Early Step In Development Research Articles

Malformation rate in fetuses and children conceived after ICSI: results of a prospective cohort study

There is an ongoing discussion regarding conflicting data on malformation rate in children born after intracytoplasmic sperm injection (ICSI). A prospective, multicentric, control cohort study was done in Germany. Fifty-nine centres prospectively recruited pregnancies before the 16th week of gestation, which were included in the study if they were ongoing beyond this time. Children were examined according to a standardized procedure. A control cohort of children conceived spontaneously was taken from a prospective birth registry (Mainzer Modell), where children were examined according to the exact same criteria as the ICSI cohort. Major malformation rate was calculated, based on data of all liveborn and stillborn children, as well as on all spontaneous and induced abortions, beginning with the 16th week of gestation. In the ICSI cohort, 8.6% of infants (291/3372), and in the control cohort 6.9% of infants (2140/30940), had a major malformation. This resulted in a crude relative risk (RR) of 1.25 (95% confidence interval 1.11–1.40). There was no influence of sperm origin on major malformation rate in children born after ICSI. There is an increased risk for a child born after ICSI to have a major malformation compared with a child that has been spontaneously conceived. Based on knowledge of the early developmental steps following ICSI, as well as on data of conventional IVF in general, it is assumed that this increased risk is due to parental factors causing the infertility, which has led to ICSI in the first place.

Kermit, a frizzled interacting protein, regulates frizzled 3 signaling in neural crest development.

Wnts are a family of secreted glycoproteins that are important for multiple steps in early development. Accumulating evidence suggests that frizzled genes encode receptors for Wnts. However, the mechanism through which frizzleds transduce a signal and the immediate downstream components that convey that signal are unclear. We have identified a new protein, Kermit, that interacts specifically with the C-terminus of Xenopus frizzled-3 (Xfz3). Kermit is a 331 amino acid protein with a central PDZ domain. Kermit mRNA is expressed throughout Xenopus development and is localized to neural tissue in a pattern that overlaps Xfz3 expression temporally and spatially. Co-expression of Xfz3 and Kermit results in a dramatic translocation of Kermit to the plasma membrane. Inhibition of Kermit function with morpholino antisense oligonucleotides directed against the 5' untranslated region of Kermit mRNA blocks neural crest induction by Xfz3, and this is rescued by co-injection of mRNA encoding the Kermit open reading frame. These observations suggest that Kermit is required for Wnt/frizzled signaling in neural crest development. To the best of our knowledge, Kermit is the first protein identified that interacts directly with the cytoplasmic portion of frizzleds to modulate their signaling activity.

Systems Engineering of Time-Triggered Architectures - The Setta Approach

SETTA addresses the systems engineering of safety-critical distributed real¬time systems with a special focus on time-triggered architectures. An innovative meth¬odology and a corresponding engineering environment is developed which aims for a higher maturity at early development steps. Key features are the support for a virtual systems integration and the tighter interconnection between the functional development process and the safety analysis process. The supporting tool components are designed and implemented in the course of the SETTA project. The methodology is evaluated by pilot applications from the automotive, aerospace, and railway domain.

Modeling the interplay of generic and genetic mechanisms in cleavage, blastulation, and gastrulation.

Early development of multicellular organisms is marked by a rapid initial increase in their cell numbers, accompanied by spectacular morphogenetic processes leading to the gradual formation of organs of characteristic shapes. During morphogenesis, through differentiation under strict genetic control, cells become more and more specialized. Morphogenesis also requires coordinated cell movement and elaborate interactions between cells, governed by fundamental physical or generic principles. As a consequence, early development must rely on an intricate interplay of generic and genetic mechanisms. We present the results of computer simulations of the first nontrivial morphogenetic transformations in the life of multicellular organisms: initial cleavages, blastula formation, and gastrulation. The same model, which is based on the physical properties of individual cells and their interactions, describes all these processes. The genetic code determines the values of the model parameters. The model accurately reproduces the major steps of early development. It predicts that physical constraints strongly influence the timing of gastrulation. Gastrulation must occur prior to the appearance of dynamical instability, which would destabilize and eventually derail normal development. Within our model, to avoid the instability, we suddenly change the values of some of the model parameters. We interpret this change as a consequence of specific gene activity. After changing the physical characteristics of some cells, normal development resumes, and gastrulation proceeds.

