The expression of twisted gastrulation in postnatal mouse brain and functional implications

Twisted gastrulation (TWSG1), an extracellular regulator of bone morphogenetic protein (BMP) signaling, is critical for embryonic brain development. Mice deficient in TWSG1 have abnormal forebrain development manifesting as holoprosencephaly. The expression and potential roles of TWSG1 in postnatal brain development are less well understood. We show that Twsg1 is expressed in the adult mouse brain in the choroid plexus (CP), hippocampus, and other regions, with the strongest expression observed in CP. TWSG1 was also detected in a human fetal brain at mid-gestation, with highest levels in the epithelium of CP. Bmp1, Bmp2, Bmp4–Bmp7 as well as BmprIA and BmprII, but not BmprIB, were expressed in CP. BMP antagonists Chordin (Chrd) and Noggin were not detected in CP, however Chrd-like 1 and brain-specific Chrd-like (Brorin) were expressed. Electrophysiological study of synaptic plasticity revealed normal paired-pulse facilitation and long-term potentiation in the CA1 region of hippocampus in Twsg1^−/− mice. Among the homozygous mutants that survive beyond the first 2 weeks, the prevalence of hydrocephalus was 4.3%, compared to 1.5% in a wild type colony (P=0.0133) between 3 and 10 weeks of life. We detected a high level of BMP signaling in CP in wild type adult mice that was 17-fold higher than in the hippocampus (P=0.005). In contrast, transforming growth factor beta (TGFβ) signaling was predominant in the hippocampus. Both BMP signaling and the expression of BMP downstream targets Msx1 and Msx2 were reduced in CP in Twsg1^−/− mice. In summary, we show that Twsg1 is expressed in the adult mouse and human fetal CP. We also show that BMP is a branch of TGFβ superfamily that is dominant in CP. This presents an interesting avenue for future research in light of the novel roles of CP in neural progenitor differentiation and neuronal repair, especially since TWSG1 appears to be the main regulator of BMP present in CP.     

The development of lymphocytes with T- or B-membrane determinants in the human foetus

The development of T- or B-membrane determinants on human foetal lymphoid cells was studied by the direct immunofluorescence technique, using a tetramethyl rhodamine isothiocyanate (TRITC) labelled horse antihuman T-cell conjugate (ATC) for the detection of T lymphocytes and a fluorescein isothiocyanate (FITC) labelled goat antihuman Fab conjugate for the demonstration of Ig-bearing B lymphocytes. Human foetal lymphocytes were also tested for spontaneous rosette formation with sheep red blood cells (SRBC).

Cell suspensions of liver, spleen, thymus, bone marrow and blood of twenty-five human foetuses of 5·5–26 weeks of gestational age have been investigated. ATC-positive lymphoid cells were first seen in the liver at 5·5 weeks; E rosette-forming cells (ERFC) and Ig-bearing lymphoid cells were first found at 9 weeks. ERFC were also present in the thymus at 9 weeks. By 12 weeks, fluorescent B and T lymphocytes were found in bone marrow and spleen. ERFC were also found in bone marrow at this age, but not in spleen. At 15 weeks, more than 80% of blood lymphoid cells had T or B determinants.

A difference in the reactivity of lymphoid cells with the ATC and their capacity to form E rosettes was observed. In liver and spleen, the ATC determinant was detectable before the SRBC receptor. In bone marrow, blood and thymus the ATC determinant was found on a higher percentage of lymphoid cells than was the SRBC receptor when those organs were first investigated. During the entire investigated period of gestation, the majority of lymphoid cells in liver and bone marrow did not react with either of the conjugates, nor did they form E rosettes. In all organs investigated, except in the thymus, lymphoid cells were occasionally seen which reacted with both conjugates. By the 16th week of foetal age, more than 90% of lymphoid cells in thymus, spleen and blood had acquired T- or B-membrane determinants.

