Notch-Mediated Segmentation and Growth Control of the Drosophila Leg

The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, we show that the Notch signaling pathway plays a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream of the action of Wingless and Decapentaplegic. Our studies further show that Notch activation is both necessary and sufficient to promote leg growth. We also identify target genes regulated both positively and negatively downstream of Notch signaling that are required for normal leg development. Together, these observations outline a regulatory hierarchy for the segmentation and growth of the leg. The Notch pathway is also deployed for segmentation during vertebrate somitogenesis, which raises the possibility of a common origin for the segmentation of these distinct tissues.     

Four-jointed interacts with dachs, abelson and enabled and feeds back onto the Notch pathway to affect growth and segmentation in the Drosophila leg

The molecular basis of segmentation and regional growth during morphogenesis of Drosophila legs is poorly understood. We show that four-jointed is not only required for these processes, but also can direct ectopic growth and joint initiation when its normal pattern of expression is disturbed. These effects are non-autonomous, consistent with our demonstration of both transmembrane and secreted forms of the protein in vivo. The similarities between four-jointed and Notch phenotypes led us to further investigate the relationships between these pathways. Surprisingly, we find that although four-jointed expression is regulated downstream of Notch activation, four-jointed can induce expression of the Notch ligands, Serrate and Delta, and may thereby participate in a feedback loop with the Notch signaling pathway. We also show that four-jointed interacts with abelson, enabled and dachs, which leads us to suggest that one target of four-jointed signaling is the actin cytoskeleton. Thus, four-jointed may bridge the gap between the signals that direct morphogenesis and those that carry it out.     

The odd-skipped family of zinc finger genes promotes Drosophila leg segmentation

Notch signaling controls formation of joints at leg segment borders and growth of the developing Drosophila leg. Here, we identify the odd-skipped gene family as a key group of genes that function downstream of the Notch receptor to promote morphological changes associated with joint formation during leg development. odd, sob, drm, and bowl are expressed in a segmental pattern in the developing leg, and their expression is regulated by Notch signaling. Ectopic expression of odd, sob, or drm can induce invaginations in the leg disc epithelium and morphological changes in the adult leg that are characteristic of endogenous invaginating joint cells. These effects are not due to an alteration in the expression of other genes of the developing joint. While odd or drm mutant clones do not affect leg segmentation, and thus appear to act redundantly, bowl mutant clones do perturb leg development. Specifically, bowl mutant clones result in a failure of joint formation from the distal tibia to tarsal segment 5, while more proximal clones cause melanotic protrusions from the leg cuticle. Together, these results indicate that the odd-skipped family of genes mediates Notch function during leg development by promoting a specific aspect of joint formation, an epithelial invagination. As the odd-skipped family genes are involved in regulating cellular morphogenesis during both embryonic segmentation and hindgut development, we suggest that they may be required in multiple developmental contexts to induce epithelial cellular changes.     

Different downstream pathways for Notch signaling are required for gliogenic and chondrogenic specification of mouse mesencephalic neural crest cells

We examined the roles of Notch signaling and fibroblast growth factors (FGFs) in the gliogenesis of mouse mesencephalic neural crest cells. The present study demonstrated that Notch activation or FGF treatment promotes the differentiation of glia expressing glial fibrillary acidic protein. Notch activation or FGF2 exposure during the first 48 h in culture was critical for glial differentiation. The promotion of gliogenesis by FGF2 was significantly suppressed by the inhibition of Notch signaling using Notch-1 siRNA. These data suggest that FGFs activate Notch signaling and that this activation promotes the gliogenic specification of mouse mesencephalic neural crest cells. Notch activation and FGF treatment have been shown to participate in the chondrogenic specification of these cells [Nakanishi, K., Chan, Y.S., Ito, K., 2007. Notch signaling is required for the chondrogenic specification of mouse mesencephalic neural crest cells. Mech. Dev. 124, 190–203]. Therefore, we analyzed whether or not there were differences between gliogenic and chondrogenic specifications in the downstream pathway of the Notch receptor. Whereas the activation of only the Deltex-mediated pathway was sufficient to promote glial specification, the activation of both RBP-J- and Deltex-dependent pathways was required for chondrogenic specification. These results suggest that the different downstream pathways of the Notch receptor participate in the gliogenic and chondrogenic specification of mouse mesencephalic neural crest cells.     

Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, we analyzed the function of the Delta (Gb'Delta) and Notch (Gb'Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb'Delta and Gb'Notch were expressed in developing legs, and that RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. Our results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, we found that Gb'Delta was expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries.     

Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

During Drosophila optic lobe development, proliferation and differentiation must be tightly modulated to reach its normal size for proper functioning. The JAK/STAT pathway plays pleiotropic roles in Drosophila development and in the larval brain, has been shown to inhibit medulla neuroblast formation. In this study, we find that JAK/STAT activity is required for the maintenance and proliferation of the neuroepithelial stem cells in the optic lobe. In loss-of-function JAK/STAT mutant brains, the neuroepithelial cells lose epithelial cell characters and differentiate prematurely while ectopic activation of this pathway is sufficient to induce neuroepithelial overgrowth in the optic lobe. We further show that Notch signaling acts downstream of JAK/STAT to control the maintenance and growth of the optic lobe neuroepithelium. Thus, in addition to its role in suppression of neuroblast formation, the JAK/STAT pathway is necessary and sufficient for optic lobe neuroepithelial growth.     

Spatial regulation of DELTA expression mediates NOTCH signalling for segmentation of Drosophila legs.

The Notch (N) signalling pathway is recruited for segregation of cell fates in a number of Drosophila tissue types. We show here that N dependent segmentation of Drosophila legs is regulated by a dynamic pattern of expression of its ligand, DELTA (DL). During third larval instar and early stages of pupation, high levels of DL expression is seen in stripes of cells in the leg imaginal discs which later form the proximal borders of leg joints. These domains also displayed heightened Dl enhancer activity. During subsequent stages of pupation, following segmentation of the leg primordium, DL expression becomes uniform throughout these segments barring the joints. We further show that regulatory Dl mutations or mis-expression of DL abolish leg segmentation. Domains of N signalling for segmentation of legs of flies are thus set up by a stringent spatial regulation of expression of its ligand at the segment border. Further, a comparable role of DL in antennal development reveals a common paradigm of DL-N signalling for segmentation of appendages in flies.     

The vertebrate segmentation clock and its role in skeletal birth defects

The segmental structure of the vertebrate body plan is most evident in the axial skeleton. The regulated generation of somites, a process called somitogenesis, underlies the vertebrate body plan and is crucial for proper skeletal development. A genetic clock regulates this process, controlling the timing of somite development. Molecular evidence for the existence of the segmentation clock was first described in the expression of Notch signaling pathway members, several of which are expressed in a cyclic fashion in the presomitic mesoderm (PSM). The Wnt and fibroblast growth factor (FGF) pathways have also recently been linked to the segmentation clock, suggesting that a complex, interconnected network of three signaling pathways regulates the timing of somitogenesis. Mutations in genes that have been linked to the clock frequently cause abnormal segmentation in model organisms. Additionally, at least two human disorders, spondylocostal dysostosis (SCDO) and Alagille syndrome (AGS), are caused by mutations in Notch pathway genes and exhibit vertebral column defects, suggesting that mutations that disrupt segmentation clock function in humans can cause congenital skeletal defects. Thus, it is clear that the correct, cyclic function of the Notch pathway within the vertebrate segmentation clock is essential for proper somitogenesis. In the future, with a large number of additional cyclic genes recently identified, the complex interactions between the various signaling pathways making up the segmentation clock will be elucidated and refined.