Bilateral asymmetry of the inositol trisphosphate-mediated calcium signaling in two-cell ascidian embryos

In ascidian oocytes, numerous calcium signaling events occur at fertilization which contribute to resume and complete meiosis, and determine the three embryonic axes. The main ooplasmic and intracellular calcium channels at work in the calcium signaling of the one-cell embryo have different roles and fates when the first mitosis begins. By whole-cell patch-clamp recording, we observed different families of these calcium channels in the blastomeres of Phallusia mammillata two-cell ascidian embryos. Membrane capacitance has been measured to evaluate the oocyte and blastomere surface area, allowing certification of the exact time of cell division. At the two-cell stage, no difference was observed in the density of voltage-dependent calcium channels in each blastomere, or in the ryanodine-sensitive calcium stores. In contrast, a bilateral asymmetry was recorded for the ooplasmic channels responsible for calcium entry after calcium store depletion: they could be activated only in the blastomere not wearing the polar bodies. The same laterality was observed in the InsP 3-induced internal calcium release. Moreover, this asymmetry included a one-way communication in the InsP 3-dependent calcium signaling between the two blastomeres. These results enhance the understanding of the early steps of development, and underscore the interest for ascidians in studies of polarity patterning.

Conserved developmental algorithms during thalamocortical circuit formation in mammals and reptiles.

The general patterns of early thalamocortical development follow a similar sequence in all mammals. Thalamocortical projections descend through the ventral thalamus, advance in the internal capsule amongst cells which already possess dorsal thalamic projections, then reach the cerebral cortex by associating with subplate cells and their early corticofugal projections. Initially, the thalamic projections pause in the internal capsule and subplate layer. The interactions of the thalamocortical projections with the early generated, largely transient cells of the subplate, marginal zone, internal capsule and ventral thalamus are believed to play a crucial role in the organized deployment of thalamic projections and establishing a functional cortical architecture. Selective fasciculation, contact guidance and release of neurotrophic factors are thought to play roles in the development of thalamocortical projections. These ideas are obtaining support from recent work on reeler and other strains of mice. The evolutionary origin of these largely transient cells and the overlying logic of early developmental steps are not understood. The behaviour of the thalamocortical and corticothalamic projections at the corticostriatal junction is particularly puzzling. The comparison of early forebrain development in mammals and reptiles is beginning to reveal highly conserved cellular and molecular interactions during early thalamocortical development and to reveal homologies between telencephalic subdivisions.

Role of the neutrophil in the development of sepsis following abdominal aortic aneurysm surgery

Abstract Background There is evidence to suggest that the neutrophil (PMN) plays a critical early step in development of the ischaemia–reperfusion syndrome and sepsis. Not all patients undergoing aortic aneurysm repair develop these postoperative complications. The PMN receptor CD16 plays an important role in phagocytosis, cell-mediated cytotoxicity and the release of free radicals and proteolytic enzymes. This study determined if there is any relationship between PMN CD16 expression, phagocytosis and the development of sepsis. Methods Some 50 patients (39 men and 11 women, median age 70 years) who underwent elective infrarenal abdominal aortic aneurysm repair were studied. Venous blood was taken before operation, throughout surgery and for 7 days afterwards. CD16 was measured, unstimulated and following further stimulation with phorbol myrisate, lipopolysaccharide and N-formyl-methionyl-leucyl-phenylalanine using flow cytometry. Phagocytosis was measured using flow cytometry and the production of elastase using an enzyme-linked immunosorbent assay. Sepsis was defined according to the ACCP/SCCM definition. Results There were 36 uncomplicated operations and 14 patients with sepsis. There was no difference between the two groups in respect of nutritional, co-morbid or technical factors. In the group that developed septic complications, the level of PMN CD16 expression was significantly higher before operation (30·2 versus 10·4 mcf; P < 0·05, Mann–Whitney U test) and throughout the postoperative period. Surgery produced no change in CD16 expression in either the septic or uncomplicated group. Stimulation of the PMNs before operation caused an increase in CD16 expression in both groups. After operation, stimulation of PMNs in the septic group resulted in a fall in CD16 expression (40·8 versus 20·4 mcf; P < 0·05, Mann–Whitney U test); surgery produced no change in the level of expression in the uncomplicated group. Phagocytic ability was comparable before operation (91·2 versus 94·2 mcf), but showed a greater reduction in the septic group after operation (73·4 versus 86·2 mcf; P < 0·05, Mann–Whitney U test). Elastase concentration was also similar before operation (48 versus 49 μg l−1); after operation, there was a greater increase in the septic group (166 versus 104 μg l−1; P < 0·05, Mann–Whitney U test). Conclusion This study provides evidence of functional differences in PMN behaviour in patients who subsequently develop sepsis following aneurysm surgery. This difference occurs early in the postoperative course and it may be possible to identify these patients before operation.

