of intracellular poly(A) mRNAs or levels of rRNAs, large neurons contained two- to 12-fold lower levels of hsp70 mRNAs than neurons of intermediate size and five- to 30-fold lower levels than glia. These results suggest that hsp70 mRNAs accumulated as rapidly in large neurons as in small neurons and glia, but that the large size of these neurons precluded intracellular hsp70 mRNA concentrations increasing as quickly. The susceptibility of large neurons to stress-induced cell death could be due, in part, to their inability to synthesize rapidly hsp70 in sufficient amounts to protect these cells from the initial molecular consequences of stress.

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat brain.

1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem. 2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstable in vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem. 3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region. 4. In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type. 5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.

Anomalous binding of radiolabeled oligonucleotide probes to plaques and tangles in Alzheimer disease hippocampus.

Several reports indicate that Alzheimer disease (AD) brain contains elevated levels of heat shock 70 proteins. To determine the cellular localization of the heat shock 70 mRNAs, specific oligonucleotide probes were in situ hybridized to AD and control brains. When oligonucleotides were in situ hybridized to brain sections with no AD neuropathology, hybridization was cell-specific and prior ribonuclease (RNase) treatment of adjacent sections resulted in no hybridization signal. However, in situ hybridization to AD hippocampus resulted in heavy grain deposition over senile plaques and neurofibrillary tangles. Despite altering a number of experimental variables, we observed a similar pattern of grain deposition with most of the oligonucleotides tested, including one oligonucleotide specific for glutamic acid decarboxylase mRNA. In situ hybridization with either an RNA probe for glutamic acid decarboxylase or an oligonucleotide probe specific for 18S rRNA did not show this pattern of grain deposition. In control studies a sense hsc70 oligonucleotide showed no grain deposition in either cerebellum or hippocampus. Sections from AD hippocampus pretreated with RNase prior to in situ hybridization demonstrated enhanced grain deposition with the majority of probes tested. Anomalous in situ hybridization to AD hippocampus was usually eliminated by removing formamide from the posthybridization washes, although post-RNase sticking often remained intense. These findings indicate that artifactual probe binding to senile plaques and neurofibrillary tangles may complicate the analysis of in situ hybridization studies using oligonucleotide probes to determine mRNA distribution in AD brain.

Effects of thymosin beta 4 and thymosin beta 10 on actin structures in living cells.

The beta-thymosins are a family of small proteins originally isolated from the thymus. Recently, two of the major mammalian isoforms, thymosin beta 4 (T beta 4) and thymosin beta 10 (T beta 10), are identified as significant actin monomer sequestering proteins which may be involved in regulating actin filament assembly. To study the cellular function of beta-thymosins, we have used isoform-specific antibodies to determine their concentration and intracellular distribution, and examined the effects of inducing overexpression of T beta 4 and T beta 10 on actin filament structures. Immunofluorescence labeling of peritoneal macrophages showed that both beta-thymosins are uniformly distributed within the cytoplasm. cDNA-mediated overexpression of beta-thymosins in CV1 fibroblasts induced extensive loss of phalloidin-stained actin stress fibers. Stress fibers in the cell center were more susceptible than those at the periphery. There was a decrease in the number of focal adhesions, as evidenced by a decrease in discrete vinculin staining and an increase in diffuse vinculin fluorescence. The majority of the transfected cells had normal shape in spite of extensive loss of actin filaments. Occasionally, cells overexpressing beta-thymosin were observed to divide. In these cells, beta-thymosin was excluded from the midbody which contains an actin filament-rich contractile ring. Our results indicate that T beta 4 and T beta 10 are functionally very similar and both are effective regulators of a large subset of actin filaments in living cells.

Alterations in actin-binding beta-thymosin expression accompany neuronal differentiation and migration in rat cerebellum.

