Different Stages Of Ontogeny Research Articles

Immunoglobulin V H gene mutational analysis suggests that blastic variant of mantle cell lymphoma derives from different stages of B-cell maturation

To characterise the nature of the cellular origin of the blastic variant of mantle cell lymphoma (MCL-BV), we analysed the immunoglobulin (Ig) heavy chain variable region (V H) genes in four cases of MCL-BV. The rearranged V H–D–J H genes were PCR-amplified, cloned and sequenced. In one case, the comparison of the rearranged V H gene sequence to known germline V H gene templates showed no somatic mutations suggesting a pre-germinal centre B-cell origin for tumour cells. In the other three cases, the V H gene sequences showed varied number of point mutations relative to the putative germline V H gene sequences but the point mutations were not associated with intraclonal diversification. In one of the mutated cases, the distribution and type of the mutations indicated that tumour cells had been selected by an antigen. Since somatically mutated Ig genes are expressed by B-cells that have reached a germinal centre/post-germinal centre stage of development, these findings suggest that the MCL-BV cell of origin may also be a germinal centre or a post-germinal centre B-cell. Taken together, our findings suggest that the development of MCL-BC may not be restricted to one stage of B-cell differentiation and that they may represent transformants of B-cells at different stages of ontogeny.

Expression and function of cell adhesion molecules on fetal liver, cord blood and bone marrow hematopoietic progenitors: Implications for anatomical localization and developmental stage specific regulation of hematopoiesis

The mechanism of localization, migration, and regulation of hematopoiesis at different stages of ontogeny is not well understood, but may relate to the specific adhesive interactions between hematopoietic stem cells and their microenvironment at different ontogenic stages. We studied the expression of cell adhesion molecules (CAM) on fetal liver (FL), umbilical cord blood (UCB) and adult bone marrow (ABM) CD34 + cells, and the adhesion of committed progenitors (CFC) from all three sources to ABM stromal layers and purified extracellular matrix proteins. Compared to ABM CFC, significantly more UCB CFC and fewer FL CFC adhered to ABM stroma. Adhesion of FL CFC to fibronectin (FN), the 75 kD RGD containing FN fragment and the 33–66 kD COOH-terminal heparin binding FN fragment was also significantly less than that of ABM CFC. Like ABM CFC, the adhesion of FL CFC was mediated through α4β1 and α5β1 integrins. Of note, more FL CD34 + cells expressed α5 integrins and the number of α4, α5 and β1 integins per cell (mean channel frequency) was similar or higher for FL CD34 + cells than ABM CD34 + cells. Further, treatment of FL CFC with a β1 integrin activating antibody (8A2), increased adhesion of FL CFC to FN to the same level as that of 8A2 treated ABM CFC. This suggests that the α4β1 and α5β1 integrins on FL CD34 + cells may be present in a low avidity/affinity state. We also show that unlike ABM, FL CD34 + cells expressed α2 and that approximately 20% FL CFC adhered to collagen IV. Further, α2β1 integrin on FL CFC was functional since their engagement, either by adhesion to collagen IV or through blocking α2 antibodies, transmitted proliferation inhibitory signals. In contrast to α4b and α5β1 integrin dependent adhesion, α2β1 dependent adhesion of FL CFC to collagen IV was not enhanced after treatment with 8A2. The reason for this is not clear but suggests that α2 integrins on FL CFC are maximally activated. This novel adhesive interaction with collagen IV, reminiscent of that described for CML progenitors, may have a role in the extramedullary localization of FL hematopoiesis or its developmental stage-specific regulation by its microenvironment. Studies to evaluate these possibilities are underway.

Ontogenetic aspects of hypertension development: analysis in the rat.

