Mice lacking link protein develop dwarfism and craniofacial abnormalities

Link protein (LP), an extracellular matrix protein in cartilage, stabilizes aggregates of aggrecan and hyaluronan, giving cartilage its tensile strength and elasticity. Cartilage provides the template for endochondral ossification and is crucial for determining the length and width of the skeleton. During endochondral bone formation, hypertrophic chondrocytes die and the cartilage is replaced with bone matrix. Here, we have generated targeted mutations in mice in the gene encoding LP (Crtl1). Homozygotes showed defects in cartilage development and delayed bone formation with short limbs and craniofacial anomalies. Most Crtl1(tm1Nid/tm1Nid) mice died shortly after birth due to respiratory failure, but some survived and developed progressive dwarfism and lordosis of the cervical spine. They showed small epiphysis, slightly flared metaphysis of long bones and flattened vertebrae, characteristic of spondyloepiphyseal dysplasias. The cartilage contained significantly reduced aggrecan depositions in the hypertrophic zone, and decreased numbers of prehypertrophic and hypertrophic chondrocytes. Reduced Indian hedgehog (Ihh) expression was observed in prehypertrophic chondrocytes, and apoptosis was inhibited in hypertrophic chondrocytes. These results indicate that LP is important for the formation of proteoglycan aggregates and normal organization of hypertrophic chondrocytes, and suggest that cartilage matrix has a role in chondrocyte differentiation and maturation.     

The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation

During vertebrate limb development, growth plate chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (-/-) cells, and analyzed cell-cell interactions in the growth plate in vivo. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic chondrocytes. Further, ectopic hypertrophic chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.     

Effect of a combined trenbolone acetate and estradiol implant on steady-state IGF-I mRNA concentrations in the liver of wethers and the longissimus muscle of steers

Endochondral ossification in growth plates proceeds through several consecutive steps of late cartilage differentiation leading to chondrocyte hypertrophy, vascular invasion, and, eventually, to replacement of the tissue by bone. It is well established that the subchondral vascular system is pivotal in the regulation of this process. Cells of subchondral blood vessels act as a source of vascular invasion and, in addition, release factors influencing growth and differentiation of chondrocytes in the avascular growth plate. To elucidate the paracrine contribution of endothelial cells we studied the hypertrophic development of resting chondrocytes from the caudal third of chick embryo sterna in co-culture with endothelial cells. The design of the experiments prevented cell-to-cell contact but allowed paracrine communication between endothelial cells and chondrocytes. Under these conditions, chondrocytes rapidly became hypertrophied in vitro and expressed the stage-specific markers collagen X and alkaline phosphatase. This development also required signaling by thyroid hormone in synergy. Conditioned media could replace the endothelial cells, indicating that diffusible factors mediated this process. By contrast, smooth muscle cells, fibroblasts, or hypertrophic chondrocytes did not secrete this activity, suggesting that the factors were specific for endothelial cells. We conclude that endochondral ossification is under the control of a mutual communication between chondrocytes and endothelial cells. A finely tuned balance between chondrocyte-derived signals repressing cartilage maturation and endothelial signals promoting late differentiation of chondrocytes is essential for normal endochondral ossification during development, growth, and repair of bone. A dysregulation of this balance in permanent joint cartilage also may be responsible for the initiation of pathological cartilage degeneration in joint diseases.     

