Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg

Segmentation plays crucial roles during morphogenesis. Drosophila legs are divided into segments along the proximal-distal axis by flexible structures called joints. Notch signaling is necessary and sufficient to promote leg growth and joint formation, and is activated in distal cells of each segment in everting prepupal leg discs. The homeobox gene defective proventriculus (dve) is expressed in regions both proximal and distal to the intersegmental folds at 4 h after puparium formation (APF). Dve-expressing region partly overlaps with the Notch-activated region, and they become a complementary pattern at 6 h APF. Interestingly, dve mutant legs resulted in extra joint formation at the center of each tarsal segment, and the forced expression of dve caused a jointless phenotype. We present evidence that Dve suppresses the potential joint-forming activity, and that Notch signaling represses Dve expression to form joints.     

The Hox gene Ultrabithorax modulates the shape and size of the third leg of Drosophila by influencing diverse mechanisms

The developmental mechanisms that regulate the relative size and shape of organs have remained obscure despite almost a century of interest in the problem and the fact that changes in relative size represent the dominant mode of evolutionary change. Here, I investigate how the Hox gene Ultrabithorax (Ubx) instructs the legs on the third thoracic segment of Drosophila melanogaster to develop with a different size and shape from the legs on the second thoracic segment. Through loss-of-function and gain-of-function experiments, I demonstrate that different segments of the leg, the femur and the first tarsal segment, and even different regions of the femur, regulate their size in response to Ubx expression through qualitatively different mechanisms. In some regions, Ubx acts autonomously to specify shape and size, whereas in other regions, Ubx influences size through nonautonomous mechanisms. Loss of Ubx autonomously reduces cell size in the T3 femur, but this reduction seems to be partially compensated by an increase in cell numbers, so that it is unclear what effect cell size and number directly have on femur size. Loss of Ubx has both autonomous and nonautonomous effects on cell number in different regions of the basitarsus, but again there is not a strong correlation between cell size or number and organ size. Total organ size appears to be regulated through mechanisms that operate at the level of the entire leg segment (femur or basitarsus) relatively independently of the behavior of individual subpopulations of cells within the segment.     

Spatial and temporal regulation of the homeotic selector gene Antennapedia is required for the establishment of leg identity in Drosophila

Antennapedia is one of the homeotic selector genes required for specification of segment identity in Drosophila. Dominant mutations that ectopically express Antennapedia cause transformation of antenna to leg. Loss-of-function mutations cause partial transformation of leg to antenna. Here we examine the role of Antennapedia in the establishment of leg identity in light of recent advances in our understanding of antennal development. In Antennapedia mutant clones in the leg disc, Homothorax and Distal-less are coexpressed and act via spineless to transform proximal femur to antenna. Antennapedia is negatively regulated during leg development by Distal-less, spineless, and dachshund and this reduced Antennapedia expression is needed for the proper development of distal leg elements. These findings suggest that the temporal and spatial regulation of the homeotic selector gene Antennapedia in the leg disc is necessary for normal leg development in Drosophila.     

The Tribolium ortholog of knirps and knirps-related is crucial for head segmentation but plays a minor role during abdominal patterning

Segment formation in the long germ insect Drosophila is dominated by overlapping gap gene domains in the syncytial blastoderm. In the short germ beetle Tribolium castaneum abdominal segments arise from a cellular growth zone, implying different patterning mechanisms. We describe here the single Tribolium ortholog of the Drosophila genes knirps and knirps-related (called Tc-knirps). Tc-knirps expression is conserved during head patterning and at later stages. However, posterior Tc-knirps expression in the ectoderm is limited to a stripe in A1, instead of a broad abdominal domain covering segment primordia A2-A5 as in Drosophila. Tc-knirps RNAi yields only mild defects in the abdomen, at a position posterior to the abdominal Tc-knirps domain. In addition, Tc-knirps RNAi larvae lack the antennal and mandibular segments. These defects are much more severe than the head defects caused by combined inactivation of Dm-knirps and Dm-knirps-related. Our findings support the notion that the role of gap gene homologs in abdominal segmentation differs fundamentally in long and short germ insects. Moreover, the pivotal role of Tc-knirps in the head suggests an ancestral role for knirps as head patterning gene. Based on this RNAi analysis, Tc-knirps functions neither in the head nor the abdomen as a canonical gap gene.     