In this review, we attempt to outline the age-dependent interactions of principal systems controlling the structure and function of the cardiovascular system in immature rats developing hypertension. We focus our attention on the cardiovascular effects of various pharmacological, nutritional, and behavioral interventions applied at different stages of ontogeny. Several distinct critical periods (developmental windows), in which particular stimuli affect the further development of the cardiovascular phenotype, are specified in the rat. It is evident that short-term transient treatment of genetically hypertensive rats with certain antihypertensive drugs in prepuberty and puberty (at the age of 4-10 wk) has long-term beneficial effects on further development of their cardiovascular apparatus. This juvenile critical period coincides with the period of high susceptibility to the hypertensive effects of increased salt intake. If the hypertensive process develops after this critical period (due to early antihypertensive treatment or late administration of certain hypertensive stimuli, e.g., high salt intake), blood pressure elevation, cardiovascular hypertrophy, connective tissue accumulation, and end-organ damage are considerably attenuated compared with rats developing hypertension during the juvenile critical period. As far as the role of various electrolytes in blood pressure modulation is concerned, prohypertensive effects of dietary Na+ and antihypertensive effects of dietary Ca2+ are enhanced in immature animals, whereas vascular protective and antihypertensive effects of dietary K+ are almost independent of age. At a given level of dietary electrolyte intake, the balance between dietary carbohydrate and fat intake can modify blood pressure even in rats with established hypertension, but dietary protein intake affects the blood pressure development in immature animals only. Dietary protein restriction during gestation, as well as altered mother-offspring interactions in the suckling period, might have important long-term hypertensive consequences. The critical periods (developmental windows) should be respected in the future pharmacological or gene therapy of human hypertension.

Immunoglobulin V H Gene Mutational Analysis Suggests that Primary Effusion Lymphomas Derive from Different Stages of B Cell Maturation

Primary effusion lymphoma (PEL) is a recently described distinct subtype of non-Hodgkin's lymphoma associated with infection by the Kaposi's sarcoma-associated herpesvirus, also called human herpesvirus-8. Most cases of PEL are also associated with the Epstein-Barr virus (EBV). In order to better characterize the cellular origin of PEL, we investigated the immunoglobulin (Ig) heavy chain variable region (VH,) genes expressed by tumor cells of the BC-1 and BC-3 cell lines derived from PELs and five original PEL specimens. In the six EBV-positive PELs examined, including the BC-1 cell line, the expressed VH gene sequences showed numerous point mutations relative to the putative germline VH gene sequences. In addition, the VH, segment of one of these cases showed intraclonal sequence heterogeneity, indicating ongoing somatic mutation. In five cases, the distribution and type of mutations indicated that tumor cells had been selected by antigen. Because somatically mutated Ig genes are expressed by B cells that have reached a germinal center/post-germinal center stage of development, these findings suggest that the PEL cell of origin is a germinal center or post-germinal center B cell in most cases. In contrast, the VH gene segment expressed by tumor cells of the BC-3 cell line, which was originated from an EBV-negative PEL obtained from an HIV-negative patient, was unmutated, suggesting a pre-germinal center B cell origin for tumor cells of this particular PEL cell line. Taken together, these findings suggest that development of PELs may not be restricted to one stage of B cell differentiation and may represent transformation of B cells at different stages of ontogeny.

60K gelatinase involved in mammary gland involution is regulated by β-oestradiol

Cell matrix interactions are critical in the expression and maintenance of differentiated functions in mammary gland. Matrix metalloproteinases (MMPs), by acting on different matrix components, contribute to the remodelling of extracellular matrix. Of the three major gelatinases found in rat mammary gland at different stages of ontogeny, 60K gelatinase, a Ca 2+ dependent neutral MMP, seems to be involved in involution, as it appears at the late stage of involution. Further investigations on its regulation by hormones which influence the mammary gland function were carried out. Administration of β-oestradiol caused the appearance of 60K gelatinase on the 2nd day of involution, while in untreated controls this activity was absent. On treatment of mammary epithelial cells of the 2nd day involuting tissue in culture with β-oestradiol, the 60K gelatinase activity appeared, while the untreated controls did not show the activity. The effect of β-oestradiol was studied further by metabolic labelling of the epithelial cells from the 2nd day involuting tissue. A concentration dependent increase in the amount of radiolabelled 60K gelatinase was found on treatment with β-oestradiol. The upregulation of the 60K gelatinase activity in vivo was also found by immunocytochemical staining of the β-oestradiol treated tissues. The effect of β-oestradiol appears to be specific for 60K as the activity of other gelatinases (130K and 68K) in the mammary gland were not affected. Furthermore, a drastic regression of the mammary gland as evidenced by histochemical analysis and a marked decrease in the milk protein production in β-oestradiol treated tissues indicated the onset of early involution. These results indicate that the 60K gelatinase which is upregulated during involution or on induction of early involution may play a key role in remodelling of extracellular matrix in mammary gland and further that this enzyme is subject to modulation by β-oestradiol.