Subtractive analysis of S. franciscanus and S. purpuratus ovary mRNA: what kinds of genes determine species-specificity?

Our goal is to identify and understand points of regulation in sperm–egg recognition as well as in steps of early development. These processes are species-specific and are the key to understanding speciation. In both vertebrates and invertebrates, the interaction of the sperm and egg displays a wide range of species-specificity. The questions we would like to answer are: What kinds of molecules determine the specificity and control the fertilisation process? Are early steps in development regulated in a species-specific manner?As an approach to identifying genes that determine species-specificity, in two different species, S. purpuratus (S. p.) and S. franciscanus (S. f.), we used a new subtractive hybridisation method known as RDA (representational difference analysis) (Lisitsyn et al., 1993; Hubank et al., 1994). Several species-specific clones were isolated from S. f. ovary mRNA by this method using mRNA from another species but the same genus of sea urchin, S. p. Four different clones were obtained and the species-specificity of the sequence was confirmed by hybridisation. One of them has four tandem EGF repeats and is homologous to the S. p. EGF-II gene (Yang et al., 1989) and A. crassispina EGIP (exogastrula inducing protein) gene (Ishihara et al., 1982). The first three EGF repeats (EGF 1–3) have 60% similarity among these species, but the fourth EGF domain (EGF 4) is highly divergent. The EGF-II protein is believed to be involved in the signalling events of early development, because purified EGF causes exogastrulation when it is added to the seawater prior to gastrulation (Ishihara et al., 1982). Recombinant EGF 3 from S. f. induces exogastrulation in both S. f. and S. p.

Angiosperm fertilisation, embryo and endosperm development in vitro

The application of micromanipulation techniques enables the isolation and in vitro fusion of female and male angiosperm gametes. In combination with tissue culture methods, which are adapted for the culture of single cells, these techniques allow individual development of zygotes and endosperm. Both in vitro produced zygotes and zygotes which are isolated after in vivo pollination develop into embryos and fertile plants in culture. Zygotes and endosperm are able to self-organise in culture independently from maternal tissue. Many steps of early development in vitro of both the embryo and endosperm are comparable to the situation in planta, making this system a useful model for studies of embryogenesis and endosperm development. Because of access to single gametes and zygotes, these tools will also facilitate studies of gamete interaction and gametic hybridisation under defined conditions. The use of these techniques together with biochemical and molecular methods, adapted to the analysis of single cells, allows one to analyse early developmental events, precisely timed after fertilisation. Recent advances in the biotechnology of fertilisation and of zygote culture are discussed as well as prospects for future studies.

Fasciclin II and Beaten path modulate intercellular adhesion in Drosophila larval visual organ development.

Previous studies demonstrated that Fasciclin II and Beaten path are necessary for regulating cell adhesion events that are important for motoneuron development in Drosophila. We observe that the cell adhesion molecule Fasciclin II and the secreted anti-adhesion molecule Beaten path have additional critical roles in the development of at least one set of sensory organs, the larval visual organs. Taken together, phenotypic analysis, genetic interactions, expression studies and rescue experiments suggest that, in normal development, secretion of Beaten path by cells of the optic lobes allows the Fasciclin II-expressing larval visual organ cells to detach from the optic lobes as a cohesive cell cluster. Our results also demonstrate that mechanisms guiding neuronal development may be shared between motoneurons and sensory organs, and provide evidence that titration of adhesion and anti-adhesion is critical for early steps in development of the larval visual system.

