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Revisión Inmunología Vol. 22 / Núm 1/ Enero-Marzo 2003: 17-26 Hedgehog proteins: expression and function in the thymus R. Sacedón1, A. Varas2, C. Hernández-López2, M.C. Gutiérrez-de Frías2, S.V. Outram3, T. Crompton3, A. Vicente1, A. Zapata2 1Department of Cell Biology, Faculty of Medicine, Complutense University. Department of Cell Biology, Faculty of Biology, Complutense University, Madrid, Spain. 3Department of Biology, Imperial College of Science, Technology, and Medicine, London. 2 PROTEÍNAS HEDGEHOG: EXPRESIÓN Y FUNCIÓN EN EL TIMO RESUMEN Evidencias crecientes demuestran que los morfógenos clásicos, altamente conservados durante la evolución y tradicionalmente implicados en el desarrollo embrionario, donde determinan diferenciación celular y patrones de desarrollo, son expresados en tejidos adultos, como son la médula ósea y el sistema inmunitario. Estas moléculas podrían ser piezas importantes del rompecabezas que orquesta la diferenciación y la homeostasis del sistema inmune, aunque sea sólo recientemente cuando estamos comenzando a entender donde encajan en el entramado del sistema inmune. En esta revisión, describimos la ruta de señalización de las proteínas Hedgehog (Hh) centrándonos en su implicación en la diferenciación de las células T y su posible conexión con otros morfógenos, como son las proteínas morfogénicas del hueso (Bone Morphogenetic Proteins, BMPs) ABSTRACT Growing evidence demonstrates that classical ancient morphogens traditionally implicated in embryonic processes, determining cell fate and developmental patterns, are expressed in adult tissues including bone marrow and immune system. They could represent important pieces of the puzzle that orchestrate immune system differentiation and homeostasis, although only recently we are beginning to understand where they fit in the immune system millieu. In this review we outline the signalling cascade of the Hedgehog (Hh) proteins, focusing on its involvement in T-cell differentiation and the possible connections with other morphogens such as Bone Morphogenetic Proteins (BMPs). KEY WORDS: Hedgehog (Hh) proteins / Bone Morphogenetic Proteins (BMPs) / T differentiation / Thymus. PALABRAS CLAVE: Proteínas Hedgehog (Hh) / Proteínas morfogénicas del hueso / Diferenciación T / Timo. THE ANCIENT HEDGEHOG FAMILY Hh genes encode a family of secreted proteins the activities of which include those of classic morphogens, orchestrating embryonic development, but also the regulation of cell survival and proliferation. Knowledge of their pleiotropic functions is profoundly complex as they elicit different or even opposed effects depending on the dose and/or target cell. Initially described as a segment polarity gene in Drosophila, hh proteins were named on the basis of the appearance of mutant larvae. Disruption of hh gene leads to Drosophila larvae covered by a continuos lawn of denticles projecting from their cuticle as spines of a hedgehog, which suggested to Nüsslein-Volhard and Wieschands(1) the name of this family of proteins. 17 HEDGEHOG PROTEINS: EXPRESSION AND FUNCTION IN THE THYMUS Sonic Hh (Shh), named after Sega hedgehog hero, and Indian and Desert Hh (Ihh and Dhh, respectively), both referred as two hedgehog species, were later identified in vertebrates(2), from fish to mammals. They are key players in most of vertebrate developmental processes and so, implicated in several types of human congenital abnormalities, including holoprosoencephalia, and cancer(3-5). The most pleiotropic of vertebrate Hh molecules is Shh. It orchestrates diverse events such as left-right asymmetry(6–8), dorso-ventral patterning of the central nervous system(2,9–12) and somites(13,14), as well as limb morphogenesis(15,16). Shh is also involved in the development of lung(17), tooth(18,19), hair(20), pancreas(21,22) and tongue(23). Haematopoietic stem cells are Shh targets (24) and, as discussed later, T cell differentiation(25) and proliferation(26) seem to be also modulated by this Hh protein. On the other hand, Ihh plays key functions in the development of bone and cartilage(27) as well as pancreatic morphogenesis(22). In addition, it activates haematopoiesis and vasculogenesis during early post-implantation development(28). Its involvement in development of the mammary gland(29) as well