Interaction of central and peripheral noradrenergic structures in rat ontogeny

Morphology and morphometrical parameters of perikaryons and transcription activity of nuclear chromatin in neurons of cranial cervical sympathetic ganglion and in locus ceruleus are studied at different stages of ontogeny under conditions of desympathization and after selective damage to the locus ceruleus neurons induced by intracysternal injection of 6-hydroxydopamine. It is shown that the magnitude and direction of changes in neuronal ultrastructure and activity of protein-synthesizing apparatus varies during ontogeny and depends on the integral state of catecholaminergic neurons.

Molecular Characterization of IgA- and/or IgG-Switched Chronic Lymphocytic Leukemia B Cells

The immunoglobulin (Ig) variable region (V) genes expressed by IgM chronic lymphocytic leukemia (CLL) B cells display little or no somatic mutations. However, preliminary findings have shown that Ig V genes of IgA and IgG CLLs may be somatically mutated, suggesting that isotype-switched CLLs may represent a "subtype" of the disease. To investigate the degree and nature of somatic mutations and the role of antigen (Ag) in the clonal selection and expansion of isotype-switched CLLs, and to determine whether specific oncogene or tumor suppressor gene mutations are associated with isotype-switched CLLs, we analyzed the expressed Ig VH gene, bcl-1 and bcl-2 proto-oncogene, and p53 tumor suppressor gene configurations of 3 IgA-, 1 IgG-, and 1 IgA/ IgG-expressing CLLs. These isotype-switched CLL B cells expressed surface HLA-DR, CD19, CD23, and CD5, and displayed no alterations of the bcl-1 and bcl-2 oncogenes and the p53 tumor-suppressor gene. The cDNA VH-D-JH gene sequence was joined with that of the C alpha gene in the B cells of the three IgA CLLs, and with that of the C gamma gene in the IgG CLL B cells. In the IgA/IgG-coexpressing CLL B cells, identical VH-D-JH cDNA sequences were spliced to either C alpha or C gamma genes. In all five CLLs, the pattern of C mu DNA probe hybridization to the digested genomic DNAs was consistent with deletion of the C mu exon from the rearranged Ig gene locus, suggesting that these CLL B cells had undergone DNA switch recombination. In one IgA CLL, the expressed VH gene was unmutated. In all other class-switched CLLs, the Ig VH segment gene was mutated, but the point mutations were not associated with intraclonal diversification. In one IgA and in the IgA/IgG-coexpressing CLL, the nature and distribution of the mutations were consistent with Ag selection. These findings suggest that IgA- and/or IgG-expressing CLLs represent, in their VH gene structure, transformants of B cells at different stages of ontogeny. They also suggest that Ag may play a role in the clonal selection of some of these isotype-switched leukemic cells, but bcl-1 and bcl-2 oncogene rearrangements and p53 tumor suppressor gene mutation are not associated with the pathogenesis of isotype-switched CLLs.

Molecular Characterization of IgA- and/or IgG-Switched Chronic Lymphocytic Leukemia B Cells

AbstractThe immunoglobulin (Ig) variable region (V) genes expressed by IgM chronic lymphocytic leukemia (CLL) B cells display little or no somatic mutations. However, preliminary findings have shown that Ig V genes of IgA and IgG CLLs may be somatically mutated, suggesting that isotype-switched CLLs may represent a “subtype” of the disease. To investigate the degree and nature of somatic mutations and the role of antigen (Ag) in the clonal selection and expansion of isotype-switched CLLs, and to determine whether specific oncogene or tumor suppressor gene mutations are associated with isotype-switched CLLs, we analyzed the expressed Ig VH gene, bcl-1 and bcl-2 proto-oncogene, and p53 tumor suppressor gene configurations of 3 IgA-, 1 IgG-, and 1 IgA/IgG-expressing CLLs. These isotype-switched CLL B cells expressed surface HLA-DR, CD19, CD23, and CD5, and displayed no alterations of the bcl-1 and bcl-2 oncogenes and the p53 tumor-suppressor gene. The cDNA VH -D-JH gene sequence was joined with that of the Cα gene in the B cells of the three IgA CLLs, and with that of the Cγ gene in the IgG CLL B cells. In the IgA/IgG-coexpressing CLL B cells, identical VH -D-JH cDNA sequences were spliced to either Cα or Cγ genes. In all five CLLs, the pattern of Cμ DNA probe hybridization to the digested genomic DNAs was consistent with deletion of the Cμ exon from the rearranged Ig gene locus, suggesting that these CLL B cells had undergone DNA switch recombination. In one IgA CLL, the expressed VH gene was unmutated. In all other class-switched CLLs, the Ig VH segment gene was mutated, but the point mutations were not associated with intraclonal diversification. In one IgA and in the IgA/IgG-coexpressing CLL, the nature and distribution of the mutations were consistent with Ag selection. These findings suggest that IgA- and/or IgG-expressing CLLs represent, in their VH gene structure, transformants of B cells at different stages of ontogeny. They also suggest that Ag may play a role in the clonal selection of some of these isotype-switched leukemic cells, but bcl-1 and bcl-2 oncogene rearrangements and p53 tumor suppressor gene mutation are not associated with the pathogenesis of isotype-switched CLLs.