Terminal differentiation of chondrocytes is arrested at distinct stages identified by their expression repertoire of marker genes

During endochondral bone formation, cells in the emerging cartilaginous model transit through a cascade of several chondrocyte differentiation stages, each characterized by a specific expression repertoire of matrix macromolecules, until, as a final step, the hypertrophic cartilage is replaced by bone. In many permanent cartilage tissues, however, late differentiation of chondrocytes does not occur, due to negative regulation by the environment of the cells. Here, addressing the reason for the difference between chondrocyte fates in the chicken embryo sternum, cells from the caudal and cranial part were cultured separately in serum-free agarose gels with complements defined earlier that either permit or prevent hypertrophic development. Total RNA was extracted using a novel protocol adapted to agarose cultures, and the temporal changes in developmental stage-specific mRNA expression were monitored by Northern hybridization and phosphor image analysis. Kinetic studies of the mRNA accumulation not only showed significant differences between the expression patterns of cranial and caudal cultures after recovery, but also revealed two checkpoints of chondrocyte differentiation in keeping with cartilage development in vivo. Terminal differentiation of caudal chondrocytes is blocked at the late proliferative stage (stage Ib), while the cranial cells can undergo hypertrophic development spontaneously. The differentiation of cranial chondrocytes is reversible, since they can re-assume an early proliferative (stage Ia) phenotype under the influence of insulin, fibroblast growth factor-2 and transforming growth factor-beta in combination. Thus, the expression pattern in the latter culture resembles that of articular chondrocytes. We also provide evidence that the capacities of caudal and sternal chondrocytes to progress from the late proliferative (stage Ib) to hypertrophic stage (stage II) correlate with their differing abilities to express the Indian hedgehog gene.     

Evidence for regulation of cartilage differentiation by the homeobox gene Hoxc-8

Homeobox genes of the Hox class are required for proper patterning of skeletal elements, but how they regulate the differentiation of specific tissues is unclear. We show here that overexpression of a Hoxc-8 transgene causes cartilage defects whose severity depends on transgene dosage. The abnormal cartilage is characterized by an accumulation of proliferating chondrocytes and reduced maturation. Since Hoxc-8 is normally expressed in chondrocytes, these results suggest that Hoxc-8 continues to regulate skeletal development well beyond pattern formation in a tissue-specific manner, presumably by controlling the progression of cells along the chondrocyte differentiation pathway. The comparison to Hoxd-4 and Isl-1 indicates that this role in chondrogenesis is specific to proteins of the Hox class. Their capacity for regulation of cartilage differentiation suggests that Hox genes could also be involved in human chondrodysplasias or other cartilage disorders.     

Morphological and cytochemical findings in 150 cases of T-lineage acute lymphoblastic leukaemia in adults. German Multicentre ALL Study Group (GMALL)

During the process of endochondral ossification chondrocytes progress through stages of terminal differentiation culminating in apoptotic death. We have developed a serum-free suspension culture that allows terminal differentiation and facilitates the investigation of factors affecting chondrocyte apoptosis. We have found that chondrocytes not committed to terminal differentiation, i.e., those from the caudal region of chick embryo sterna, a region that remains cartilaginous for some months after the chick hatches, maintained high viability in serum-free suspension culture. A strong dependence of viability on culture density and sensitivity to induction of apoptosis with the protein kinase inhibitor, staurosporine, was consistent with the proposal that these chondrocytes, like nearly all cells, require intercellular communication for survival. Chondrocytes that were committed to terminal differentiation, i.e., those from the cephalic region of chick embryo sterna, a region that is replaced by bone before the chick hatches, expressed the hypertrophic phenotype but maintained their viability in culture for only approximately 6 days. Subsequent cell death was very consistent between cultures and shown to occur by an apoptotic process by analysis of DNA fragmentation and cell morphology. Short-term viability of hypertrophic chondrocytes was independent of culture density and relatively resistant to treatment with staurosporine. Induction of the hypertrophic phenotype in immature chondrocytes committed them to cell death and prevention of expression of the hypertrophic phenotype prevented cell death. We conclude that commitment of chondrocytes to terminal differentiation is associated with a commitment to apoptosis and apoptosis of hypertrophic chondrocytes in growth cartilage does not require initiation by external signals.     

End labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosis in terminally differentiated chondrocytes

The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTag and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate for apoptosis. Dual parameteric flow cytometric contour plots of Hoechst and 7-amino-actinomycin D fluorescence showed that abut 8% of cells in the plate were apoptotic. Most of these cells were in hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate the number of cells undergoing this event, the data lend credence to the view that cells are removed from the epiphysis through apoptosis. If this is the case, then chondrocytes probably enter the terminal phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death.     

Notch1-induced delay of human hematopoietic progenitor cell differentiation is associated with altered cell cycle kinetics

Hematopoiesis is a balance between proliferation and differentiation that may be modulated by environmental signals. Notch receptors and their ligands are highly conserved during evolution and have been shown to regulate cell fate decisions in multiple developmental systems. To assess whether Notch1 signaling may regulate human hematopoiesis to maintain cells in an immature state, we transduced a vesicular stomatitis virus G-protein (VSV-G) pseudo-typed bicistronic murine stem cell virus (MSCV)-based retroviral vector expressing a constitutively active form of Notch1 (ICN) and green fluorescence protein into the differentiation competent HL-60 cell line and primary cord blood-derived CD34(+) cells. In addition, we observed endogenous Notch1 expression on the surface of both HL-60 cells and primary CD34(+) cells, and therefore exposed cells to Notch ligand Jagged2, expressed on NIH3T3 cells. Both ligand-independent and ligand-dependent activation of Notch resulted in delayed acquisition of differentiation markers by HL-60 cells and cord blood CD34(+) cells. In addition, primary CD34(+) cells retained their ability to form immature colonies, colony-forming unit-mix (CFU-mix), whereas control cells lost this capacity. Activation of Notch1 correlated with a decrease in the fraction of HL-60 cells that were in G0/G1 phase before acquisition of a mature cell phenotype. This enhanced progression through G1 was noted despite preservation of the proliferative rate of the cells and the overall length of the cell cycle. These findings show that Notch1 activation delays human hematopoietic differentiation and suggest a link of Notch differentiation effects with altered cell cycle kinetics.     

The Notch signalling pathway in hair growth

The Notch signalling pathway is an important mediator of cell fate selection whose involvement in epidermal appendage formation is now becoming recognised. Hair follicle development and hair formation involve the co-ordinated differentiation of several different cell types in which Notch appears to have a role. We report intricate expression patterns for the Notch-1 receptor and three ligands, Delta-1, Jagged-1 and Jagged-2 in the hair follicle. Notch-1 is expressed in ectodermal-derived cells of the follicle, in the inner cells of the embryonic placode and the follicle bulb, and in the suprabasal cells of the mature outer root sheath. Delta-1 is only expressed during embryonic follicle development and is exclusive to the mesenchymal cells of the pre-papilla located beneath the follicle placode. Expression of Jagged-1 or Jagged-2 overlaps Notch-1 expression at all stages. In mature follicles, Jagged-1 and Jagged-2 are expressed in complementary patterns in the follicle bulb and outer root sheath, Jagged-1 in suprabasal cells and Jagged-2 predominantly in basal cells. In the follicle bulb, Jagged-2 is localised to the inner (basal) bulb cells next to the dermal papilla which do not express Notch-1, whereas Jagged-1 expression in the upper follicle bulb overlaps Notch-1 expression and correlates with bulb cell differentiation into hair shaft cortical and cuticle keratinocytes.     

Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation

Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent gene-targeting and disruption experiments have implicated PTHrP as a developmental regulatory molecule. Apparent PTHrP functions include the regulation of endochondral bone development, of hair follicle formation, and of branching morphogenesis in the breast. Herein, we report that overexpression of PTHrP in chondrocytes using the mouse type II collagen promoter induces a novel form of chondrodysplasia characterized by short-limbed dwarfism and a delay in endochondral ossification. This features a delay in chondrocyte differentiation and in bone collar formation and is sufficiently marked that the mice are born with a cartilaginous endochondral skeleton. In addition to the delay, chondrocytes in the transgenic mice initially become hypertrophic at the periphery of the developing long bones rather than in the middle, leading to a seeming reversal in the pattern of chondrocyte differentiation and ossification. By 7 weeks, the delays in chondrocyte differentiation and ossification have largely corrected, leaving foreshortened and misshapen but histologically near-normal bones. These findings confirm a role for PTHrP as an inhibitor of the program of chondrocyte differentiation. PTHrP may function in this regard to maintain the stepwise differentiation of chondrocytes that initiates endochondral ossification in the midsection of endochondral bones early in development and that also permits linear growth at the growth plate later in development.     