as in differentiation of visceral endoderm(30) has been also suggested. Dhh function seems to be more restricted. Its implication in the development of testicles and external genitalia(31-33) and in the formation of peripheral nerve ensheathment(33,34) has been demonstrated. All the described developmental processes are regulated by Hh molecules not only because of their capacity to determine cell fate but also by their ability to modulate cell survival and proliferation. The first evidence supporting a role for Hh in cell proliferation raises from the evidence that mutations that activate the Hh cascade lead to several types of cancer such as basal cell carcinoma, medulloblastoma, rhabdomyosarcoma(3-5). Nevertheless, the molecular basis that link Hh signalling to cell cycle progression remains unclear and only a few recent studies have approached this question. For example, in cerebellar granule neurone precursors, Shh promotes cell division regulating levels of proteins implicated in G1-S restriction point, including cyclins D1, D2 and E(35). Similar results are observed in Drosophila eye development where Hh induces the expression of cyclins D and E(36). Furthermore, the interaction of the Hh receptor with the phosphorylated form of cyclin B, and thereby its cytoplasmic retention, has also related the Hh cascade with the regulation of G2-M cell cycle progression, although its physiological significance is not yet understood(37). Not only a tightly controlled cell proliferation but also programmed cell death are essential to proper organogenesis. On this regard, Shh elicits apoptotic or antiapoptotic effects 18 VOL. 22 NUM. 1/ 2003 in different organs. Shh survival actions in somites(38), neurons(39), neural crest cells(40) and muscle cells of the limb(41,42) contrast with its apoptotic effect in posterior limb bud cells(43). Again, further investigations are required to a better knowledge of the molecular basis of these dual roles. HEDGEHOG SIGNALLING CASCADE Hh proteins are synthesised in a precursor form that is autocatalitically processed rendering an active N-terminal fragment that is secreted(44). Concomitantly to this posttranslational cleavage, the signalling molecules are covalently coupled to cholesterol at their COOH-terminal ends, which is necessary for their movement across the target field(45,46). A second lipid modification, palmitoylation, occurs in the NH2-terminal end of a part of the proteins increasing their signalling activity in vitro(47) and in vivo(48). Both modifications participate in tethering the proteins to the cell membrane in synthesising cells, although they are neither necessary for receptor recognition nor affect receptor binding affinity. On the other hand, the Drosophila model provides evidence that Hh target expression requires the active release of lipid modified Hh from the surface membrane of the secreting cell. This is mediated by a membrane molecule called Dispatched(49,50). All Hh proteins share an apparently exclusive signalling pathway (Fig. 1) and differences among the particular roles of Shh, Ihh and Dhh during vertebrate development are really provided, and delimited by their distinct temporal and spatial expression domains (31). Pathi et al. (51), also demonstrated that all three proteins can elicit similar biological responses, but that their relative potencies differ in an assaydependent manner. In most cases, data revealed a stronger activity of Shh followed by Ihh and Dhh, with the lowest potency. However, these quantitative differences are not based on their ability to bind to the receptor. Additional components of the Hh cascade, which modulate the responses of the three Hh proteins as well as the requirement of different range of required signalling potencies for each particular biological event, could explain the existence of three different Hh proteins in vertebrates. Patched (Ptc), a politopic transmembrane protein with homology to transporter or channel proteins(52-54), and Smoothened (Smo), a serpentine transmembrane protein reminiscent of G-protein coupled receptors(55,56), represent the membrane components of the Hh cascade (Fig. 1). Ptc is the ligand binder and Smo transduces the intracellular signal. In the absence of ligand, Ptc exerts an inhibitory effect on Smo activity that is abrogated after Hh binding. INMUNOLOGÍA