The beta 4- and beta 10-thymosins, recently identified as actin monomer-sequestering proteins, are developmentally regulated in brain. Using specific mRNA and protein probes, we have used in situ hybridization and immunohistochemical techniques to investigate the distribution of the beta-thymosin mRNAs and their proteins in developing rat cerebellum. Early in postnatal development, both beta-thymosin mRNAs were expressed at highest levels in the postmitotic, premigratory granule cells of the external granular layer; expression diminished as granule cells migrated to and differentiated within the developing internal granular layer. In addition, both beta-thymosin proteins were present in bundles of cerebellar afferent fibers in the white matter at this time. Throughout the maturation period, both proteins were present in elongating parallel fibers in the upper portion of the molecular layer. Later in cerebellar development, thymosin beta 4, but not thymosin beta 10, was expressed in Golgi epithelial cells and Bergmann processes. Thymosin beta 4 was expressed in a small population of cells with microglial morphology scattered throughout the gray and white matter. Thymosin beta 10 was detected in an even smaller population of glia. Expression of thymosin beta 4 and thymosin beta 10 in premigratory granule cells and in growing neuronal processes is consistent with the possibility that both beta-thymosins are involved in the dynamics of actin polymerization during migration and process extension of neurons.

Plasticity of astroglial glutamate and gamma-aminobutyric acid uptake in cell cultures derived from postnatal mouse cerebellum.

The plasticity of astroglial glutamate and gamma-aminobutyric acid (GABA) uptakes was investigated using mouse cerebellar cell cultures. The influence of external factors, such as different sera and/or the presence of neurons, was examined. Control autoradiography experiments showed that after short-term exposure to radioactive amino acids, granule cells took up neither glutamate nor GABA, and beta-alanine predominantly inhibited astroglial GABA uptake. Astroglial uptake was quantified by measuring the radioactivity taken up by the cells in the culture and relating this measurement to the number of glial fibrillary acidic protein-positive cells present. Glutamate uptake was investigated in astroglial cultures and subcultures and in neuronal-astroglial cultures derived from postnatal day 4 mouse cerebella. In the absence of neurons, glutamate uptake increased during the first 9 days after plating and then leveled off. At 14 days in vitro in horse serum, which favors the differentiation of fibrous-like astrocytes, glutamate uptake related to astrocyte number was twice as high as in fetal calf serum. In the presence of cerebellar neurons, this rate was even higher. The specificity of the responsiveness of astrocytes to neurons with respect to glutamate uptake was investigated by comparing GABA uptake in the different culture conditions. Neurons also increased the rate of GABA uptake by astrocytes. Another component of the astroglial plasma membrane, the density of beta-adrenergic receptors, was, however, not markedly affected by the presence of neurons. Hence, these results showed that in astrocytes plated from postnatal day 4 mouse cerebella, the level of neurotransmitter uptake can be regulated in vitro by factors present in sera and by cerebellar neurons in the culture. However, this plasticity declined during development because astrocytes plated from postnatal day 8 cerebella and cultured under identical conditions were less active in glutamate uptake and were insensitive to the presence of horse serum. The latter observation suggested that the metabolic plasticity of astrocytes is restricted to a period defined early in cerebellar development and is no longer evident by postnatal day 8.

Thymosin beta 10 and thymosin beta 4 are both actin monomer sequestering proteins.

The beta-thymosins are a family of related peptides. Recently, thymosin beta 4 was identified as a significant actin monomer sequestering protein in cells. To determine if other beta-thymosins also bind actin, and how they may participate in the regulation of actin polymerization, we expressed thymosin beta 4 and its major homolog, thymosin beta 10, in bacteria and characterized their interactions with actin. Equilibrium sedimentation studies showed that thymosin beta 4 behaved as a monomeric protein in solution. Both beta-thymosins bound skeletal muscle actin and inhibited actin polymerization with similar Kd values (between 0.7-1 microM). They were not inhibited by polyphosphoinositides. Kinetic measurements showed that at high ratios of beta-thymosin to actin, beta-thymosin decreased the rate of barbed end filament growth. However, in spite of a close agreement between the kinetic and steady state Kd values, the rate of barbed end filament growth was slightly, but reproducibly, larger than expected, and this deviation was particularly noticeable at lower ratios of beta-thymosin to actin. We conclude that unlike profilin, beta-thymosins are primarily actin monomer sequestering proteins, although some aspects of their interactions with actin are still not completely understood.

Hsp70 mRNA induction is reduced in neurons of aged rat hippocampus after thermal stress.