Distinct roles of type I bone morphogenetic protein receptors in the formation and differentiation of cartilage

The bone morphogenetic proteins (BMPs), TGF beta superfamily members, play diverse roles in embryogenesis, but how the BMPs exert their action is unclear and how different BMP receptors (BMPRs) contribute to this process is not known. Here we demonstrate that the two type I BMPRs, BMPR-IA and BMPR-IB, regulate distinct processes during chick limb development. BmpR-IB expression in the embryonic limb prefigures the future cartilage primordium, and its activity is necessary for the initial steps of chondrogenesis. During later chondrogenesis, BmpR-IA is specifically expressed in prehypertrophic chondrocytes. BMPR-IA regulates chondrocyte differentiation, serving as a downstream mediator of Indian Hedgehog (IHH) function in both a local signaling loop and a longer-range relay system to PTHrP. BMPR-IB also regulates apoptosis: Expression of activated BMPR-IB results in increased cell death, and we showed previously that dominant-negative BMPR-IB inhibits apoptosis. Our studies indicate that in TGF beta signaling systems, different type I receptor isoforms are dedicated to specific functions during embryogenesis.     

Targeted expression of constitutively active receptors for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormone-related peptide

Mice in which the genes encoding the parathyroid hormone (PTH)-related peptide (PTHrP) or the PTH/PTHrP receptor have been ablated by homologous recombination show skeletal dysplasia due to accelerated endochondral bone formation, and die at birth or in utero, respectively. Skeletal abnormalities due to decelerated chondrocyte maturation are observed in transgenic mice where PTHrP expression is targeted to the growth plate, and in patients with Jansen metaphyseal chondrodysplasia, a rare genetic disorder caused by constitutively active PTH/PTHrP receptors. These and other findings thus indicate that PTHrP and its receptor are essential for chondrocyte differentiation. To further explore the role of the PTH/PTHrP receptor in this process, we generated transgenic mice in which expression of a constitutively active receptor, HKrk-H223R, was targeted to the growth plate by the rat alpha1 (II) collagen promoter. Two major goals were pursued: (i) to investigate how constitutively active PTH/PTHrP receptors affect the program of chondrocyte maturation; and (ii) to determine whether expression of the mutant receptor would correct the severe growth plate abnormalities of PTHrP-ablated mice (PTHrP-/-). The targeted expression of constitutively active PTH/PTHrP receptors led to delayed mineralization, decelerated conversion of proliferative chondrocytes into hypertrophic cells in skeletal segments that are formed by the endochondral process, and prolonged presence of hypertrophic chondrocytes with delay of vascular invasion. Furthermore, it corrected at birth the growth plate abnormalities of PTHrP-/- mice and allowed their prolonged survival. "Rescued" animals lacked tooth eruption and showed premature epiphyseal closure, indicating that both processes involve PTHrP. These findings suggest that rescued PTHrP-/- mice may gain considerable importance for studying the diverse, possibly tissue-specific role(s) of PTHrP in postnatal development.     

