A Delta-Notch signaling border regulated by Engrailed/Invected repression specifies boundary cells in the Drosophila hindgut

The Drosophila hindgut develops three morphologically distinct regions along its anteroposterior axis: small intestine, large intestine and rectum. Single-cell rings of 'boundary cells' delimit the large intestine from the small intestine at the anterior, and the rectum at the posterior. The large intestine also forms distinct dorsal and ventral regions; these are separated by two single-cell rows of boundary cells. Boundary cells are distinguished by their elongated morphology, high level of both apical and cytoplasmic Crb protein, and gene expression program. During embryogenesis, the boundary cell rows arise at the juxtaposition of a domain of Engrailed (En)- plus Invected (Inv)-expressing cells with a domain of Delta (Dl)-expressing cells. Analysis of loss-of-function and ectopic expression phenotypes shows that the domain of Dl-expressing cells is defined by En/Inv repression. Further, Notch pathway signaling, specifically the juxtaposition of Dl-expressing and Dl-non-expressing cells, is required to specify the rows of boundary cells. This Notch-induced cell specification is distinguished by the fact that it does not appear to utilize the ligand Serrate and the modulator Fringe.     

Cell-fate choice and boundary formation by combined action of Notch and engrailed in the Drosophila hindgut

The Drosophila large intestine is initially subdivided into dorsal and ventral domains with distinct cell types, and a one-cell-wide strand of boundary cells is induced between them. Here we show that cell identity and localization of the boundary cells are determined by the combined action of Delta, Notch, and engrailed genes. The prospective dorsal domain of the hindgut primordium expresses engrailed. Engrailed represses Delta, which is ubiquitously expressed throughout the prospective hindgut region in early blastodermal stages, in the dorsal domain, and thus generates a Delta-positive/negative prepattern. Expression of Engrailed protein determines the dorsal domain, while an Engrailed-negative (Delta-positive) region is differentiated into the ventral domain. Delta-positive ventral cells activate a Notch cascade in abutting dorsal cells, and thus induce their differentiation into boundary cells. Mis-expression of a constitutively active Notch intracellular domain causes the entire large intestine to develop as boundary cells. It was also found that the transducing activity of a transmembrane form of activated Notch, which requires further proteolytic processing to generate intracellular fragments, is suppressed in the Delta-positive domain. Delta acts in two distinct ways: it activates the Notch signaling pathway in adjacent Delta-negative cells, and, at the same time, autonomously blocks Notch signaling in Delta-positive cells by affecting Notch processing.     

A re-evaluation of the contributions of Apterous and Notch to the dorsoventral lineage restriction boundary in the Drosophila wing

The Drosophila limb primordia are subdivided into compartments: cell populations that do not mix during development. The wing is subdivided into dorsal (D) and ventral (V) compartments by the activity of the selector gene apterous in D cells. Apterous causes segregation of D and V cell populations by at least two distinct mechanisms. The LRR transmembrane proteins Capricious and Tartan are transiently expressed in D cells and contribute to initial segregation of D and V cells. Signaling between D and V cells mediated by Notch and Fringe contributes to the maintenance of the DV affinity boundary. Given that Notch is activated symmetrically, in D and V cells adjacent to the boundary, its role in boundary formation remains somewhat unclear. We re-examine the roles of Apterous and Fringe activities in DV boundary formation and present evidence that Fringe cannot, by itself, generate an affinity difference between D and V cells. Although not sufficient, Fringe is required via Notch activation for expression of an Apterous-dependent affinity difference. We propose that Apterous controls expression of surface proteins that confer an affinity difference in conjunction with activated Notch. Thus, we view Apterous as instructive and Notch activity as essential, but permissive.     

Apical ectodermal ridge induction by the transplantation of En-1-overexpressing ectoderm in chick limb bud

In the early chick embryo, the dorsal–ventral (DV) boundary organizes the apical ectodermal ridge (AER) structure in the limb bud field. Here it is reported that Engrailed-1 ( En-1 ), a homolog of the Drosophila segment polarity gene engrailed expressed in the ventral limb ectoderm, participates in AER formation at the DV boundary of the limb bud. Restricted ectopic expression of En-1 in the dorsal side of the limb bud by transplantation of En-1 -overexpressing ectoderm induces ectopic AER at the boundary of En-1 -positive and -negative cells. The results suggest that En-1 is involved in AER formation at the DV boundary of the limb bud.     