Dominant maternal interactions with Drosophila segmentation genes

Summary A systematic search for X chromosome loci showing a dominant maternal interaction with the segmentation genes Krüppel, hunchback, knirps and hairy was performed using deficiencies spanning 65% of the X chromosome. No interaction with the knirps gene was observed, but five regions of the X chromosome showed a maternal dominant interaction with the Krüppel gene. Two of these regions also show a maternal dominant interaction with either hunchback (region 10A7–10A8) or hairy (region 10E1–10F3). In all of these interactions dead embryos were observed which showed the same defects as embryos homozygous for the segmentation gene tested. These results suggest that a complex repartition of maternal products necessary for subsequent segmentation may occur in the Drosophila egg.     

The function of Notch signalling in segment formation in the crustacean Daphnia magna (Branchiopoda)

Ten years ago we showed for the first time that Notch signalling is required in segmentation in spiders, indicating the existence of similar mechanisms in arthropod and vertebrate segmentation. However, conflicting results in various arthropod groups hampered our understanding of the ancestral function of Notch in arthropod segmentation. Here we fill a crucial data gap in arthropods and analyse segmentation in a crustacean embryo. We analyse the expression of homologues of the Drosophila and vertebrate segmentation genes and show that members of the Notch signalling pathway are expressed at the same time as the pair-rule genes. Furthermore, inactivation of Notch signalling results in irregular boundaries of the odd-skipped-like expression domains and affects the formation of segments. In severe cases embryos appear unsegmented. We suggest two scenarios for the function of Notch signalling in segmentation. The first scenario agrees with a segmentation clock involving Notch signalling, while the second scenario discusses an alternative mechanism of Notch function which is integrated into a hierarchical segmentation cascade.     

The zinc finger homeodomain-2 gene of Drosophila controls Notch targets and regulates apoptosis in the tarsal segments

The development of the Drosophila leg is a good model to study processes of pattern formation, cell death and segmentation. Such processes require the coordinate activity of different genes and signaling pathways that progressively subdivide the leg territory into smaller domains. One of the main pathways needed for leg development is the Notch pathway, required for determining the proximo-distal axis of the leg and for the formation of the joints that separate different leg segments. The mechanisms required to coordinate such events are largely unknown. We describe here that the zinc finger homeodomain-2 (zfh-2) gene is highly expressed in cells that will form the leg joints and needed to establish a correct size and pattern in the distal leg. There is an early requirement of zfh-2 to establish the correct proximo-distal axis, but zfh-2 is also needed at late third instar to form the joint between the fourth and fifth tarsal segments. The expression of zfh-2 requires Notch activity but zfh-2 is necessary, in turn, to activate Notch targets such as Enhancer of split and big brain. zfh-2 is controlled by the Drosophila activator protein 2 gene and regulates the late expression of tarsal-less. In the absence of zfh-2 many cells ectopically express the pro-apoptotic gene head involution defective, activate caspase-3 and are positive for acridine orange, indicating they undergo apoptosis. Our results demonstrate the key role of zfh-2 in the control of cell death and Notch signaling during leg development.     

Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis

While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, we noted interesting and unexpected mutant phenotypes in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch (Pu) and pale (ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up (Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. We also demonstrate expression of Punch, Ple, Catsup and dopamine in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, we show that these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally we show that dopamine regulates endocytosis via controlling the dynamin protein level.     

Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis

The Drosophila melanogaster ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from each other. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. Here we show that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment.     

Posterior skeletal development and the segmentation clock period are sensitive to Lfng dosage during somitogenesis

The segmental structure of the axial skeleton is formed during somitogenesis. During this process, paired somites bud from the presomitic mesoderm (PSM), in a process regulated by a genetic clock called the segmentation clock. The Notch pathway and the Notch modulator Lunatic fringe (Lfng) play multiple roles during segmentation. Lfng oscillates in the posterior PSM as part of the segmentation clock, but is stably expressed in the anterior PSM during presomite patterning. We previously found that mice lacking overt oscillatory Lfng expression in the posterior PSM (Lfng^?FCE) exhibit abnormal anterior development but relatively normal posterior development. This suggests distinct requirements for segmentation clock activity during the formation of the anterior skeleton (primary body formation), compared to the posterior skeleton and tail (secondary body formation). To build on these findings, we created an allelic series that progressively lowers Lfng levels in the PSM. Interestingly, we find that further reduction of Lfng expression levels in the PSM does not increase disruption of anterior development. However tail development is increasingly compromised as Lfng levels are reduced, suggesting that primary body formation is more sensitive to Lfng dosage than is secondary body formation. Further, we find that while low levels of oscillatory Lfng in the posterior PSM are sufficient to support relatively normal posterior development, the period of the segmentation clock is increased when the amplitude of Lfng oscillations is low. These data support the hypothesis that there are differential requirements for oscillatory Lfng during primary and secondary body formation and that posterior development is less sensitive to overall Lfng levels. Further, they suggest that modulation of the Notch signaling by Lfng affects the clock period during development.     