     

Bone morphogenetic protein-2/4 signalling pathway components are expressed in the human thymus and inhibit early T-cell development

T-cell differentiation is driven by a complex network of signals mainly derived from the thymic epithelium. In this study we demonstrate in the human thymus that cortical epithelial cells produce bone morphogenetic protein 2 (BMP2) and BMP4 and that both thymocytes and thymic epithelium express all the molecular machinery required for a response to these proteins. BMP receptors, BMPRIA and BMPRII, are mainly expressed by cortical thymocytes while BMPRIB is expressed in the majority of the human thymocytes. Some thymic epithelial cells from cortical and medullary areas express BMP receptors, being also cell targets for in vivo BMP2/4 signalling. The treatment with BMP4 of chimeric human-mouse fetal thymic organ cultures seeded with CD34+ human thymic progenitors results in reduced cell recovery and inhibition of the differentiation of human thymocytes from CD4- CD8- to CD4+ CD8+ cell stages. These results support a role for BMP2/4 signalling in human T-cell differentiation.     

Autocrine expression of interleukin-7 rescues lymphoid expansion in interleukin-7-deficient mice.

The murine interleukin-7 (IL-7) gene was disrupted to examine the role of IL-7 in the lymphoid system. Expansion of lymphoid cells is sharply curtailed in IL-7-deficient mice. This is evident in a dramatic reduction but not elimination of lymphoid cells in the thymus, bone marrow and spleen. The few thymocytes present express CD4 and/or CD8 markers associated with T-cell maturation. Similarly, a limited number of B cells detected in the bone marrow rearrange and express immunoglobulin genes. Small but distinct populations of B and T cells are found in the spleens of IL-7-deficient mice. Thus the signal transmitted by IL-7 plays a central role in the expansion of lymphocytes while it is not absolutely required for their maturation. A transgene that directs expression of IL-7 to lymphoid cells was found to restore the numbers of thymocytes, bone marrow B-cell progenitors and splenic lymphocytes of IL-7-deficient mice to approximately normal levels. This genetic complementation confirms that the lymphoid defect is specifically due to the absence of IL-7 and demonstrates that the expansion of lymphoid cells in an organism is regulated by their exposure to IL-7.     

Selective expression of asialo GM1 on maturational subsets of lymphocytes in normal and athymic mice

In an attempt to clarify the restriction of asialo GM1 expression, the presence of asialo GM1 positive cells in the lymphoid tissues of normal and athymic mice was examined using affinity-purified monospecific anti-asialo GM1 antibodies and a FACS-II. The results presented herein demonstrate that a significant frequency of asialo GM1-positive cells can be detected in the primary lymphoid tissues (bone marrow and thymus) as well as in the secondary lymphoid tissues (spleen and lymph nodes). The observed expression of asialo GM1 within the T-cell lineage is, in general, consistent with the previously proposed restriction of asialo GM1 expression to mature T cells. However, the demonstration that a significant frequency of asialo GM1-positive cells is detectable in bone marrow of normal mice, as well as in spleen and bone marrow of athymic mice, establishes that a substantial number of lymphocytes not committed to the T-cell lineage and/or pre-T cells also express the asialo Gm1 antigen. These results indicate that the asialo GM1 marker may be an important tool for following T differentiation from the multipotential stem cell to the functionally mature T cell.     

The bone marrow: a nest for migratory memory T cells

It has been known for a long time that T-cell precursors generated in the bone marrow migrate to the thymus, where T-cell development occurs. However, a fact often neglected is that, under physiological conditions, mature CD4 and CD8 lymphocytes undergo extensive migration from the blood to the bone marrow and vice versa. Here, we first review several observations showing that the bone marrow can function as a secondary lymphoid organ for both CD4 and CD8 cells, as well as a preferential homing site for memory T cells. Second, we discuss evidence that, a long time after priming, memory CD8 cells proliferate more extensively in the bone marrow than they do in either secondary lymphoid or extra-lymphoid organs. Finally, we propose that the bone marrow is a central organ in mature T-cell traffic and contributes greatly to long-term cytotoxic memory, which has implications for adoptive immunotherapy and vaccine design.     

Cell-fate decisions in early T cell development: regulation by cytokine receptors and the pre-TCR

During lymphocyte development, cell-fate decisions are determined by a myriad of signals produced by the micro- environment of the thymus and the bone marrow. These yet to be fully defined developmental cues regulate stage-specific gene expression, and the extraordinarily well-characterized stages of T and B cell development have provided attractive model systems for studying regulation of cellular differentiation. In particular, studies on the contribution of both antigen receptors and cytokine receptors to lymphoid development have illuminated essential signalling pathways in early T and B cells. Here, we review investigations supporting an obligatory role for the IL-7 receptor pathway in early T cell development. IL-7 is produced by both thymus and bone marrow stromal cells, and its potential contribution to survival, differentiation and proliferation of pro-T cells is discussed. We also address the contribution of the pre-T cell receptor (pre-TCR) to differentiation past the pro-T cell stage, and recent advances in deciphering the composition and function of the pre-TCR complex are discussed. Finally, we suggest future directions in this field that may serve to reveal whether and how signals initiated by the cytokine receptors and pre-TCR may intersect, and to define which down-stream molecular events are regulated by these receptors.     