Fibroblast growth factor receptor 3 mutations promote apoptosis but do not alter chondrocyte proliferation in thanatophoric dysplasia

Thanatophoric dysplasia (TD) is a lethal skeletal disorder caused by recurrent mutations in the fibroblast growth factor receptor 3 (FGFR 3) gene. The mitogenic response of fetal TD I chondrocytes in primary cultures upon stimulation by either FGF 2 or FGF 9 did not significantly differ from controls. Although the levels of FGFR 3 mRNAs in cultured TD chondrocytes were similar to controls, an abundant immunoreactive material was observed at the perinuclear level using an anti-FGFR 3 antibody in TD cells. Transduction signaling via the mitogen-activated protein kinase pathway was assessed by measuring extracellular signal-regulated kinase activity (ERK 1 and ERK 2). Early ERKs activation following FGF 9 supplementation was observed in TD chondrocytes (2 min) as compared with controls (5 min) but no signal was detected in the absence of ligand. By contrast ligand-independent activation of the STAT signaling pathway was demonstrated in cultured TD cells and confirmed by immunodetection of Stat 1 in the nuclei of hypertrophic TD chondrocytes. Moreover, the presence of an increased number of apoptotic chondrocytes in TD fetuses was associated with a higher expression of Bax and the simultaneous decrease of Bcl-2 levels. Taken together, these results indicate that FGFR 3 mutations in TD I fetuses do not hamper chondrocyte proliferation but rather alter their differentiation by triggering premature apoptosis through activation of the STAT signaling pathway.     

Parathyroid hormone-related protein is required for tooth eruption

Parathyroid hormone (PTH)-related protein (PTHrP)-knockout mice die at birth with a chondrodystrophic phenotype characterized by premature chondrocyte differentiation and accelerated bone formation, whereas overexpression of PTHrP in the chondrocytes of transgenic mice produces a delay in chondrocyte maturation and endochondral ossification. Replacement of PTHrP expression in the chondrocytes of PTHrP-knockout mice using a procollagen II-driven transgene results in the correction of the lethal skeletal abnormalities and generates animals that are effectively PTHrP-null in all sites other than cartilage. These rescued PTHrP-knockout mice survive to at least 6 months of age but are small in stature and display a number of developmental defects, including cranial chondrodystrophy and a failure of tooth eruption. Teeth appear to develop normally but become trapped by the surrounding bone and undergo progressive impaction. Localization of PTHrP mRNA during normal tooth development by in situ hybridization reveals increasing levels of expression in the enamel epithelium before the formation of the eruption pathway. The type I PTH/PTHrP receptor is expressed in both the adjacent dental mesenchyme and in the alveolar bone. The replacement of PTHrP expression in the enamel epithelium with a keratin 14-driven transgene corrects the defect in bone resorption and restores the normal program of tooth eruption. PTHrP therefore represents an essential signal in the formation of the eruption pathway.     

Cis-interactions between Delta and Notch modulate neurogenic signalling in Drosophila

We find that ectopic expression of Delta or Serrate in neurons within developing bristle organs is capable of non-autonomously inducing the transformation of the pre-trichogen cell into a tormogen cell in a wide variety of developmental contexts. The frequencies at which Delta can induce these transformations are dependent on the level of ectopic Delta expression and the levels of endogenous Notch signalling pathway components. The pre-trichogen cell becomes more responsive to Delta- or Serrate-mediated transformation when the level of endogenous Delta is reduced and less responsive when the dosage of endogenous Delta is increased, supporting the hypothesis that Delta interferes autonomously with the ability of a cell to receive either signal. We also find that a dominant-negative form of Notch, ECN, is capable of autonomously interfering with the ability of a cell to generate the Delta signal. When the region of Notch that mediates trans-interactions between Delta and the Notch extracellular domain is removed from ECN, the ability of Delta to signal is restored. Our findings imply that cell-autonomous interactions between Delta and Notch can affect the ability of a cell to generate and to transduce a Delta-mediated signal. Finally, we present evidence that the Fringe protein can interfere with Delta- and Serrate-mediated signalling within developing bristle organs, in contrast to previous reports of the converse effects of Fringe on Delta signalling in the developing wing.     