Levels of heat-shock 70 mRNAs, relative to those of 18S rRNA, were quantitated in specific cell types of hippocampus of adult and aged rats subjected to identical heat shock regimens. Body temperature changes in response to the heat stress were no different in adult and aged rats. In control rats, as well as 3 h after initiation of heat shock in both adult and aged rats, relative levels of the constitutively synthesized heat-shock cognate 70 (hsc70) mRNA were highest in hippocampal neurons and much lower in glia. No heat-shock protein 70 (hsp70) mRNAs were present in any cell type of control adult or aged rats. In heat-shocked adult rats, the relative levels of the heat-shock-inducible hsp70 mRNAs were highest in a subpopulation of glia, intermediate in granule cells of the dentate gyrus, and lowest in pyramidal cells of Ammon's horn. Relative levels of hsp70 mRNA were several-fold lower in the dentate gyrus granule cells of aged rats compared to relative levels in controls and were also reduced in many pyramidal cells of the hippocampus but not in hippocampal glia. These findings suggest that some neuronal populations in the hippocampus may be at increased risk for stress-related injury in the aged animal.

Brain non-adenylated mRNAs.

Most eukaryotic messenger RNA (mRNA) species contain a 3'-poly(A) tract. The histone mRNAs are a notable exception although a subclass of histone-encoding mRNAs is polyadenylated. A class of mRNAs lacking a poly(A) tail would be expected to be less stable than poly(A)+ mRNAs and might, like the histones, have a half-life that varied in response to changes in the intracellular milieu. Brain mRNA exhibits an unusually high degree of sequence complexity; studies published ten years ago suggested that a large component of this complexity might be present in a poly(A)- mRNA population that was expressed postnatally. The question of the existence of a complex class of poly(A)- brain mRNAs is particularly tantalizing in light of the heterogeneity of brain cells and the possibility that the stability of these poly(A)- mRNAs might vary with changes in synaptic function, changing hormonal stimulation or with other modulations of neuronal function. The mRNA complexity analyses, although intriguing, did not prove the existence of the complex class of poly(A)- brain mRNAs. The observed mRNA complexity could have resulted from a variety of artifacts, discussed in more detail below. Several attempts have been made to clone members of this class of mRNA. This search for specific poly(A)- brain mRNAs has met with only limited success. Changes in mRNA polyadenylation state do occur in brain in response to specific physiologic stimuli; however, both the role of polyadenylation and de-adenylation in specific neuronal activities and the existence and significance of poly(A)- mRNAs in brain remain unclear.

Cloning and characterization of a testis-specific thymosin beta 10 cDNA. Expression in post-meiotic male germ cells.

Thymosin beta 10 is one of a small family of proteins closely related in sequence to thymosin beta 4, recently identified as an actin-sequestering protein. A single molecular weight species of thymosin beta 10 mRNA is present in a number of rat tissues. In adult rat testis, an additional thymosin beta 10 mRNA of higher molecular weight was identified. Nucleotide sequencing of cDNA clones complementary to the testis-specific thymosin mRNA indicated that this mRNA differed from the ubiquitous thymosin beta 10 mRNA only in its 5'-untranslated region, beginning 14 nucleotides upstream of the translation initiation codon. These results, together with primer extension experiments, suggest that the two thymosin beta 10 mRNAs are transcribed from the same gene through a combination of differential promoter utilization and alternative splicing. The novel thymosin beta 10 mRNA could be detected only in RNA isolated from sexually mature rat testis. Both mRNAs were present in pachytene spermatocytes; only the testis-specific mRNA was detected in postmeiotic haploid spermatids. Immunoblot analysis using specific antibodies showed that the thymosin beta 10 protein synthesized in adult testis was identical in size to that synthesized in brain. Immunohistochemical analysis showed that the protein was present in differentiating spermatids, suggesting that the testis-specific thymosin beta 10 mRNA is translated in haploid male germ cells.

Quantitative in situ hybridization analysis of glutamic acid decarboxylase messenger RNA in developing rat cerebellum.