The proteasome immunosubunit multicatalytic endopeptidase complex-like 1 is a T-cell-intrinsic factor influencing homeostatic expansion

Homeostatic regulatory mechanisms maintain the constant ratios between different lymphocyte subsets in the secondary lymphoid organs. How this dynamic equilibrium is achieved, in particular following the clonal expansion and subsequent contraction of different cells after infection, remains poorly understood. Expression of the proteasome immunosubunits has been shown to influence not only major histocompatibility complex class I (MHC-I) antigen processing and thereby T-cell responses, but also the CD4/CD8 T-cell ratios in lymphoid organs. We examined the relationships between these different immunosubunit-mediated effects in mice of various proteasome subunit compositions during infection with Listeria monocytogenes. Mice that lacked the immunosubunit multicatalytic endopeptidase complex-like 1 (MECL-1) maintained enhanced CD4/CD8 T-cell ratios during infection, while MHC-I surface levels resembled those in wild-type (wt) mice. LMP7 gene-deficient mice, on the other hand, showed reduced MHC-I expression, while their splenic CD4/CD8 ratios were similar to those in wt mice. Remarkably, analysis of bone marrow-chimeric immunosubunit gene-deficient mice, reconstituted with a mixture of wt and LMP7- plus MECL-1-deficient bone marrow, revealed that the LMP7- plus MECL-1-deficient T-cell population maintained a higher CD4/CD8 T-cell ratio than the wt T-cell population before, during, and after infection and T-cell memory formation. Since in these mice the immunosubunit-positive and immunosubunit-negative T-cell populations were selected in the same thymus and expanded in the same lymphoid environments, our findings indicate that MECL-1 influences the homeostatic equilibrium between T-cell subsets, not through indirect extracellular signals, such as MHC-I expression or the cytokine milieu, but through direct effects on T-cell-intrinsic processes.     

Quantitative analysis of tissue distribution of Ly10-like murine alloantigen(s) with antisera and monoclonal antibodies

Tissue distribution of non-Lyt1.1 ("Ly10-like") antigen or antigens encoded by short chromosomal segment differentiating B6-Ly-1a congenic strain from B6 strain of mice was studied by quantitative absorption of (BALB/c X B6)F1 anti B6-Ly-1a antiserum and by direct cytotoxicity of Ly-10-132-12-26 monoclonal antibody on lymphoid cell populations. Identical strain but not tissue distribution pattern does not allow to conclude whether antiserum and monoclonal antibody detect the same or closely linked antigens. Absorption experiments revealed the highest antigen content in the brain tissue, lower in testis and kidney, still lower in lymphoid organs and the lowest in liver and lung. Among lymphoid cells, bone marrow cells had highest absorbing capacity, followed by thymus, spleen and lymph nodes. Monoclonal antibody lysed almost 100% of thymocytes, 30% bone marrow cells and 10-20% of spleen and lymph node cells (both T-cell and B-cell enriched populations contained the same proportion of positive cells). Cortisone resistant thymocytes showed the same sensitivity as cortical thymocytes to Ly-10-132-12-26 antibody which is distinguishable characteristics of medullary thymocytes from peripheral T cells. Mitogen activated lymphocytes exhibited significantly higher expression of Ly10-like antigen than resting peripheral lymphocytes.     

Bmp7 mediates early signaling events during induction of chick epidermal organs.

