Ubiquitin Regulation of Trk Receptor Trafficking and Degradation.

The regulation of Trk receptors is critical for orchestrating multiple signalling pathways required for developing and maintaining neuronal networks. Activation of Trk receptors results in signalling, internalisation and subsequent degradation of the protein. Although ubiquitination of TrkA by Nedd4-2 has been identified as an important degradation pathway, much less is known about the pathways regulating the degradation of TrkB and TrkC. Critical to the interaction between TrkA and Nedd4-2 is a PPxY motif present within TrkA but absent in TrkB and TrkC. Given the absence of this interaction motif, it remains to be determined how TrkB and TrkC are ubiquitinated. Here we report that the adaptor protein Ndfip1 can interact with all three Trk receptors and show for TrkB the recruitment of Nedd4-2 through PPxY motifs present in Ndfip1. Ndfip1 mediates the ubiquitination of TrkB, resulting in receptor trafficking predominantly on Rab7 containing late endosomes, highlighting a pathway for TrkB degradation at the lysosome. In vitro, overexpression of Ndfip1 increased TrkB ubiquitination and decreased viability of BDNF-dependent primary neurons. In vivo, conditional genetic deletion of Ndfip1 increased TrkB in the brain and resulted in enlargement of the granular cell layer of the dentate gyrus.

Genome-wide association study of shared components of reading disability and language impairment.

Written and verbal languages are neurobehavioral traits vital to the development of communication skills. Unfortunately, disorders involving these traits-specifically reading disability (RD) and language impairment (LI)-are common and prevent affected individuals from developing adequate communication skills, leaving them at risk for adverse academic, socioeconomic and psychiatric outcomes. Both RD and LI are complex traits that frequently co-occur, leading us to hypothesize that these disorders share genetic etiologies. To test this, we performed a genome-wide association study on individuals affected with both RD and LI in the Avon Longitudinal Study of Parents and Children. The strongest associations were seen with markers in ZNF385D (OR = 1.81, P = 5.45 × 10(-7) ) and COL4A2 (OR = 1.71, P = 7.59 × 10(-7) ). Markers within NDST4 showed the strongest associations with LI individually (OR = 1.827, P = 1.40 × 10(-7) ). We replicated association of ZNF385D using receptive vocabulary measures in the Pediatric Imaging Neurocognitive Genetics study (P = 0.00245). We then used diffusion tensor imaging fiber tract volume data on 16 fiber tracts to examine the implications of replicated markers. ZNF385D was a predictor of overall fiber tract volumes in both hemispheres, as well as global brain volume. Here, we present evidence for ZNF385D as a candidate gene for RD and LI. The implication of transcription factor ZNF385D in RD and LI underscores the importance of transcriptional regulation in the development of higher order neurocognitive traits. Further study is necessary to discern target genes of ZNF385D and how it functions within neural development of fluent language.

Common genetic variation near the connexin-43 gene is associated with resting heart rate in African Americans: a genome-wide association study of 13,372 participants.

BACKGROUND: Genome-wide association studies have identified several genetic loci associated with variation in resting heart rate in European and Asian populations. No study has evaluated genetic variants associated with heart rate in African Americans. OBJECTIVE: To identify novel genetic variants associated with resting heart rate in African Americans. METHODS: Ten cohort studies participating in the Candidate-gene Association Resource and Continental Origins and Genetic Epidemiology Network consortia performed genome-wide genotyping of single nucleotide polymorphisms (SNPs) and imputed 2,954,965 SNPs using HapMap YRI and CEU panels in 13,372 participants of African ancestry. Each study measured the RR interval (ms) from 10-second resting 12-lead electrocardiograms and estimated RR-SNP associations using covariate-adjusted linear regression. Random-effects meta-analysis was used to combine cohort-specific measures of association and identify genome-wide significant loci (P≤2.5×10(-8)). RESULTS: Fourteen SNPs on chromosome 6q22 exceeded the genome-wide significance threshold. The most significant association was for rs9320841 (+13 ms per minor allele; P = 4.98×10(-15)). This SNP was approximately 350 kb downstream of GJA1, a locus previously identified as harboring SNPs associated with heart rate in Europeans. Adjustment for rs9320841 also attenuated the association between the remaining 13 SNPs in this region and heart rate. In addition, SNPs in MYH6, which have been identified in European genome-wide association study, were associated with similar changes in the resting heart rate as this population of African Americans. CONCLUSIONS: An intergenic region downstream of GJA1 (the gene encoding connexin 43, the major protein of the human myocardial gap junction) and an intragenic region within MYH6 are associated with variation in resting heart rate in African Americans as well as in populations of European and Asian origin.