The appearance and relative amounts of GAD mRNA in rat cerebellar neurons during postnatal development was studied by in situ hybridization. GAD mRNA content within all GABAergic neurons increased during the first month of postnatal development, but the degree and time course of the increase varied among different neuronal types. In newborn rats, GAD mRNA was present only in the prenatally-formed Purkinje and Golgi cells. GAD mRNA in Golgi cells had reached adult levels by postnatal day 14, while GAD mRNA levels in Purkinje cells reached adult levels one week later. Most basket cells expressed GAD mRNA by postnatal day 14, and final levels were attained one week later. Stellate cells in the bottom two-thirds of the molecular layer attained their final GAD mRNA content by postnatal day 21 whereas stellate cells in close proximity to the pial surface were not yet mature at this age. No GAD mRNA was detected within the external granular layer at any time during development. In adult rat, approximately 40% of cerebellar GAD mRNA was contained within the Purkinje cell population, 38% within the stellate cells, 17% within the basket cells, and only 5% within the Golgi cells. Increases in GAD mRNA within GABAergic neurons during cerebellar development correlated with the timing of neuronal maturation and synaptogenesis in these cell populations, suggesting that synaptic activity affects GAD gene expression in developing cerebellum.

Expression of heat shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or amphetamine treatment.

The expression of strictly inducible hsp70 mRNAs and constitutively expressed hsc70 mRNAs was compared in cerebellum and cerebral cortex of control rats, heat-shocked rats, and rats made hyperthermic with amphetamine. An hsc70-specific oligonucleotide probe identified a 2.55-kb mRNA in cerebellum and cerebral cortex of all rats. An hsp70-specific oligonucleotide probe identified a 3.05-kb mRNA and a 3.53-kb mRNA in cerebellum and cerebral cortex of heat-shocked and amphetamine-treated rats, but not in control rats. Quantitation demonstrated that both hsp70 and hsc70 mRNA levels, relative to 18S rRNA levels, were increased following each treatment. The relative levels of both mRNAs were higher in cerebellum than in cerebral cortex. In amphetamine-treated rats, hsc70 mRNA relative levels increased at body temperatures greater than 39 degrees C, whereas hsp70 mRNA synthesis was induced at temperatures greater than 40 degrees C. Total thermal response values and relative levels of both mRNAs were compared. The results suggested that both the transcription and turnover of hsp70 mRNAs differed between cerebellum and cerebral cortex. At equivalent total thermal response values, amphetamine-treated rats had higher relative levels of hsp70 mRNAs than heat-shocked rats, suggesting that amphetamine enhanced the induction of hsp70 mRNAs.

Developmental expression of mRNAs encoding thymosins beta 4 and beta 10 in rat brain and other tissues.

In the course of screening a fetal rat cerebellum cDNA library for developmentally regulated sequences, we have identified a cDNA clone identical in sequence to that encoding a protein originally isolated from thymus, thymosin beta 10. Based on northern hybridization analyses with gene-specific oligonucleotide probes derived from the 3'-untranslated regions of thymosin beta 10 mRNA and the closely related beta 4 mRNA, we showed that both thymosin mRNAs were present at highest levels in fetal cortex and cerebellum but also were present at varying levels in all other fetal tissues examined (thymus, spleen, lung, kidney, adrenal, heart, and liver). Steady-state levels of thymosin beta 10 mRNA in cerebellum declined to negligible levels after day 14 of postnatal development. Its levels exhibited a similar pattern in developing cortex, although the adult cortex had slightly higher thymosin beta 10 mRNA levels. These results suggest that thymosin beta 10 mRNA is subject to strong developmental regulation in the rat central nervous system. Reduction of thymosin beta 10 mRNA levels also was seen during development of kidney, heart, and liver. Levels of both thymosin beta 10 and beta 4 mRNAs remained relatively constant during development of thymus, spleen, and lung. Thymosin beta 4 mRNA levels dropped much less sharply during brain development than did levels of the beta 10 mRNA. Testis and ovary contained the highest relative levels of thymosin beta 10 mRNA among adult tissues, but little thymosin beta 4 mRNA. A novel thymosin beta 10 mRNA species unique to adult testis was detected. These results indicate that both thymosins must function in the development of brain and many other organs, as well as in different subsets of organs in the adult.