Patched, the receptor of Hedgehog, is a lipoprotein receptor

The Hedgehog (Hh) family of secreted signaling proteins has a broad variety of functions during metazoan development and implications in human disease. Despite Hh being modified by two lipophilic adducts, Hh migrates far from its site of synthesis and programs cellular outcomes depending on its local concentrations. Recently, lipoproteins were suggested to act as carriers to mediate Hh transport in Drosophila. Here, we examine the role of lipophorins (Lp), the Drosophila lipoproteins, in Hh signaling in the wing imaginal disk, a tissue that does not express Lp but obtains it through the hemolymph. We use the up-regulation of the Lp receptor 2 (LpR2), the main Lp receptor expressed in the imaginal disk cells, to increase Lp endocytosis and locally reduce the amount of available free extracellular Lp in the wing disk epithelium. Under this condition, secreted Hh is not stabilized in the extracellular matrix. We obtain similar results after a generalized knock-down of hemolymph Lp levels. These data suggest that Hh must be packaged with Lp in the producing cells for proper spreading. Interestingly, we also show that Patched (Ptc), the Hh receptor, is a lipoprotein receptor; Ptc actively internalizes Lp into the endocytic compartment in a Hh-independent manner and physically interacts with Lp. Ptc, as a lipoprotein receptor, can affect intracellular lipid homeostasis in imaginal disk cells. However, by using different Ptc mutants, we show that Lp internalization does not play a major role in Hh signal transduction but does in Hh gradient formation.     

The human granulocyte/macrophage colony-stimulating factor receptor alpha2 isoform influences haemopoietic lineage commitment and divergence

A number of alternatively spliced isoforms of haemopoietic growth factor receptors (HGFRs) have been described, but their role in human haemopoiesis remains undetermined. We have investigated the relative expression of the alpha1 and alpha2 isoforms of human granulocyte/macrophage colony-stimulating factor receptor (hGM-CSFR) during haemopoietic cell differentiation, and have shown that both subunits are independently regulated during differentiation of CD34+ human haemopoietic progenitor cells. To further investigate these ex-vivo observations, we established a series of murine FDCP mix cell lines, which, as a consequence of the ectopic expression of alpha1 or alpha2 hGM-CSFR, demonstrated differential differentiation responses to hGM-CSF. In this model system, hGM-CSFR-alpha2-expressing cells showed increased hGM-CSF-mediated erythroid/megakaryocytic differentiation compared with hGM-CSFR-alpha1-expressing cells.     

p63 regulates commitment to the prostate cell lineage

Molecular mechanisms underlying prostate and urothelial development remain unclear. This situation presents major limitations in identifying the cell type(s) and molecular events involved in the development of prostate and bladder cancer. It has been shown that mice lacking the basal cell marker p63 present several epithelial defects, including epidermis and prostate buds agenesis and urothelial abnormalities. Here, we use the p63-/- mouse as a tool to define cell lineages in the prostate epithelium and urothelium. By complementing p63-/- blastocysts with p63+/+ beta-galactosidase (beta-gal)-positive ES cells, we show that secretory cells of the prostate originate from p63-positive basal progenitor cells. Importantly, our urogenital sinus transplantation studies demonstrate that p63 prevents intestinal differentiation of the urogenital sinus endoderm and is therefore required to maintain commitment to the prostate cell lineage. Finally, in contrast with the prostate findings, analysis of the urothelium from rescued p63-/- chimeras shows that umbrella (superficial) cells can develop and be maintained independently from p63-positive basal and intermediate cells.     