Regulation of gene expression in the distal region of the Drosophila leg by the Hox11 homolog, C15

The distal region of the Drosophila leg, the tarsus, is divided into five segments (ta I-V) and terminates in the pretarsus, which is characterized by a pair of claws. Several homeobox genes are expressed in distinct regions of the tarsus, including aristaless (al) and lim1 in the pretarsus, Bar (B) in ta IV and V, and apterous (ap) in ta IV. This pattern is governed by regulatory interactions between these genes; for example, Al and B are mutually antagonistic resulting in exclusion of B expression from the pretarsus. Although Al is necessary, it is not sufficient to repress B, indicating another factor is required. Here, this factor is identified as the product of the C15 gene, which is another homeodomain protein, a homolog of the human Hox11 oncogene. C15 is expressed in the same cells as al and, together, C15 and Al appear to directly repress B. C15/Al also act indirectly to repress ap in ta V, i.e., in surrounding cells. To do this, C15/Al autonomously repress expression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus, restricting Dl to ta V and creating a Dl+/Dl- border at the interface between ta V and the pretarsus. This results in upregulation of Notch signaling, which induces expression of the bowl gene, the product of which represses ap.     

Evidence that nervy, the Drosophila homolog of ETO/MTG8, promotes mechanosensory organ development by enhancing Notch signaling

In the imaginal tissue of developing fruit flies, achaete (ac) and scute (sc) expression defines a group of neurally-competent cells called the proneural cluster (PNC). From the PNC, a single cell, the sensory organ precursor (SOP), is selected as the adult mechanosensory organ precursor. The SOP expresses high levels of ac and sc and sends a strong Delta (Dl) signal, which activates the Notch (N) receptor in neighboring cells, preventing them from also adopting a neural fate. Previous work has determined how ac and sc expression in the PNC and SOP is regulated, but less is known about SOP-specific factors that promote SOP fate. Here, we describe the role of nervy (nvy), the Drosophila homolog of the mammalian proto-oncogene ETO, in mechanosensory organ formation. Nvy is specifically expressed in the SOP, where it interacts with the Ac and Sc DNA binding partner Daughterless (Da) and affects the expression of Ac and Sc targets. nvy loss- and gain-of-function experiments suggest that nvy reinforces, but is not absolutely required for, the SOP fate. We propose a model in which nvy acts downstream of ac and sc to promote the SOP fate by transiently strengthening the Dl signal emanating from the SOP.     

Interaction between X-Delta-2 and Hox genes regulates segmentation and patterning of the anteroposterior axis

In vertebrates, the paraxial mesoderm already exhibits a complex Hox gene pattern by the time that segmentation occurs and somites are formed. The anterior boundaries of the Hox genes are always maintained at the same somite number, suggesting coordination between somite formation and Hox expression. To study this interaction, we used morpholinos to knockdown either the somitogenesis gene X-Delta-2 or the complete Hox paralogous group 1 (PG1) in Xenopus laevis. When X-Delta-2 is knocked down, Hox genes from different paralogous groups are downregulated from the beginning of their expression at gastrula stages. This effect is not via the canonical Notch pathway, as it is independent of the Notch effector Su(H). We also reveal for the first time a clear role for Hox genes in somitogenesis, as loss of PG1 gene function results in the perturbation of somite formation and downregulation of the X-Delta-2 expression in the PSM. This effect on X-Delta-2 expression is also observed during neurula stages, before the somites are formed. These results show that somitogenesis and patterning of the anteroposterior axis are closely linked via a feedback loop involving Hox genes and X-Delta-2, suggesting the existence of a coordination mechanism between somite formation and anteroposterior patterning. Such a mechanism is likely to be functional during gastrulation, before the formation of the first pair of somites, as suggested by the early X-Delta-2 regulation of the Hox genes.