Light and electron microscopic demonstration of cholesterol distribution in membrane structures of the rat auditory cortex

As part of our investigations on the changes in the cortex of different stages of ontogeny, the aim of this study was to analyse the cholesterol distribution in the auditory cortex of adult rats. The light microscopic Schultz reaction as well as electron microscopic thin sections and freeze-etching combined with a cholesterol specific marker were applied to cholesterol demonstration. High and low cholesterol areas were found in the plasma membrane and membranes of some organelles. A low cholesterol content was observed in the membranes of the Golgi apparatus, mitochondria and junctional contacts. A very low cholesterol content was found in the pre- and postsynaptic membranes. High cholesterol contents were present in the neuronal and glial non-junctional plasma membranes. The cholesterol distribution in the membranes of the endoplasmic reticulum and nuclear envelope appeared to be different.

Consumption rates and the evolution of diet-induced plasticity in the head morphology of Melanoplus femurrubrum (Orthoptera: Acrididae).

Phenotypic plasticity may be an ecologically important evolutionary response to natural selection in multiple environments. I have determined the effect of diet-induced developmental plasticity in the head size of grasshoppers (Melanoplus femurrubrum) onfeeding performance on two types of plants. Full-sib families were divided and raised on either red clover, Trifolium repens, or rye grass, Lolium perenne. In three different stages of ontogeny, grasshoppers raised on rye grass had significantly larger heads, relative to body size, than full-sibs raised on clover. A principal components analysis indicated that two to five relative head size characters covaried as a block in their plastic response to the feeding environment. Regressions of adjusted consumption rates (mg/sec) against relative head size revealed that larger head sizes, induced by the rye grass diet, enhanced consumption rates of rye grass, but not clover. Unexpectedly, a similar positive association was observed between head size and consumption rate for grasshoppers raised on clover when they were feeding on clover. These results support the inference that grasshoppers exhibit adaptive phenotypic plasticity. However, the unexpected influence of head size on consumption rates of clover indicates that the functional relationship between head morphology and feeding performance is complex and that variation in this relationship among plant environments is not sufficient to explain the evolution of diet-induced phenotypic plasticity.

Nonrandom use of J alpha gene segments. Influence of V alpha and J alpha gene location.

TCR genes (alpha, beta, gamma, and delta) undergo rearrangement during development in the thymus. The repertoire of alpha beta and gamma delta cells is shaped first by genetic processes that control rearrangement and expression and then by intercellular processes that "select" cells expressing only particular receptors. In this report, alpha chain transcripts from different stages of ontogeny were examined to determine the influence of V and J gene location on the thymic repertoire. J alpha gene segments were not used equally by V alpha genes. The frequency of J alpha use, and thus the alpha chain repertoire, was influenced by at least three factors: the location of the V gene, the location of the J gene, and the age of the animal. V genes that are proximal to the J alpha region preferentially use the most 5' J alpha gene segments. In contrast, a V alpha gene that is distal to the J alpha region was rarely joined to these same 5' J alpha gene segments in thymocytes from adult animals. We suggest that the data are most easily explained by the frequent occurrence of secondary rearrangements in which a V alpha J alpha rearranged gene would be replaced by the joining of a flanking V alpha gene to a flanking J alpha gene segment. Finally, the evidence suggests that there may be differences in the extent of secondary rearrangements between fetal and adult animals.