R. SACEDÓN ET AL. Figure 1. Hh Signalling cascade in vertebrates. Hh proteins (Shh, Ihh, Dhh) are synthesised and secreted but they can also attach to the cell membrane through cholesterol or palmitoylation modification. Target field establishment is promoted by cell to cell transfer and through the building of multimers, possibly mediated by a homologue of the Drosophila membrane protein, Dispatched. Heparan sulfate associated to proteoglycans, such as syndecans, and/or other extracellular matrix proteins modulate or promote Hh binding to its receptor. Patched represents the Hh ligand binder while Smoothened initiates the Hh signalling cascade that activates transcriptional activities of Gli proteins. PKA inhibits Smoothened function. Additionally, other cell membrane proteins such as Hip, Megalin and Gas1 modulate Hh levels or even could initiate new intracellular cascades (see text for more details). In the absence of Ptc, Smo exhibits a tonic activity(55,57,58). The mechanism underlying Ptc inhibitory function is under debate, although recent evidence reinforces the idea that the permanent physical interaction between Ptc and Smo is not necessary. On the contrary, this inhibition would involve the covalent modification of Smo, combined with the regulation of the vesicular trafficking(59-63). Direct protein kinase A (PKA) phosphorylation of Smo in its COOHterminal domain has been also proposed to inhibit Hh cascade targeting Smo to degradation (Fig. 1)(59). The intracellular Hh transduction pathway has been also more extensively studied in Drosophila. Smo derepression leads to gene expression regulation, modifying the activity of a transcription factor, Cubitus interruptus (Ci)(64). In the fly, in the absence of Hh, Ci is retained in the cytosol forming a complex with several segment polarity proteins, the kinase fused, costal- 2 and suppresser of fused (SuFu). These proteins mediate and regulate Ci anchorage to cytoskeletal microtubules and, therefore, cytoplasmicnuclear shuttling(65-69). Ci is phosphorylated by PKA and other kinases. PKA phosphorylation of Ci is required for Ci to be cleaved yielding a transcriptional repressor. In fact, PKA mutant cells show constitutive activation of Hh target genes(70). Hh signalling leads to phosphorylation of Fused and dephosphorylation of Ci. The change in phosphorylation pattern leads to the inhibition of repressor formation. Then, Ci is translocated to the nucleus where, alone or in collaboration with CREB Binding Protein or SuFu, it interacts with DNA and activates Hh target genes(71). In vertebrates three gli genes (gli1, gli2, gli3) are the Ci homologous (Fig. 1). Nevertheless, the functions of the three Gli transcription factors are not redundant, since they exhibit different expression patterns during development and their cellular activities are significantly distinct(72). Gli1 is a Hh-dependent activator of Hh target genes whereas Gli 3 is mainly a Hh-dependent repressor. PKA activity also promotes Gli 3 repression by inducing its cleavage, which yields a repressor form, as described for Ci(72,73). All three Gli proteins are Hh target genes but whereas gli-1 expression is induced by Hh, the expression of gli-3 is repressed(74,75). On the contrary, Gli2 can function as repressor or activator of Hh target genes: it acts as activator in a Hh-dependent manner or as repressor independently of Hh(76-79). Not all Gli functions are associated with the Hh cascade. Gli 2 and Gli3 are expressed in regions, such as the dorsal neural tube, that require absence of Hh signalling(75,80-82). This may correlate with their Hh-independent inhibitory activities. Moreover, Hh signalling cascade is not restricted to Gli function(83). There is a dramatic in vitro impairment of neural crest cell migration induced by Shh that is insensitive both to inhibition of protein synthesis or to cyclopamine treatment, a plant steroid alkaloid that blocks Smo function(60,84). On this basis, Testaz et al.