T-cell lineage commitment and cytokine responses of thymic progenitors

The earliest steps of intrathymic differentiation recently have been elucidated. It has been reported that both CD4lo (CD44+ CD25- c-kit+ CD3- CD4lo CD8-) and pro-T cells (CD44+ CD25+ c-kit+ CD3- CD4- CD8-, representing the next step in maturation) exhibit germline T-cell receptor beta and gamma loci, suggesting that neither population is exclusively committed to the T-cell lineage. Several groups have shown that CD4lo cells retain the capacity to generate multiple lymphoid lineages in vivo; however, the lineage commitment status of pro-T cells is unknown. To determine when T-cell lineage commitment occurs, we examined the ability of sorted CD4lo and pro-T cells to generate lymphoid lineage cells in vivo or in fetal thymic organ cultures (FTOCs). When intravenously injected into scid mice, CD4lo cells generated both T and B cells, whereas the progeny of pro-T cells contained T cells exclusively. Fetal thymic organ cultures repopulated with CD4lo cells contained both T and natural killer (NK) cells, whereas cultures repopulated with pro-T cells contained T cells almost exclusively. These observations strongly suggest that T-cell lineage commitment occurs during the transition of CD4lo to pro-T cells. Because it is likely that the thymic microenvironment plays a critical role in T-cell commitment, we compared the responses of CD4lo and pro-T cells to various cytokine combinations in vitro, as well as the ability of the cultured cells to repopulate organ cultures. Cytokine combinations that maintained T-cell repopulation potential for both CD4lo and pro-T cells were found. CD4lo cells proliferated best in response to the combination containing interleukin-1 (IL-1), IL-3, IL- 6, IL-7, and stem cell factor (SCF). Unlike CD4lo cells, pro-T cells were much more dependent upon IL-7 for proliferation and FTOC repopulation. However, combinations of cytokines lacking IL-7 were found that maintained the T-cell repopulating potential of pro-T cells, suggesting that, whereas this cytokine is clearly very important for normal pro-T cell function, it is not an absolute necessity during early T-cell expansion and differentiation.     

Cardiopoietic factors: extracellular signals for cardiac lineage commitment

Cardiac muscle creation during embryogenesis requires extracellular instructive signals that are regulated precisely in time and space, intersecting with intracellular genetic programs that confer or fashion the ability of the cells to respond. Unmasking the essential signals for cardiac lineage decisions has paramount importance for cardiac development and regenerative medicine, including the directed differentiation of progenitor and stem cells to a cardiac muscle fate.     

Patched dependence receptor triggers apoptosis through ubiquitination of caspase-9

Patched (Ptc), the main receptor for Sonic Hedgehog, is a tumor suppressor. Ptc has been shown to be a dependence receptor, and as such triggers apoptosis in the absence of its ligand. This apoptosis induction occurs through the recruitment by the Ptc intracellular domain of a caspase-activating complex, which includes the adaptor proteins DRAL and TUCAN, and the apical caspase-9. We show here that this caspase-activating complex also includes the E3 ubiquitin ligase NEDD4. We demonstrate that Ptc-mediated apoptosis and Ptc-induced caspase-9 activation require NEDD4. We show that Ptc, but not Bax, the prototypical inducer of the intrinsic cell-death pathway, triggers polyubiquitination of caspase-9. Moreover, a caspase-9 mutant that could not be ubiquitinated failed to mediate Ptc-induced apoptosis. Taken together, these data support the view that the Ptc dependence receptor specifically allows the activation of caspase-9 via its ubiquitination, which occurs via the recruitment by Ptc of NEDD4.     

Disruption of the SCL locus in T-lymphoid malignancies correlates with commitment to the T-cell receptor alpha beta lineage.

The SCL/tal-1 gene on chromosome 1 is disrupted in up to 30% of immature T-cell malignancies, thus representing the most commonly recognized chromosomal abnormality in this disorder. Abnormalities of the gene occur rarely by chromosomal translocation into the T-cell receptor (TCR) delta locus and commonly by a site-specific 95-kb deletion, SIL-SCL (tald). Analysis of the SIL-SCL deletion by Southern blotting and polymerase chain reaction (PCR) in a series of 52 immature T-cell malignancies showed a type A deletion in 21% of cases, but no type B deletions. The type A deletion correlated with malignancies of the TCR alpha beta lineage, either on the basis of TCR alpha beta expression or bilateral TCR delta deletion. Fifty percent (5 of 10) of TCR alpha beta-expressing cells demonstrated the abnormality, whereas 0% (0 of 11) of TCR gamma delta-expressing cells did so. Six of eight SIL-SCL type A cases had undergone bilateral delta deletion, whereas only one of 31 cases with an apparently normal SCL gene had done so. These data demonstrate an association between SCL disruption and TCR alpha beta lineage differentiation and suggest that the SIL-SCL deletion occurs at the same stage of ontogeny as TCR delta deletion.