Genome-wide association study of bipolar disorder in European American and African American individuals.

To identify bipolar disorder (BD) genetic susceptibility factors, we conducted two genome-wide association (GWA) studies: one involving a sample of individuals of European ancestry (EA; n=1001 cases; n=1033 controls), and one involving a sample of individuals of African ancestry (AA; n=345 cases; n=670 controls). For the EA sample, single-nucleotide polymorphisms (SNPs) with the strongest statistical evidence for association included rs5907577 in an intergenic region at Xq27.1 (P=1.6 x 10(-6)) and rs10193871 in NAP5 at 2q21.2 (P=9.8 x 10(-6)). For the AA sample, SNPs with the strongest statistical evidence for association included rs2111504 in DPY19L3 at 19q13.11 (P=1.5 x 10(-6)) and rs2769605 in NTRK2 at 9q21.33 (P=4.5 x 10(-5)). We also investigated whether we could provide support for three regions previously associated with BD, and we showed that the ANK3 region replicates in our sample, along with some support for C15Orf53; other evidence implicates BD candidate genes such as SLITRK2. We also tested the hypothesis that BD susceptibility variants exhibit genetic background-dependent effects. SNPs with the strongest statistical evidence for genetic background effects included rs11208285 in ROR1 at 1p31.3 (P=1.4 x 10(-6)), rs4657247 in RGS5 at 1q23.3 (P=4.1 x 10(-6)), and rs7078071 in BTBD16 at 10q26.13 (P=4.5 x 10(-6)). This study is the first to conduct GWA of BD in individuals of AA and suggests that genetic variations that contribute to BD may vary as a function of ancestry.

The IL-10R1 S138G loss-of-function allele and ulcerative colitis.

Genetic predisposition is a risk factor for the development of inflammatory bowel diseases (IBDs). Disruption of the interleukin (IL)-10 pathway in mice causes intestinal inflammation similar to human IBD. Two common non-synonymous IL-10R1 variants, S138G and G330R, were cloned and expressed in HeLa and Ba/F3. A reduction in IL-10-induced STAT1 and STAT3 activation was seen for IL-10R1-S138G (but not IL-10R1-G330R) by phosphospecific western blotting in both cell types. When analyzing 52 world populations for the presence of IL-10R1 variants, a strong dissimilarity was found between major geographical regions. In addition, when 182 IBD-parent trios were genotyped for both variants, a reduced transmission of haplotype -7 (carrying the S138G variant allele) to offspring with ulcerative colitis (UC) was observed. This UC-protective effect of S138G was confirmed in a Hungarian cohort (n=185, allele frequency 11.6 versus 17.5%; P=0.017) but not in an independent Belgian cohort (n=666, allele frequency 15.9 versus 15.5%; P=0.8). In conclusion, the IL-10R1 S138G variant is a loss-of-function allele for IL-10-induced STAT1 and STAT3 activation but does not protect from UC susceptibility.

Holes a plenty: oral health status a major issue for newly arrived refugees in Australia.