(83) demonstrated a biological Shh 19 HEDGEHOG PROTEINS: EXPRESSION AND FUNCTION IN THE THYMUS action independent of the classic Ptc-Smo-Gli signalling pathway, although they did not propose any alternative. MORE THAN A SECRETED PROTEIN THAT BINDS TO ITS RECEPTOR Proper Hh function requires a precise regulation in time and/or space of its signalling. This is achieved in vivo by a fairly complex system that would work by different mechanisms: a) determining the levels of available Hh molecules in a target field not only regulating their secretion and diffusion, but also their cell membrane sequestration and intracellular degradation; b) modulating the cell responsiveness at receptor and transducing levels. As any morphogen, Hh proteins are able to influence cells located in short or long distances from secreting cells. The appearance of a gradient may be originated by passive diffusion and/or facilitated transport mediated by a cell to cell membrane transfer or even a transcytosis mechanism, although this last issue remains obscure(85,86). It is known that cholesterol tethering to the membrane is required for the establishment of Shh long range field of action(46). In a recent work, Zhen et al.(86) demonstrated the existence of a freely diffusive multimeric form of cholesterol modified Shh. This Hh multimer hides the lipid attachments in its interior, increasing its diffusion field. The authors proposed that concentration in lipid rafts of Hh cholesterol modified molecules and the implication of a possible vertebrate homologue of Dispatched, could lead to the building of such a multimer (Fig. 1). On the other hand, Hh binding to Ptc has a dual role limiting Hh diffusion and reducing its levels by endocytosis, although a transduction role of such endocytosis could not be discarded (Fig. 1). Thus, the increase of Ptc levels after Hh signalling represents a negative feedback loop(87-89). McCarthy et al.(90) have proposed the existence of a new molecule that may participate in Hh function, an endocytic receptor belonging to the low density lipoprotein receptor family, megalin. This molecule mediates ligand endocytosis in the apical surface of different epithelia, and subsequently, their lysosomal degradation or, alternatively, their transcytosis. However, it is not possible to discard that megalin can also initiate a signalling cascade after Shh binding (Fig. 1). Another protein that also binds Hh molecules is the Hhinteracting protein (Hip). Hip is a glycosilated membrane protein that is expressed next to cells that produce Hh, and binds Hh proteins with the same affinity than Ptc. No signalling cascade has been demonstrated after Hip-Hh interaction, indicating that the main function of Hip would be to attenuate Hh activities(91) (Fig. 1). 20 VOL. 22 NUM. 1/ 2003 Recently, Gas 1 has been demonstrated to bind Shh and block the growth-stimulating activity of moderate doses of Shh(92). In cultured cells, Gas 1 exerts a p53-dependent growth cell arrest apparently in the absence of any known ligand(93), although in other systems this molecule does not cause cell proliferation blockage(93). This evidence suggests the existence of a Shh-independent role of Gas 1, which may be more than a Shh sequester; after Shh binding, Gas1 could originate a new Ptc/Smo-independent intracellular cascade (Fig. 1). Modulation of cell responsiveness is also crucial to proper Hh function. As described in other signalling pathways, positive and negative feedback loops are established. Ptc and Gli1 levels increase in response to Hh. On the contrary, Gli3 and Shh levels are inhibited. Membrane Smo levels have been also demonstrated to increase after Hh signalling. However, it is not clear whether this is merely an effect of Ptc derepression without a real increase in Smo transcription(59-63). In addition, two extracellular matrix glycoproteins, laminin and vitronectin, have been demonstrated to modulate proliferative and differentiation responses to Shh (94,95). Disruption of heparan sulphate proteoglycans (HSPG) synthesising enzyme Tout-velu in Drosophila suggests a role for these extracellular matrix molecules in long range Hh activities (96,97). Rubin et al. (98) extended these results to vertebrates and demonstrated that Shh-HSPG binding significantly increases proliferative responses to Hh without affecting Ptc binding affinity (Fig. 1). HH SIGNALLING REGULATES T CELL DIFFERENTIATION Cytokine and pre-TCR are considered essential signalling pathways for early T cell maturation. We have recently reported the involvement of Hh molecules in T cell development, demonstrating that Shh plays a key role regulating early T cell proliferation and differentiation(25). Shh is the main Hh