5 Digging out Roots: Pattern Formation, Cell Division, and Morphogenesis in Plants

The analysis of plant development by genetic, molecular, and surgical approaches has accumulated a large body of data, and yet it remains a challenge to uncover the basic mechanisms that are operating. Early steps of development, when the zygote and its daughter cells organize the embryonic plant, are poorly understood despite considerable efforts toward the identification of relevant genes. Reported cases of genetic redundancy suggest that the difficulty in uncovering patterning genes may reflect overlapping gene activities. Our current knowledge on plant embryo development still leaves open whether mechanisms for axis formation and subsequent pattern formation are fundamentally different in animals and plants. Axis formation may follow the general principle of establishing a peripheral asymmetric cue and mobilizing the cytoskeleton toward this cue--in the case of plants possibly located in the cell wall--but the molecules involved may be entirely different. Embryonic pattern formation involves the establishment of different domains, but although there are candidates, it is not clear whether genes that define these domains are identified yet. Pattern formation continues postembryonically in the meristem, and the flexibility of this process may be explained by a feed-forward system of patterning cues originating from more mature cells. Control of cell division and differentiation, which is important in the meristems--regions of continuous development--has been studied intensively and appears to involve short-range signaling and transmembrane receptor kinase activation. Finally, although high importance of control of cell division rates and planes for plant morphogenesis have been often inferred, recent genetic studies as well as comparative morphological data point to a less decisive role of cell division and to global controls of as yet unknown nature.

RPE, a plant gene involved in early developmental steps of nematode feeding cells.

Sedentary plant-parasitic nematodes are able to induce the redifferentiation of root cells into multinucleate nematode feeding sites (NFSs). We have isolated by promoter trapping an Arabidopsis thaliana gene that is essential for the early steps of NFS formation induced by the root-knot nematode Meloidogyne incognita. Its pattern of expression is similar to that of key regulators of the cell cycle, but it is not observed with the cyst nematode. Later in NFS development, this gene is induced by both root-knot and cyst nematodes. It encodes a protein similar to the D-ribulose-5-phosphate 3-epimerase (RPE) (EC 5.1.3.1), a key enzyme in the reductive Calvin cycle and the oxidative pentose phosphate pathway (OPPP). Quantitative RT-PCR showed the accumulation of RPE transcripts in potato, as in Arabidopsis NFS. Homozygous rpe plants have a germination mutant phenotype that can be rescued in dwarf plants on sucrose-supplemented medium. During root development, this gene is expressed in the meristems and initiation sites of lateral roots. These results suggest that the genetic control of NFSs and the first stages of meristem formation share common steps and confirms the previous cytological observations which indicate that root cells undergo metabolic reprogramming when they turn into NFSs.

Methylation of FrzCD defines a discrete step in the developmental program of Myxococcus xanthus.

Myxococcus xanthus is a gram-negative soil bacterium which undergoes fruiting body formation during starvation. The frz signal transduction system has been found to play an important role in this process. FrzCD, a methyl-accepting taxis protein homologue, shows modulated methylation during cellular aggregation, which is thought to be part of an adaptation response to an aggregation signal. In this study, we assayed FrzCD methylation in many known and newly isolated mutants defective in fruiting body formation to determine a possible relationship between the methylation response and fruiting morphology. The results of our analysis indicated that the developmental mutants could be divided into two groups based on their ability to show normal FrzCD methylation during development. Many mutants blocked early in development, i.e., nonaggregating or abnormally aggregating mutants, showed poor FrzCD methylation. The well-characterized asg, bsg, csg, and esg mutants were found to be of this type. The defects in FrzCD methylation of these signaling mutants could be partially rescued by extracellular complementation with wild-type cells or addition of chemicals which restore their fruiting body formation. Mutants blocked in late development, i.e., translucent mounds, showed normal FrzCD methylation. Surprisingly, some mutants blocked in early development also exhibited a normal level of FrzCD methylation. The characterized mutants in this group were found to be defective in social motility. This indicates that FrzCD methylation defines a discrete step in the development of M. xanthus and that social motility mutants are not blocked in these early developmental steps.

Thrombopoietin Requires Additional Megakaryocyte-Active Cytokines for Optimal Ex Vivo Expansion of Megakaryocyte Precursor Cells