BACKGROUND: Dental health needs of newly arrived refugees are much greater than for the wider Australian community. This paper identifies the disparities and highlights major dental health issues for Australia's growing and constantly changing refugee population. METHODS: Using available data and the decayed, missing and filled teeth (DMFT) index as a measure of oral health, the reported oral health status of refugee groups in Australia was compared with that of the general population, Indigenous Australians, recipients of public dental services, special needs groups in Australia and other refugee groups outside Australia. RESULTS: The reported oral health status of Australian refugees compared poorly with the comparison groups. Of particular concern was the number of reported untreated decayed teeth (D). This ranged from a mean of 2.0 to 5.2 compared with 0.6 to 1.4 for the general Australian population. Refugee groups also reported fewer filled teeth (1.0 to 5.8) compared with the general population (4.1 to 9.3). Similar results were found when reported D, M and F teeth for refugees were compared to Indigenous Australians, public dental service recipients, immigrants and special needs groups in Australia. CONCLUSIONS: Dental health of refugees, particularly untreated decay, compared poorly to that of Indigenous Australians, and special needs populations in Australia who all have known worse dental health than the general population. There is an urgent need for the inclusion of this at risk population among targeted dental services. In addition, sources of health related data must clearly identify refugees to enable appropriate comparisons with other population groups. Recommendations for refugees are made regarding on-arrival dental assessment and treatment, and community based oral health programmes.

High-resolution x-ray imaging of a globular cluster core: compact binaries in 47Tuc.

We have obtained high-resolution (approximately 1") deep x-ray images of the globular cluster 47Tucanae (NGC 104) with the Chandra X-ray Observatory to study the population of compact binaries in the high stellar density core. A 70-kilosecond exposure of the cluster reveals a centrally concentrated population of faint (Lx approximately 10(30-33) ergs per second) x-ray sources, with at least 108 located within the central 2' x 2.5' and greater, similar half with Lx approximately 10(30.5) ergs per second. All 15 millisecond pulsars (MSPs) recently located precisely by radio observations are identified, though 2 are unresolved by Chandra. The x-ray spectral and temporal characteristics, as well as initial optical identifications with the Hubble Space Telescope, suggest that greater, similar50 percent are MSPs, about 30 percent are accreting white dwarfs, about 15 percent are main-sequence binaries in flare outbursts, and only two to three are quiescent low-mass x-ray binaries containing neutron stars, the conventional progenitors of MSPs. An upper limit of about 470 times the mass of the sun is derived for the mass of an accreting central black hole in the cluster. These observations provide the first x-ray "color-magnitude" diagram for a globular cluster and census of its compact object and binary population.

Effects of dietary restriction on appendicular bone in the SENCAR mouse.

Peptide hormones, cytokines, and growth factors regulate cellular metabolism by stimulating second messenger signal transduction cascades in target tissues. A mutation in the regulatory domain of protein kinase C (PKC) in SENCAR (sensitive to carcinogenesis) mice renders them extremely sensitive to diacylglycerol and phorbol esters, resulting in rapid growth, high free radical generation, carcinogenesis, and metabolic bone disease. Dietary restriction (DR) normalizes PKC and ameliorates adverse downstream effects, including carcinogenesis, in SENCAR mice. We hypothesized that DR sufficient to ameliorate carcinogenesis would prevent or delay the early onset of metabolic bone disease in SENCAR mice. Male mice were assigned to 1 of 4 feeding groups from 10 to 16 weeks of age (the critical period when metabolic bone disease develops): ad libitum (AL)-fed; AL antioxidant (0.07% thioproline)-fed; 40% DR; or 40% DR antioxidant-fed. Femoral bone mass was determined gravimetrically. Tibial total, cortical, and trabecular bone mineral density (BMD) were determined by quantitative computed tomography. Body weight, femoral bone mass, and tibial cortical BMD were lower in DR than in AL mice. However, tibial total and trabecular BMD were higher in DR than in AL mice. Serum calcitonin, the hormone that inhibits the osteoclastic bone resorption that is most notable in trabecular bone, was 2-fold higher in DR than in AL-fed mice. Dietary thioproline had no major effects. Thus, DR sufficient to ameliorate carcinogenesis in SENCAR mice did not prevent early-onset metabolic bone disease, but it had a beneficial effect on tibial trabecular BMD that occurred at the apparent expense of cortical BMD. DR in SENCAR mice was also associated with elevated serum calcitonin, which may inhibit osteoclastic resorption and account for trabecular bone conservation in this model. In conclusion, PKC or the downstream metabolic processes regulated by it appear to play previously unrecognized roles in the regulation of tibial trabecular BMD and serum calcitonin in SENCAR mice.