Characterization of Human Lymphocytes Separated from Fetal Liver and Spleen at Different Stages of Ontogeny

Membrane markers on human lymphocytes separated from fetal liver and spleen were studied. Depending on the period of intrauterine development, a growing percentage of T- and Blymphocytes (up to 16 % and 45 %, respectively) among spleen cells was seen, but in liver the number was low independent of the gestational age (T cells < 10 % and B cells < 15 %).The majority of early CD3+ spleen cells (21st–28th week) expressed TCRαβ but not TCRγδ, although a significant proportion of these cells was still lacking CD4, CD8, and CD5 differentiation antigens, suggesting their immaturity.Later spleen T cells (29th–36th week) expressed the phenotype as mature adult-type T cells (CD3 +TCRαβ +CD4/8 +CD5 +).During ontogeny in fetal spleen, a growing number of B cells could be estimated without any changes in the proportion of subsets, expressing the different light and heavy chains.However, the proportion of CD5 + B cells decreased with gestational age. The results suggest that the functional immaturity of antenatal splenocytes could not be caused by dramatic phenotypical differences in comparison with adult-type splenic lymphocytes.

Comparison of D, JH, and junctional diversity in the fetal, adult, and aged B cell repertoires.

Polymerase chain reaction-amplified cDNA libraries of the IgH genes of fetal, young adult, and aged BALB/c mice were sequenced so that the complimentarity determining region 3 (CDR3) in each could be analyzed. The results show extensive diversity in the CDR3 region in all three libraries examined. A prominent feature of the fetal repertoire is the lack of nucleotide region additions and shorter germline-derived D segments compared with the adult repertoires. Also of interest were distinct differences in D family and JH usage in the three libraries representing different stages of ontogeny. The absence of DFL16.2 in the fetal sequences analyzed was of particular note. Also of note was a substantial underutilization of the largest D family, DSP2, in the aged repertoire. The study provides further evidence that the Ig repertoire is developmentally regulated. In addition, the results indicate that several aspects of the recombination process are different in adult and fetal B lineage cells, suggesting that B cells present early in ontogeny are distinct from those present in the adult.

Organization of the APUD system in mammalian lungs at different stages of ontogeny

Organization of the APUD system in mammalian lungs at different stages of ontogeny

Correlation between resistance of rats at different stages of ontogeny to seizures and hypoxia

It was shown in experiments on Wistar male rats that ethyl, 3/2, ethyl, 2/2, dimethylhydrazine propionate iodate (EDIHYP), a synthetic acetylcholine analogue, eliminates in situ the fall of the ventricular fibrillation threshold and the extrasystole observed on the background of vagal bradycardia in experimental myocardial infarction and postinfarction cardiosclerosis. The elimination of disturbed heart electric stability was not accompanied by cholinergic, negative chronotropic effect of the drug. In isolated heart, high concentrations of EDIHYP (10(-4) M) had negative chronotropic effect but lacked antiarrhythmic effect in local ischemia and reperfusion. The bradycardia induced by EDIHYP was absent and the antiarrhythmic effect was strikingly pronounced on the background of muscarinic receptors blockade with atropine. Thus EDIHYP realizes its antiarrhythmic effect not via muscarinic receptors but by some other way which requires studying by methods of molecular pharmacology.

Polyclonal stimulation of human B lymphocytes derived from fetal liver and spleen cells at different stages of ontogeny

The functional capacity of human lymphocytes derived from fetal liver and spleen at different stages of ontogeny (16-34 weeks of gestation) was studied using in vitro models (increase in cell volume, [3H]thymidine incorporation, Ig secretion) reflecting various stages of activation induced by mitogens (LPS, PWM) in vitro. Lymphocytes differed in their reactivity to LPS depending on the period of intrauterine development: cells from the early liver could respond with enhanced IgM production whereas lymphocytes derived from this organ after more than 25 weeks failed. The opposite was found to apply to spleen cells: only lymphocytes derived from the organ after more than 25 weeks showed significant LPS-induced in vitro differentiation. These data were in correlation with the proliferative response to LPS. It was clear that the number of CD20-positive mature B cells in the lymphocyte preparations was not responsible for these results, since comparable yields were found throughout the period of fetal development of the liver studied, whereas in the spleen increasing numbers of B cells were seen.