molecule secreted in the fetal and adult mouse thymus, and is produced by the thymic epithelium. Ihh expression is mainly associated to the blood vessels from the medullary region in the adult thymus, and Dhh is not detected in the organ(25). Further studies demonstrate that mRNA of both components of the Hh receptor, Ptc and Smo, are also present in fetal and adult thymocytes. The flow cytometric analysis of Smo expression shows that most of CD4–CD8– double negative (DN) thymocytes express high levels of the signalling component of the Hh receptor, whereas cell surface expression of Smo is downregulated in the CD4+CD8+ double positive R. SACEDÓN ET AL. INMUNOLOGÍA a b Percentage of positive cells – – + + CD4 CD8 Percentage of positive cells + – + + 10 + – + CD44 CD25 CD44 CD25 MFI = 7 MFI = 48 CD4 CD8 CD44 CD25 – CD4 CD8 CD44 CD25 + MFI = 29 + CD4 CD8 – MFI = 21 – MFI = 16 CD44 CD25 0 10 20 30 40 50 60 0 20 40 60 80 100 Figure 2. Smo expression in adult thymocytes. (a) Flow cytometric analysis shows the highest levels of Smo receptor expression in DN (CD4–CD8–) thymocytes. Adult thymocytes were analysed for Smo levels combined with CD4/CD8 staining and bars represent the percentage of Smo positive cells in gated subpopulations defined by CD4/CD8 expression. Results are the mean (±SEM) of six different experiments. (b) Further analysis of isolated adult DN thymocytes according to CD44/CD25 cell markers demonstrates the highest expression of Smo receptor in CD44+CD25+DN thymocytes decreasing in subsequent stages as DN cells differentiate. Bars represent the percentages of positive cells (mean ± SEM), and their mean fluorescence intensities (MFI) are shown. (DP) and CD4+CD8–/CD4–CD8+ single positive (SP) thymocyte subpopulations (Fig. 2). The analysis of the DN thymocyte subsets defined according to CD44 and CD25 antigen expression shows that CD44+CD25– DN cells, including the earliest thymic precursor cells which are not committed to the T cell lineage(99), do not express significant levels of Smo. In the next compartment, CD44+CD25+ pro-T cells, Smo expression is upregulated, whereas in the subsequent CD44–CD25+ and CD44–CD25– DN subpopulations, both the proportions of positive cells and the levels of expression, gradually decrease (Fig. 2). Therefore, among thymocyte precursors, the main responders to Shh are, presumptively, the CD25+ DN cells. This cell subset is undergoing the rearrangement of the TCRβ chain gene that, if successful, will allow the expression of a functional pre-TCR, which will signal inducing both proliferation and differentiation of T-cell precursors, as well as allelic exclusion of the TCRβ locus(100-102). The molecular transduction machinery of the hedgehog cascade, including the transcriptional factors Gli1, Gli2 and Gli3, are also expressed in the murine thymus. Interestingly, the relative levels of gli1, gli2 and gli3 mRNA found in fetal and adult thymus are different. Whereas in the adult thymus gli1>gli2>gli3, inverse proportions are found in the fetal samples, which suggests that the contribution of Hh cascade changes during development(25). In contrast to the murine model, all Hh proteins are expressed in the human thymus (Sacedón et al, manuscript in preparation). By using double immunofluorescence, Shh expression is found to be restricted to epithelial cells located in the subcapsullar and medullar regions, whereas Ihh-positive and Dhh-positive epithelial cells are scattered at random throughout the thymic cortex and medulla (Fig. 3). The components of the Hh receptor are expressed by both thymocytes and epithelial cells in all thymic compartments, although Ptc is expressed by a higher number of cells than Smo. In contrast, the expression of Ptc2, a Ptc homologue, is restricted to epithelial cells which appear dispersed throughout the thymic parenchyma (Fig. 3). When the expression of the Hh receptor is analysed in the different human thymocyte subsets, thymocyte precursors express the highest levels of Smo, which is downregulated in DP and SP cell subsets, as previously shown for mice. Moreover, our results indicate a role for Shh in increasing the survival of CD34 thymic precursors as well as in decreasing their proliferation and differentiation (Vicente et al, manuscript in preparation). From this evidence a simple question is derived: Is Smo