Systemic administration of antisense p75(NTR) oligodeoxynucleotides rescues axotomised spinal motor neurons.

The 75 kD low-affinity neurotrophin receptor (p75(NTR)) is expressed in developing and axotomised spinal motor neurons. There is now convincing evidence that p75(NTR) can, under some circumstances, become cytotoxic and promote neuronal cell death. We report here that a single application of antisense p75(NTR) oligodeoxynucleotides to the proximal nerve stumps of neonatal rats significantly reduces the loss of axotomised motor neurons compared to controls treated with nonsense oligodeoxynucleotides or phosphate-buffered saline. Our investigations also show that daily systemic intraperitoneal injections of antisense p75(NTR) oligodeoxynucleotides for 14 days significantly reduce the loss of axotomised motor neurons compared to controls. Furthermore, we found that systemic delivery over a similar period continues to be effective following axotomy when intraperitoneal injections were 1) administered after a delay of 24 hr, 2) limited to the first 7 days, or 3) administered every third day. In addition, p75(NTR) protein levels were reduced in spinal motor neurons following treatment with antisense p75(NTR) oligodeoxynucleotides. There were also no obvious side effects associated with antisense p75(NTR) oligodeoxynucleotide treatments as determined by behavioural observations and postnatal weight gain. Our findings indicate that antisense-based strategies could be a novel approach for the prevention of motor neuron degeneration associated with injuries or disease.

A potential role for the p75 low-affinity neurotrophin receptor in spinal motor neuron degeneration in murine and human amyotrophic lateral sclerosis.

INTRODUCTION: The p75 neurotrophin receptor has been recognized as a death-signalling molecule under certain circumstances. Its role in motor neuron degeneration in amyotrophic lateral sclerosis (ALS) was analysed in SOD1-G93A transgenic mice and in spinal cords from human amyotrophic lateral sclerosis. METHOD: The precise loss of motor neurons in SOD1-G93A transgenic mice from birth to adulthood was established using the unbiased fractionator/optical dissector neuronal counting technique. RESULTS: This study showed an early trend in the loss of lumbar motor neurons in SOD1-G93A mice, beginning at birth and progressing to a massive 80% reduction by 4 months of age, when the disease is severe. This study also found that the p75 neurotrophin receptor was expressed in lumbar motor neurons in symptomatic SOD1-G93A mice and in motor neurons in the cervical spinal cords of patients with ALS. CONCLUSIONS: The murine and human ALS data suggest that the p75 neurotrophin receptor may play a death-signalling role in the pathogenesis of motor neuron degeneration. The precise mechanism by which this receptor drives the apoptotic process, both in murine SOD1-G93A motor neuron degeneration and in human amyotrophic lateral sclerosis, remains to be determined.

Expression of leukemia inhibitory factor receptor mRNA in sensory dorsal root ganglion and spinal motor neurons of the neonatal rat.

Previous studies have shown that the application of leukemia inhibitory factor to the proximal nerve stump prevents the degeneration of axotomized sensory neurons in the dorsal root ganglion and motor neurons in the spinal cord of newborn rats. This study investigated the expression of leukemia inhibitory factor receptor mRNA in these neurons using in situ hybridization. Leukemia inhibitory factor receptor mRNA was detected both in sensory neurons within the dorsal root ganglion and motor neurons of the cervical spinal cord. Twenty-four hours after axotomy these neurons continue to express leukemia inhibitory factor receptor mRNA. This pattern of leukemia inhibitory factor receptor expression provides a mechanism by which endogenous and exogenous leukemia inhibitory factor could act on injured sensory and motor neurons.