The conformational equilibrium of human growth hormone

To examine the role of bone morphogenetic protein (BMP) signaling in chondrocytes during endochondral ossification, the dominant negative (DN) forms of BMP receptors were introduced into immature and mature chondrocytes isolated from lower and upper portions of chick embryo sternum, respectively. We found that control sternal chondrocyte populations expressed type IA, IB, and II BMP receptors as well as BMP-4 and -7. Expression of a DN-type II BMP receptor (termed DN-BMPR-II) in immature lower sternal (LS) chondrocytes led to a loss of differentiated functions; compared with control cells, the DN-BMPR- II-expressing LS chondrocytes proliferated more rapidly, acquired a fibroblastic morphology, showed little expression of type II collagen and aggrecan genes, and upregulated type I collagen gene expression. Expression of DN-BMPR-II in mature hypertrophic upper sternal (US) chondrocytes caused similar effects. In addition, the DN-BMPR-II-expressing US cells exhibited little alkaline phosphatase activity and type X collagen gene expression, while the control US cells produced both alkaline phosphatase and type X collagen. Both DN-BMPR-II-expressing US and LS chondrocytes failed to respond to treatment with BMP-2 . When we examined the effects of DN forms of types IA and IB BMP receptors, we found that DN-BMPR-IA had little effect, while DN-BMPR-IB had similar but weaker effects compared with those of DN-BMPR-II. We conclude that BMP signaling, particularly that mediated by the type II BMP receptor, is required for maintenance of the differentiated phenotype, control of cell proliferation, and expression of hypertrophic phenotype.     

Delta-notch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feedback regulation

Recent data suggest that dental cells utilize the evolutonarily conserved Notch-mediated intercellular signaling pathway to regulate their fates. Here we report on the expression and regulation of Delta1, a transmembrane ligand of the Notch receptors, during mouse odontogenesis. Delta1 is weakly expressed in dental epithelium during tooth initiation and morphogenesis, but during cytodifferentiation, expression is upregulated in the epithelium-derived ameloblasts and the mesenchyme-derived odontoblasts. The expression pattern of Delta1 in ameloblasts and odontoblasts is complementary to Notch1, Notch2, and Notch3 expression in adjacent epithelial and mesenchymal cells. Notch1 and Notch2 are upregulated in explants of dental mesenchyme adjacent to implanted cells expressing Delta1, suggesting that feedback regulation by Delta-Notch signaling ensures the spatial segregation of Notch receptors and ligands. TGFbeta1 and BMPs induce Delta1 expression in dental mesenchyme explants at the stage at which Delta1 is upregulated in vivo, but not at earlier stages. In contrast to the Notch family receptors and their ligand Jagged1, expression of Delta1 in the tooth germ is not affected by epithelial-mesenchymal interactions, showing that the Notch receptors and their two ligands Jagged1 and Delta1 are subject to different regulations.     

Complex proteolytic processing acts on Delta, a transmembrane ligand for Notch, during Drosophila development

Delta functions as a cell nonautonomous membrane-bound ligand that binds to Notch, a cell-autonomous receptor, during cell fate specification. Interaction between Delta and Notch leads to signal transduction and elicitation of cellular responses. During our investigations to further understand the biochemical mechanism by which Delta signaling is regulated, we have identified four Delta isoforms in Drosophila embryonic and larval extracts. We have demonstrated that at least one of the smaller isoforms, Delta S, results from proteolysis. Using antibodies to the Delta extracellular and intracellular domains in colocalization experiments, we have found that at least three Delta isoforms exist in vivo, providing the first evidence that multiple forms of Delta exist during development. Finally, we demonstrate that Delta is a transmembrane ligand that can be taken up by Notch-expressing Drosophila cultured cells. Cell culture experiments imply that full-length Delta is taken up by Notch-expressing cells. We present evidence that suggests this uptake occurs by a nonphagocytic mechanism.