The induction and specification of a large number of vertebrate organs require reciprocal signaling between an epithelium and subjacent mesenchyme. In the formation of integumentary organs, the initial inductive signaling events leading to the formation of the organ primordia stem from the mesenchyme. However, the epithelium must have the capacity to respond to these signals. We demonstrate that bone morphogenetic protein 7 (Bmp7) is an early molecular marker for epidermal organ development during development of feathers and scales of the chick. Bmp7 is expressed broadly in the preplacode epidermis and subsequently becomes localized to the forming placodes of feathers and scales. An examination of Bmp7 expression in the scaleless mutant chicken integument indicates that Bmp7 expression in the epidermis is associated with the ability to form epidermal organs. We show that BMP7 function is necessary for the formation of epidermal placodes in both feather and scale forming epidermis. In addition, precocious expression of Bmp7 in the metatarsal epidermis of the Silkie mutant or treatment of the metatarsus with ectopic BMP7 protein results in feather development from scale forming integument. From these data, we propose that Bmp7 is necessary and sufficient, in a developmental context, to mediate the competence of an epithelium to respond to inductive signals from the underlying mesenchyme to form epidermal organs in the chick. We propose that regulation of Bmp7 in localized areas of the embryonic epidermis facilitates the development of regional formation of integumentary organs.     

T-cell development, doing it in a dish

Summary:  The thymus provides a unique environment for the development of T lymphocytes from bone marrow-derived progenitor cells. Several environmental factors have been identified that influence the development of T cells in the thymus. In particular, the Notch pathway has emerged as critical for the induction of T-lineage commitment and differentiation. Until recently, however, the precise nature of the thymus-derived signals that drive T-cell development were unclear, and the only reliable in vitro culture system that supported T-cell differentiation required the use of thymus organ cultures. Here, we discuss recent advances in the identification of critical Notch receptor ligands that have facilitated the development of a simple in vitro model for the differentiation of T cells 'in a dish', providing an alternate approach for studying T lymphopoiesis.     

Immunological capacity of the chicken embryo. I. Relationship between the maturation of lymphoid tissues and the occurrence of cell-mediated immunity in the developing chicken embryo.

In an investigation of the ontogeny of lymphoid tissue in chick embryos to relate maturation of lymphocytes with immunological competence, the numbers and sizes of lymphocytes were determined in the thymus, bursa of Fabricius, spleen, femoral marrow and peripheral blood of embryos from the 12th to 21st day of incubation, and in 6-day-old chicks. Results showed the thymus to be the first fully developed and most active lymphocytopoietic organ, followed by the bursa. The bone marrow was not lymphocytopoietic; the spleen and bone marrow were mainly granulocytopoietic and erythropoietic; some morphological differences between thymic and bursal lymphocytes were shown by light microscopy. It appears that in embryos and young chicks the lymphocytes are derived from the thymus and bursa, but not the bone marrow. In tests of immunological competency, cells of the thymus, bursa, spleen, bone marrow and peripheral blood from 12--21-day-old embryos and 6-day-old chicks were transferred to chorioallantoic membranes of 12-day-old recipient embryos. There were distinct differences between the ability of various lymphoid tissues to induce formation of chorioallantoic pocks or splenic enlargement. The thymus, spleen and peripheral blood elicited both lymphocytic pocks and splenomegaly, the bursa elicited splenomegaly only, and the bone marrow was ineffective. The bone marrow, however, induced formation of nonlymphocytic pocks. It is concluded that the immunological activity of the chicken embryo is primarily effected by the thymus and bursa and that cell-mediated immunity appears in the 2nd week of incubation.     

Early lymphoid progenitors in mouse and man are highly sensitive to glucocorticoids

Glucocorticoids are extensively used in anti-inflammatory therapy and may contribute to the normal regulation of lymphopoiesis. This study utilized new information about the early stages of lymphopoiesis in mouse and man to determine precisely which cell types are hormone sensitive. Cycling B lineage precursors were depleted in dexamethasone-treated mice, while mature, non-dividing CD45R(Hi) CD19(Hi) lymphocytes, myeloid progenitors and stem cells with the potential for lymphocyte generation on transplantation were spared. Lineage marker-negative (Lin(-)) IL-7R(+) Flk-2(+) pro-lymphocytes also declined, but not as rapidly as the terminal deoxynucleotidyl transferase-positive cells within an early Lin(-) c-kit(Hi) Sca-1(Hi) fraction of bone marrow. Hormone-sensitive cells with additional properties of early lymphoid progenitors (ELP) were identified within the same Lin(-) c-kit(Hi) Sca-1(Hi) subset using human mu transgenic mice and recombination-activating gene 1 (RAG1)/green fluorescent protein knock-in animals. Furthermore, cells with a recent history of RAG1 expression were more glucocorticoid sensitive than mature lymphocytes in marrow and spleen. Lymphocyte progenitors in mice bearing a human bcl-2 transgene were protected from dexamethasone treatment. However, isolated progenitors from either wild-type or bcl-2 transgenic mice were directly sensitive to the hormone in stromal cell-free cultures, suggesting that additional factors must determine vulnerability to glucocorticoids. B lineage lymphocyte precursors were found to be abnormally elevated in the bone marrow of adrenalectomized or RU486-treated mice. This suggests that glucocorticoids may normally contribute to steady-state regulation of lymphopoiesis. Finally, parallel studies revealed that the earliest events in human lymphopoiesis are susceptible to injury during glucocorticoid therapy.     