Role of neurotrophin receptor p75NTR in mediating neuronal cell death following injury.

1. The neurotrophin receptor p75NTR has been shown to mediate neuronal cell death after nerve injury. 2. Down-regulation of p75NTR by antisense oligonucleotides is able to inhibit both sensory and motor neuron death and this treatment is more effective than treatment with growth factors. 3. p75NTR induces cell death by a unique death signalling pathway involving transcription factors (nuclear factor kappa B and c-jun), Bcl-2 family members and caspases.

Differential loss of spinal sensory but not motor neurons in the p75NTR knockout mouse.

Sensory neurons in the dorsal root ganglia (DRG) and motor neurons in the spinal cord express the 75 kDa low-affinity neurotrophin receptor (p75NTR) during prenatal development. The p75NTR gene knockout mouse provides a unique opportunity to assess the role of p75NTR during this period. Quantitative analysis of the p75NTR knockout mouse revealed a significant developmental loss of sensory neurons. In the cervico-thoracic ganglia approximately 75% of the neurons are lost, while in the lumbar ganglia the loss is approximately 50%. In contrast, motor neurons were not lost in either the cervical or lumbar spinal cord. These data suggest that p75NTR is essential for the prenatal survival of a significant number of sensory, but not motor neurons.

The ubiquitin-proteasome system and cellular proliferation and regulation in osteoblastic cells.

The 26S proteasome is the macromolecular assembly that mediates ATP- and ubiquitin-dependent extralysosomal intracellular protein degradation in eukaryotes. However, its contribution to the regulation of osteoblast proliferation and hormonal regulation remains poorly defined. Treating osteoblasts with MG-132 or lactacystin (membrane-permeable proteasome inhibitors) attenuates proliferation. Three proteasome activities (peptidylglutamyl-peptide bond hydrolase-, chymotrypsin-, and trypsin-like) were detected in osteoblasts. Catabolic doses of PTH stim-ulated these activities, and cotreatment with PTH and MG-132 blocked stimulation. The proteasome alpha- and beta-subunits, polyubiquitins, and large ubiquitin-protein conjugates were detected by Western blotting. A 90-min treatment with 10 nM PTH had no effect on the amount of proteasome alpha or beta subunit protein, but increased the relative amount of large ubiquitin-protein conjugates by 200%. MG-132 inhibited deubiquitination of large ubiquitin-protein conjugates. The protein kinase A inhibitor SQ22536 blocked much of the PTH-induced stimulation of MCP activities, while dibutyryl cAMP stimulated it, suggesting that protein kinase A-dependent phosphorylation is important in PTH stimulation of proteasome activities. In conclusion, the ubiquitin-proteasome system is essential for osteoblast proliferation under control and PTH-treated conditions. PTH mediates its metabolic effects on the osteoblast, in part, by enhancing ubiquitinylation of protein substrates and stimulating three major proteasome activities by a cAMP-dependent mechanism.

Leukemia inhibitory factor maintains choline acetyltransferase expression in vivo.

Following axotomy most medial septal neurons in the adult rat brain have dramatically reduced numbers of choline acetyltransferase (ChAT) positive neurons. Since leukemia inhibitory factor (LIF) promotes cholinergic expression in several neuronal populations, the aim of this study was to determine if LIF would continue to support cholinergic expression in axotomized medial septal neurons. Mini-osmotic pumps were used to infuse saline or LIF into the lateral cerebral ventricle. Counts of ChAT and low-affinity nerve growth factor (p75NGFR) immunostained neurons indicated that LIF-treated animals retained ChAT expression in > 90% of axotomized neurons whereas in saline-infused animals this was < 30%. Also, LIF was equally effective in maintaining p75NGFR expression levels in axotomized medial septal neurons.