downregulation a mere consequence of T cell differentiation or a requirement for thymocytes to progress into T cell differentiation program?. The involvement of Hh in T cell differentiation was addressed in vitro using mouse foetal thymus organ cultures (FTOC) that were maintained for three days in the presence of growing doses of Shh or the monoclonal antibody 5E1, which specifically blocks Shh binding to Ptc. Such experiments 21 HEDGEHOG PROTEINS: EXPRESSION AND FUNCTION IN THE THYMUS VOL. 22 NUM. 1/ 2003 A B Figure 3. Expression of Hh proteins in the human thymus. Diagram indicating the distribution of expressed Hh proteins in the human thymus as demonstrated by immunofluorescence. Note the restricted expression of Hh ligands (Shh, Ihh and Dhh) and Ptc2 receptor to thymic epithelial cells while Smo and Ptc receptors are widely distributed (Sc, subcapsule; C, cortex; M, medulla). reveal the capacity of exogenous Shh to block thymocyte development at the CD25+ DN stage (Fig. 4), after initiation of TCRβ chain gene rearrangement, whereas neutralisation of endogenous Hh leads to an earlier appearance of high numbers of DP thymocytes (Fig. 4). These increased numbers of DP cells are actually reflecting an acceleration of thymocyte differentiation since there is a concomitant increase of the more mature subpopulation of DN cells, CD44–CD25– (Fig. 4), and because neither proliferation nor survival are induced in the DP cell subset arising in anti-Shh treated FTOC(25). Although Shh neutralisation promotes differentiation of DN to DP cells, it is unable to replace a pre-TCR signal. This is consistent with the fact that the extra DP thymocytes produced in the presence of the anti-Shh antibody show a normal expression of TCRβ chain, and the inability of antiShh treatment to overcome the developmental arrest at the CD25 + DN stage exhibited by Rag1 –/– thymocytes (25). Nevertheless, Shh signalling abrogation does enhance the differentiation of Rag1–/– thymocytes induced by anti-CD3 stimulation, which mimics the physiological pre-TCR signal(25). This indicates that Shh signalling inhibition can only promote thymocyte development after the pre-TCR complex has instructed CD25+ DN cells to progress to DP thymocytes. As mentioned above, ligation of CD3, and therefore preTCR signalling, determines cell cycle progression, differentiation from DN to DP cells and allelic exclusion of the TCRβ locus, but also induces the downregulation of both smo RNA and cell surface Smo expression and promotes on developing DN thymocytes a refractory state to Hh signalling within 2 days of receiving the pre-TCR signal(25). 22 Figure 4. Exogenously added Shh arrests thymocyte differentiation while Shh neutralization stimulates thymocyte maturation. (A) CD4/CD8 expression in E14 FTOC maintained in the presence either of Shh or an anti Shh mAb. (B) Phenotype of DN (CD4–CD8–) thymocytes obtained from cultures above described and analysed for CD44/CD25 expression. This indicates a direct link between the pre-TCR and Hh signalling pathways and, indirectly, points out that preTCR complex could affect the Hh signaling. Therefore, Shh might function to maintain CD25+ DN cells as non-proliferating cells while they are rearranging their TCRβ chain genes, since neutralisation of Hh signalling increases the proliferative rate of CD25+ DN cells(25). The successful TCRβ gene rearrangement and the subsequent expression and signalling of the pre-TCR has an immediate consequence that is the downregulation of Smo. Consequently, Shh can no longer signals and DN cells are released to proliferate and differentiate to the DP cell stage. Thus, termination of Hh signalling seems to be a pre-requisite for differentiation of DN to DP cells(25). The effects of Hh proteins on T cells have been recently extended to periphery(26). CD4 T lymphocytes have been demonstrated to produce Shh, and anti-CD3 stimulation increases that production. Resting and stimulated CD4 T cells express Smo and Ptc receptors as well as the transcription factor Gli1. This expression increases after stimulation and Hh cascade reduces apoptosis and induces cell growth after suboptimal anti-CD3/CD28 in vitro stimulation. Neutralisation of Shh in the cultures with high doses of 5E1 reduces cell proliferation but not cell survival(26). R. SACEDÓN ET AL. INMUNOLOGÍA Figure 5. BMP4 