Pregnancy: a clue to normal regulation of B lymphopoiesis

The large-scale production of lymphocytes in the bone marrow reflects a delicate balance between positive and negative regulatory signals. For instance, interleukin 7 (IL-7) provides a positive signal, and appears to be both essential and limiting in the mouse. However, much less is understood concerning the negative molecular signals that may limit the output of lymphocytes. Here, Paul Kincade and colleagues discuss how a chance observation with pregnant mice revealed that sex steroids can act as negative regulators of B lymphopoiesis, and may do so under normal steady-state conditions.     

Thymus specific antigen on electrophoretically separated rat lymphocytes. Tracing of the differentiation pathway of bone marrow-derived thymocytes by use of a surface marker

Rat lymphocytes from different lymphoid organs were separated by means of free-flow electrophoresis and the separated cells subdivided into classes of high (H lymphocytes) and low (L lymphocytes) electrophoretic mobility. A rabbit antiserum against thymocytcs of low electrophoretic mobility was prepared and its cytotoxic activity tested with H and L lymphocytes from spleen, lymph node, thoracic duct lymph, bone marrow and thymus. The cytotoxic activity was bound to the IgC immunoglobulins of the antiserum. An agglutination reaction could be observed with all L lymphocytes, but not with H lymphocytes, and was not further investigated. Cytotoxicity was obtained with bone marrow L lymphocytes and H and L thymocytes, but not with lymphocytes of other sources. It was suggested that this antigen resembled the rat “thymus specific” antigen. The finding that this antigen appeared on L lymphocytes of bone marrow and thymus and diminished on thymus H lymphocytes with be treated by following the differentiation of bone marrow derived thymocytes.     

Cells involved in the immune response: XXVII. The demonstration of appendix-specific antigens in the rabbit

Rabbit appendix cells were injected into horses at weekly intervals, after which the horses were bled and the sera tested for cytotoxic activity. The unabsorbed antiserum was cytotoxic to the lymphocytes of all the lymphoid organs tested. Following absorption with bone marrow and thymus cells, the antiserum lost all cytotoxic activity directed toward these cells but displayed almost undiminished cytotoxic activity toward the cells of the SAPP organs (sacculus rotundus, appendix and Peyer's patches). The absorbed antiserum also demonstrated cytotoxic activity toward certain lymphocytes present in the spleen, lymph node and circulation. Thus, the antibodies directed toward the antigens common to all lymphoid cells were removed by the thymus and bone marrow cells, leaving behind cytotoxic antibodies directed toward antigens present on appendix and appendix-derived cells only. We have therefore succeeded in preparing antisera specific for rabbit thymus, bone marrow and appendix cells, respectively.     

Induction of a T-cell specific antigen on bone marrow lymphocytes with thymus RNA.

Expression of a rabbit T-cell specific antigen can be induced on bone marrow lymphocytes following exposure to an RNA extract obtained from the thymuses of young rabbits. The presence of the antigen was demonstrated using goat anti-rabbit T-cell serum in a complement-dependent cytotoxicity assay. The T-cell antigen first appeared 3 h after addition of the thymus RNA to bone marrow cell cultures and the maximum number of cells expressing the T-cell antigen was observed within 24 h. RNA obtained from a source other than the thymus was found to be ineffective in inducing expression of the T-cell antigen. The induction of the antigen appears to be dependent on the presence of intact thymus RNA, as RNase treatment but not trypsin treatment, destroyed the ability of the RNA to induce the T-cell antigen.