AbstractLittle is known concerning the interaction of thrombopoietin (TPO) with other megakaryocyte-active cytokines in directing the early events of megakaryocyte development. Culture of CD34+ cells in interleukins (IL) -1, -6, -11, plus stem cell factor (SCF; S) results in a 10- to 12-fold expansion in total cell numbers, whereas total CD41+ megakaryocytes are expanded ∼120-fold over input levels. Addition of TPO to IL-1, -6, -11, S generates a biphasic proliferation of CD41+ cells, accelerates their rate of production, and results in an ex vivo expansion of more than 200-fold. The addition of Flt-3 ligand (FL) increases CD41+ cell expansion to ∼380-fold over input levels. In the absence of TPO, ∼95% of the expanded cells show the phenotype of promegakaryoblasts; TPO and/or FL addition increases CD41 antigen density and ploidy in a subpopulation of promegakaryoblasts. A moderate (approximately sevenfold) expansion of megakaryocyte progenitor cells (colony-forming unit-megakaryocyte) occurs in the presence of IL-1, -6, -11, S, and the addition of TPO to this cocktail yields an ∼17-fold expansion. We conclude that early proliferative events in megakaryocyte development in vitro are regulated by multiple cytokines, and that TPO markedly affects these early developmental steps. However, by itself, TPO is neither necessary nor sufficient to generate a full proliferative/maturational in vitro response within the megakaryocyte compartment. TPO clearly affects terminal differentiation and the development of (some) high-ploidy human megakaryocytes. However, its limited in vitro actions on human cell polyploidization suggest that additional megakaryocyte-active cytokines or other signals are essential for the maximal development of human megakaryocytes.

Thrombopoietin Requires Additional Megakaryocyte-Active Cytokines for Optimal Ex Vivo Expansion of Megakaryocyte Precursor Cells

Little is known concerning the interaction of thrombopoietin (TPO) with other megakaryocyte-active cytokines in directing the early events of megakaryocyte development. Culture of CD34+ cells in interleukins (IL) -1, -6, -11, plus stem cell factor (SCF; S) results in a 10- to 12-fold expansion in total cell numbers, whereas total CD41+ megakaryocytes are expanded ∼120-fold over input levels. Addition of TPO to IL-1, -6, -11, S generates a biphasic proliferation of CD41+ cells, accelerates their rate of production, and results in an ex vivo expansion of more than 200-fold. The addition of Flt-3 ligand (FL) increases CD41+ cell expansion to ∼380-fold over input levels. In the absence of TPO, ∼95% of the expanded cells show the phenotype of promegakaryoblasts; TPO and/or FL addition increases CD41 antigen density and ploidy in a subpopulation of promegakaryoblasts. A moderate (approximately sevenfold) expansion of megakaryocyte progenitor cells (colony-forming unit-megakaryocyte) occurs in the presence of IL-1, -6, -11, S, and the addition of TPO to this cocktail yields an ∼17-fold expansion. We conclude that early proliferative events in megakaryocyte development in vitro are regulated by multiple cytokines, and that TPO markedly affects these early developmental steps. However, by itself, TPO is neither necessary nor sufficient to generate a full proliferative/maturational in vitro response within the megakaryocyte compartment. TPO clearly affects terminal differentiation and the development of (some) high-ploidy human megakaryocytes. However, its limited in vitro actions on human cell polyploidization suggest that additional megakaryocyte-active cytokines or other signals are essential for the maximal development of human megakaryocytes.

The Role of DFA in Product Development - An Industrial Case Study

In the paper the role of design for assembly (DFA) is discussed on an industrial example. The product under development is a family of valve actuators for heating control. The applied DFA method is the Boothroyd-Dewhurst method supported by a software tool. First, the existing product family was analyzed to identify design weaknesses. The results were implemented in design of the improved product. DFA is one of engineering aspects to be considered during design. Because of its cost saving potential it has a high priority. However, other design aspects have to be considered as well, and may be in collision with the DFA principles. Therefore, the final design should be a compromise giving the overall optimum solution. Some principles of DFA are of generic nature and can be implemented in early development steps. A short discussion of these considerations is given.

DNA-mediated Folding and Assembly of MyoD-E47 Heterodimers

Basic region helix-loop-helix (bHLH) transcription factors regulate key steps in early development by binding to regulatory DNA sites as heterodimers consisting of a tissue-specific factor and a widely expressed factor. We have examined the folding, dimerization, and DNA binding properties of the muscle-specific bHLH protein MyoD and its partner E47, to understand why these proteins preferentially associate in heterodimeric complexes with DNA. In the absence of DNA, the E47 bHLH domain forms a very stable homodimer, whereas MyoD is unfolded and monomeric. Fluorescence quenching experiments show that MyoD does not dimerize with E47 under dilute conditions in the absence of DNA. Residues in and around the loop of the E47 bHLH domain contribute to its markedly greater stability. An altered MyoD bHLH substituted with the loop segment from E47 folds in the absence of DNA, and it readily dimerizes with E47. In the presence of a specific DNA binding site, MyoD and E47 both form homodimeric complexes with DNA that have similar dissociation constants, despite the very different stabilities of these protein dimers off DNA. A 1:1 mixture of these bHLH domains forms almost exclusively heterodimeric complexes on DNA. Assembly of these bHLH-DNA complexes is apparently governed by the strength of each subunit's interaction with the DNA and not by the strength of protein-protein interactions at the dimer interface. These findings suggest that preferential association of MyoD with E47 in DNA complexes results from more favorable DNA contacts made by one or both subunits of the heterodimer in comparison with either homodimeric complex.