production in the murine thymus. Frozen sections from mouse thymus were double stained with anti-BMP4 and anti-cytokeratin antibodies for demonstrating the association of BMP4 production with cytokeratin-positive epithelial cells. Positive staining is marked with short arrows in the subcapsular (SC) epithelial cells and with long arrows in the cortical region (C). x 300. BMP/HH RELATIONSHIPS AND T-CELL DIFFERENTIATION Bi-directional influences between Bone Morphogenetic Protein (BMP) and Hh cascades are marked during development. BMP inhibitors, as Chordin, can sensitise neuroepithelial cells to notochord-derived Shh, facilitating the ability of Shh to induce the floor plate that is inhibited by BMP family(103,104). During anlagen hair cycle epithelial Shh acts on the follicular epithelium to promote proliferation and down-growth of the anlagen hair follicle while BMPs antagonise with Shh expression. Noggin secreted by dermal papila, probably under Shh control, abrogates Shh blockade through BMPs, that close the feedback loop inhibiting BMPs(105). On the contrary, BMP and Ihh signalling positively interact to co-ordinate chondrocyte proliferation and differentiation. Ihh, produced by hiperthrophic chondrocytes, promotes proliferation of adjacent chondrocytes and, in addition, induces the expression of several BMP genes in the perichondrium and in the proliferating chondrocytes. On the other hand, BMPs activate the expression of Ihh and negatively regulate the differentiation of terminal hyperthrophic chondrocytes(106). Recently, it has been proposed that Shh functions as a regulator of pluripotent hematopoietic stem cells via mechanisms that are dependent on downstream BMP4 signals(24). The expression of BMPs in the thymus has also been described. BMP4 expression is found in thymic epithelial cells located in the subcapsular and cortical areas (Fig. 5), whereas BMP receptors are mainly expressed by thymocytes. The specific downstream effector molecules for the BMP signalling pathway (Smad 1, 4, 5 and 8), as well as the extracellular inhibitors regulating BMP signalling (Noggin, Chordin and Twisted Gastrulation) are also expressed in the thymus(107, 108). BMP4 inhibits thymocyte proliferation, enhances thymocyte survival and arrests thymocyte differentiation at the CD44+CD25– DN stage, before commitment to T cell lineage. Conversely, neutralisation of endogenous BMP2/4 by treatment of FTOC with the antagonist Noggin promotes thymocyte differentiation increasing the numbers of DP cells, although again it is unable to overcome pre-TCR signalling, as also commented for the anti-Hh treatment(107). Therefore, as BMP4 arrests thymocyte development at an earlier developmental stage than Shh, it seems evident than BMP signalling must be previous to Shh signalling in the regulation of thymocyte development. BMP4 signalling might be involved in the specification of T cell lineage commitment, and the timing and speed of the differentiation of thymocyte precursors along the T cell lineage. However, a later influence of BMPs in thymocyte development has been also described(108). This involves a blockade in the progression from DN and immature single positive (ISP) to DP stage, which is abrogated after pre-TCR signalling that induces upregulation of Twisted gastrulation. This protein is secreted by thymocytes and synergizes with endogenous Chordin, which exerts similar activities as Noggin, enhancing its capacity to block BMP actions. It is unlikely again that Shh uses BMP4 signalling to exert its blockade at the CD44–CD25+ cell stage, but a role of Shh in BMP4 pathway in the thymus should be addressed. It will be, therefore, interesting to further assess these proposed functions of Hh and BMP4 as regulators of early thymocyte development by analysing the relationships between these two signalling pathways and those with other patterning genes also functioning in the thymus. ACKNOWLEDGMENTS This work was supported by grants PB97-0332, PM990060, BMC2001-2025 from the Spanish Ministry of Science and Technology and CAM08.3/0041/2000 and CAM 08.3/0018.1/2001 from the Comunidad Autónoma de Madrid. CORRESPONDENCE TO: Dr. Agustín G. Zapata Departamento de Biología Celular Facultad de Biología Universidad Complutense 28040 Madrid, Spain. Phone:34-91-3944979. 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