Quantitative analysis of CD5 antigen modulation by 12- O-tetradecanoylphorbol-13-acetate in T-lymphoblastic leukemia cells: Individual response patterns and their relationships with both maturation and protein kinase C content

Modulation of CD5 expression by TPA was investigated on T-leukemic cell lines corresponding to different stages of ontogeny. These CD5 changes have been analyzed simultaneously with modifications of cell growth, cell cycle, cell surface phenotype, and PKC content. CD5 expression was found 6- to 17-fold increased by TPA in a dose-dependent manner on phenotypically mature T-cells (Jurkat, JM, and T-CLL) while T-cells from earlier stages of differentiation (CEM III, CEM 95, and CEM 44) were found unresponsive. CD5 upregulation on TPA-sensitive JM cells appears correlated with inhibition of cell growth, blockage in Gl phase, and phenotypic maturation (downregulation of CD7 and CD1 antigens) and seemed to be related to PKC activation since DiC 8 (a PKC activator) mimicked this TPA effect and H7 (a PKC inhibitor) partially reduced it. On the other hand, on CEM III cells TPA induced no modulation of CD5 antigen, a less dramatic effect on cell growth and cell cycle, but a CD7 downregulation. TPA appeared fully effective in binding and translocating PKC in both CEM III and JM cells, although the PKC activity level was three times higher in the latter. Finally, our study suggests that CD5 expression is at least partially under control of PKC in phenotypically mature neoplastic T-cells while PKC could not be directly involved in the regulation of CD5 antigen in leukemic cells arrested at earlier stages of differentiation.

Expression of T cell receptor Vγ5 in the adult thymus of irradiated mice after transplantation with fetal liver cells

T cell receptor (TcR) gamma/delta displays limited diversity and its diversity is distinct in different stages of ontogeny and in different anatomical sites. The V gamma 5 and V delta 1 gene products are preferentially expressed on the early fetal thymocytes and on Thy-1+ dendritic epidermal cells, whereas the V gamma 4 and V delta 5 gene products are abundantly expressed on the adult thymocytes. To elucidate whether the developmentally ordered appearance of thymocytes expressing TcR gamma/delta is dependent on the source of T cell precursors or is controlled by the thymic environment where T cells develop, we compared the expression of V gamma 5 on the early-appearing thymocytes between irradiated mice after transplantation with fetal liver (FL) cells and those after transplantation with bone marrow (BM) cells. Sequential appearance of thymocyte subpopulations was observed in the thymus of radiation FL chimeras similar to that seen in radiation BM chimeras. A substantial number of thymocytes bearing V gamma 5 appeared in the thymus at the early stage of radiation FL chimeras, whereas few, if any, of such V gamma 5-bearing thymocytes were detected in the thymus at any stage of radiation BM chimeras. These results suggested that the ordered expression of V gamma repertoire may depend on the origin of the T cell precursors but not on the thymic environment.

Expression of the human carbonic anhydrase I gene is activated late in fetal erythroid development and regulated by stage‐specific trans‐acting factors

Using flow cytometric analysis of red cells from different stages of ontogeny with anti-CAI antibody, it was shown that the human carbonic anhydrase I (HCAI) gene product appears in a developmental stage-specific manner. Virtually no CAI protein was detectable in fetal red cells prior to birth. However, at about the time of normal delivery (40 weeks gestation) CAI production is switched on. The proportion of cells containing CAI reaches adult levels during the second half of the first year of life. Northern analysis suggests that the appearance of CAI protein results directly from the activation of the gene and the production of new mRNA. A transient heterokaryon system was set up by fusing the erythroleukaemic cell lines MEL C88 (a mouse cell line in which CAI is expressed) and K562 SAI (a human cell line with an embryonic/fetal phenotype, not expressing CAI). SP6 RNAase mapping of RNA from the fused cells showed activation of the human CAI gene. This shows the developmental stage-specific expression of HCAI to be regulated by trans-acting factors.

Expression of antibody V-regions is genetically and developmentally controlled and modulated by the B lymphocyte environment

In this study we performed a comprehensive analysis of VH family usage in the emergent, available and actual repertoires of neonatal and adult BALB/c and C57BL/6 (B6) mouse strains. For this purpose we used an in situ hybridization technique that allows the detection of VH-gene expression at a single cell level. We have found that VH gene expression in neonatal mice is determined by a non-random position-dependent process which favours the utilization of the most D-proximal VH 7183 family. The preferential usage of the 7183 family is also characteristic of early differentiating bone marrow B cells of adult BALB/c mice. At different stages of ontogeny and B cell development VH family repertoires evolve in a strain-specific manner, with significantly higher utilization of the VH J558 family in B6 mice. In the peripheral immunocompetent cell pool, local environmental factors can further modulate VH family expression and lead to increased representation of the VH J558 family in peripheral lymph nodes and of the VH X-24 family in intestinal Peyer's patches. In conclusion, our present results indicate that VH family usage is controlled by genetic, developmental and environmental factors and suggest that selection of antibody repertoires can occur at multiple stages in B cell development.