A Human Homologue of the Drosophila melanogaster diaphanous Gene Is Disrupted in a Patient with Premature Ovarian Failure: Evidence for Conserved Function in Oogenesis and Implications for Human Sterility

Premature ovarian failure (POF) is a defect of ovarian development and is characterized by primary or secondary amenorrhea, with elevated levels of serum gonadotropins, or by early menopause. The disorder has been attributed to various causes, including rearrangements of a large "critical region" in the long arm of the X chromosome. Here we report identification, in a family with POF, of a gene that is disrupted by a breakpoint. The gene is the human homologue of the Drosophila melanogaster diaphanous gene; mutated alleles of this gene affect spermatogenesis or oogenesis and lead to sterility. The protein (DIA) encoded by the human gene (DIA) is the first human member of the growing FH1/FH2 protein family. Members of this protein family affect cytokinesis and other actin-mediated morphogenetic processes that are required in early steps of development. We propose that the human DIA gene is one of the genes responsible for POF and that it affects the cell divisions that lead to ovarian follicle formation.

Differential developmental expression of the rep B and rep D xeroderma pigmentosum related DNA helicase genes from Dictyostelium discoideum.

DNA helicases are essential to many cellular processes including recombination, replication and transcription, and some helicases function in multiple processes. The helicases encoded by the Xeroderma pigmentosum (XP) B and D genes function in both nucleotide excision repair and transcription initiation. Mutations that affect the repair function of these proteins result in XP while mutations affecting transcription result in neurological and developmental abnormalities, although the underlying molecular and cellular basis for these phenotypes is not well understood. To better understand the developmental roles of these genes, we have now identified and characterized the rep B and rep D genes from the cellular slime mold Dictyostelium discoideum . Both genes encode DNA helicases of the SF2 superfamily of helicases. The rep D gene contains no introns and the rep B gene contains only one intron, which makes their genomic structures dramatically different from the corresponding genes in mammals and fish. However the predicted Dictyostelium proteins share high homology with the human XPB and XPD proteins. The single copy of the rep B and D genes map to chromosomes 3 and 1, respectively. The expression of rep B and D (and the previously isolated rep E) genes during multicellular development was examined, and it was determined that each rep gene has a unique pattern of expression, consistent with the idea that they have specific roles in development. The pattern and extent of expression of these genes was not affected by the growth history of the cells, implying that the expression of these genes is tightly regulated by the developmental program. The expression of the rep genes is a very early step in development and may well represent a key event in the initiation of development in this organism.

Thymocytopoiesis in aging: the bone marrow-thymus axis.

Manifestations of aging in the mature T lymphocyte compartment have been attributed, to a major extent, to effects of the involuted thymus, at the thymic microenvironment level. However, since generation of T lymphocytes starts from hemopoietic stem cells that settle in the thymus and differentiate there, aging effects on the stem cells, and as a consequence, on the bone marrow (BM)-thymus axis, may also have an impact on patterns of thymocytopoiesis and on age-related thymus remodeling. This communication reviews our studies designed to determine whether BM cells manifest any aging effects that become overt in the resulting thymocytes. The experiments were performed by seeding of BM cells onto lymphoid-depleted fetal thymus (FT) explants, to enable distinguishing between processes that occur in the BM and those that are caused by the aging thymic microenvironment. The data show changes in the developmental potential of BM-derived cells, as reflected from the kinetics of cell cycle and intermediate steps from stem cell settling in the thymus to an early stage at the transition from CD4(-)CD8(-), double negative (DN), to CD4(+)CD8(+), double positive (DP) thymocytes. In addition, we have demonstrated that these early developmental steps of thymocytopoiesis are subject to feedback regulation by mature T cells, and the extent of regulation may be altered in old age. The pattern of T lymphocyte generation in aging is thus a result of dynamic changes in thymic, as well as extrathymic functions, along the sequential developmental steps from the stem cell to the ultimate mature cell.