E64d, a membrane-permeable cysteine protease inhibitor, attenuates the effects of parathyroid hormone on osteoblasts in vitro.

Parathyroid hormone (PTH) activates calpains I and II (calcium-activated papain-like proteases) and stimulates the synthesis and secretion of cathepsin B (a lysosomal cysteine protease) in osteoblastic cells. Anabolic doses of PTH also stimulate osteoprogenitor cell proliferation and differentiation into mature, fully functional osteoblasts capable of elaborating bone matrix, whereas catabolic doses of PTH stimulate calcium mobilization and matrix turnover. Previous investigations in other cell types have demonstrated that calcium-activated calpains play a major role in regulating proliferation and differentiation by catalyzing limited regulatory proteolysis of nuclear proteins, transcription factors, and enzymes. We tested the hypothesis that inhibition of intracellular cysteine proteases such as the calpains will ablate PTH-mediated osteoblast proliferation and differentiation, two fundamental indices of bone anabolism. A brief preincubation with the membrane-permeable, irreversible cysteine protease inhibitor E64d (10 micrograms/mL) before short-term PTH treatment blunted PTH-induced cell proliferation in subconfluent cultures and also attenuated proliferation and inhibited differentiation in longer-term confluent cultures. This confirms the hypothesis that cysteine proteases such as the calpains are important in mediating the proliferative and prodifferentiating or anabolic effects of PTH on MC3T3-E1 cells in culture. Immunofluorescent localization demonstrated that calpain I, calpain II, and calpastatin (the endogenous calpain inhibitor) are abundant and widely distributed within actively proliferating MC3T3-E1 preosteoblasts. Since the calpains are active and stable at neutral intracellular pH levels in osteoblasts, whereas cathepsins are not, our results support a role for these calcium-activated regulatory proteases in mediating the anabolic effects of PTH in bone.

Effect of low dietary calcium on bone metabolism in the SENCAR mouse.

The SENCAR (sensitive to carcinogenesis) mouse is a unique tool for investigating the interaction between a specific defect in intracellular signaling, dietary calcium, and metabolic bone disease. The SENCAR mouse was developed by selective breeding for enhanced sensitivity to two-stage carcinogenesis. Its major genetic defect, which renders it exquisitely sensitive to stimulation with diacylglycerol or phorbol esters, is in the regulatory domain of protein kinase C, one of the primary intracellular mediators of hormonal effects. At sexual maturity, SENCAR mice are large and have big bones, but our previous pharmacokinetic studies showed that they accumulate less calcium under normal conditions and lose more calcium under adverse conditions than do other, standard strains of mice. To histologically define the effect of low dietary calcium on bone metabolism, we performed histomorphometric analysis of tetracycline-labeled sections of femoral bone from male SENCAR mice maintained on calcium-sufficient and calcium-deficient diets during the critical period from 10 to 14 weeks of age. The bone volume, absolute osteoid volume, and mineral apposition rate were lower at 14 than at 10 weeks of age in SENCAR mice fed 0.02 or 0.6% calcium diets. Calcium deficiency increased the architectural disarray and the probability of observing focal discontinuities in the growth plate. Thus, characteristic features of impaired bone metabolism (low bone volume and apposition rate) develop early in SENCAR mice and are exacerbated by low dietary calcium. Detailed examinations of the histology and biochemistry of SENCAR mouse bone will provide insights into the mechanisms by which specific defects in the signal transduction of protein kinase C contribute to impaired bone metabolism.

The calpain-calpastatin system and cellular proliferation and differentiation in rodent osteoblastic cells.

The calpain-calpastatin system, which consists of calpains I and II (two ubiquitously distributed calcium-activated papain-like cysteine proteases), as well as calpastatin (the endogenous calpain inhibitor), plays an important role in cell proliferation and differentiation in many tissues. However, its contribution to the regulation of osteoprogenitor or pluripotent stem cell proliferation and differentiation into osteoblasts remains poorly defined. In these studies, rat pluripotent mesodermal cells (ROB-C26) and mouse MC3T3-E1 preosteoblasts were induced to differentiate into osteoblasts by long-term culture or in response to bone morphogenetic protein (BMP). The occurrence and distribution of calpain-calpastatin system proteins were determined by immunofluorescent microscopy, measurement of calcium-dependent proteolytic activity, and Western blotting. Treatment of intact MC3T3-E1 cells with an irreversible, membrane-permeable cysteine protease inhibitor attenuated proliferation and alkaline phosphatase upregulation under differentiation-enhancing conditions. Calpain II activity increased during differentiation of MC3T3-E1 cells in postconfluent culture. When ROB-C26 cells were maintained in long-term culture, neutral protease, calpain I, and calpain II activities increased 2- to 3-fold in the absence of BMP. In the presence of partially purified native BMP, neutral protease and calpain I activities also increased similarly, but calpain II activity increased by 10-fold in 3 days. The maximal increase in alkaline phosphatase occurred 4 to 11 days after the calpain II activity had peaked. Induction of differentiation in long-term MC3T3-E1 cultures was associated with higher calpain II and 70- and 110-kDa calpastatin protein levels and lower 17-kDa calpastatin degradation product levels. In conclusion, cysteine protease activity is essential for preosteoblastic proliferation and differentiation. The calpain-calpastatin system is regulated during osteoprogenitor proliferation and differentiation, as it is in other cells, and bone morphogenetic protein is a specific regulator of calpain II.

Endochondral and intramembranous fetal bone development: osteoblastic cell proliferation, and expression of alkaline phosphatase, m-twist, and histone H4.

We have previously studied the expression of alkaline phosphatase (ALP) and alpha2(I) collagen (two phenotypic markers of osteoblastic cell differentiation) during development of the rat mandible, and the spatial and temporal distribution of the respective transcripts. Our current studies utilize the rat mandible and hind foot as in vivo model systems to investigate the relationship between osteoblastic differentiation and proliferation during intramembranous and endochondral bone formation. Pregnant rats, at 15.17, and 19 days of gestation were intraperitoneally injected with various doses of [3H]-thymidine, and sacrificed at various time intervals in order to label dividing embryonic osteoblastic and preosteoblastic cells. Cross sections through the mid-body of 15-day embryos showed [3H-thymidine dose-dependent labeling of a relatively high percentage of cells in the liver (49 +/- 8% at 440 muCi) and a lower percentage of cells of the developing vertebral cartilage (29 +/- 6% at 440 muCi). ALP-positive condensed mesenchyme--consisting of mandibular preosteoblast (15 days of gestation) showed a relatively high (32 +/- 5%) level of [3H]-thymidine labeling, compared to surrounding ALP-negative loose mesenchymal cells (22 +/- 1%). Similar results were observed in the developing hind foot of 19-day embryos for ALP-positive cells (15 +/- 6%) and surrounding ALP-negative cells (13 +/- 5%). In both the hind foot and the mandible an overall decrease in labeling was observed during bone development. RNA samples from these tissues show increasing amounts of ALP mRNA, and decreasing amounts of histone H4 mRNA between days 15 and 19 of gestation. These data indicate that a general inverse correlation between osteoblastic differentiation and proliferation, similar to the correlation previously described in cultured osteogenic cells, is also present in developing bones in vivo. However, these results indicate that ALP-positive preosteoblasts, committed to the osteoblastic lineage, maintain their proliferative capacity. In an attempt to elucidate underlying molecular mechanisms, we further investigated the levels of expression of m-twist in these tissues. This member of the basic helix-loop-helix family of transcription regulators has been previously implied as playing a role in osteoblast differentiation in culture. Our results demonstrate a decrease in m-twist levels during bone development in both the mandible and the hind foot.