Lobe mediates Notch signaling to control domain-specific growth in the Drosophila eye disc

Notch (N) activation at the dorsoventral (DV) boundary of the Drosophila eye is required for early eye primordium growth. Despite the apparent DV mirror symmetry, some mutations cause a preferential loss of the ventral domain, suggesting that the growth of individual domains is asymmetrically regulated. We show that the Lobe (L) gene is required non-autonomously for ventral growth but not dorsal growth, and that it mediates the proliferative effect of midline N signaling in a ventral-specific manner. L encodes a novel protein with a conserved domain. Loss of L suppresses the overproliferation phenotype of constitutive N activation in the ventral, but not in the dorsal eye, and gain of L rescues ventral tissue loss in N mutant background. Furthermore, L is necessary and sufficient for the ventral expression of a N ligand, Serrate (Ser), which affects ventral growth. Our data suggest that the control of ventral Ser expression by L represents a molecular mechanism that governs asymmetrical eye growth.     

Role of Notch pathway in terminal follicle cell differentiation during Drosophila oogenesis

 During Drosophila oogenesis the body axes are determined by signaling between the oocyte and the somatic follicle cells that surround the egg chamber. A key event in the establishment of oocyte anterior-posterior polarity is the differential patterning of the follicle cell epithelium along the anterior-posterior axis. Both the Notch and epithelial growth factor (EGF) receptor pathways are required for this patterning. To understand how these pathways act in the process we have analyzed markers for anterior and posterior follicle cells accompanying constitutive activation of the EGF receptor, loss of Notch function, and ectopic expression of Delta. We find that a constitutively active EGF receptor can induce posterior fate in anterior but not in lateral follicle cells, showing that the EGF receptor pathway can act only on predetermined terminal cells. Furthermore, Notch function is required at both termini for appropriate expression of anterior and posterior markers, while loss of both the EGF receptor and Notch pathways mimic the Notch loss-of-function phenotype. Ectopic expression of the Notch ligand, Delta, disturbs EGF receptor dependent posterior follicle cell differentiation and anterior-posterior polarity of the oocyte. Our data are consistent with a model in which the Notch pathway is required for early follicle cell differentiation at both termini, but is then repressed at the posterior for proper determination of the posterior follicle cells by the EGF receptor pathway. Received: 5 November 1998 / Accepted: 14 December 1998     

Notch and Wingless modulate the response of cells to Hedgehog signalling in the Drosophila wing

During Drosophila wing development, Hedgehog (Hh) signalling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signalling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. Here, we describe a functional hierarchy of these signalling pathways that highlights the importance of competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc revealed that collier/knot (col/kn), patched (ptc), and decapentaplegic (dpp) are repressed at the DV boundary by N signalling. Attenuation of Hh signalling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. We show that this down-regulation depends on activity of the canonical Wg signalling pathway. We propose that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. Our findings suggest a paradigm of repression of the Hh response by N and/or Wnt signalling that may be applicable to signal integration in vertebrate appendages.     

Notch signaling controls proliferation through cell-autonomous and non-autonomous mechanisms in the Drosophila eye

During Drosophila eye development, localized Notch signaling at the dorsal ventral (DV)-midline promotes growth of the entire eye field. This long-range action of Notch signaling may be mediated through the diffusible ligand of the Jak/STAT pathway, Unpaired (Upd), which was recently identified as a downstream target of Notch. However, Notch activity has not been shown to be cell-autonomously required for Upd expression and therefore yet another diffusible signal may be required for Notch activation of Upd. Our results clarify the Notch requirement, demonstrating that Notch activity at the DV-midline leads to cell-autonomous expression of Upd as monitored in loss and gain-of-function Notch clones. In addition, mutations in the Jak/STAT pathway interact genetically with the Notch pathway by suppressing Notch mediated overgrowth. N(act) clones show non-autonomous effects on the cell cycle anterior to the furrow, indicating function of the Jak/STAT pathway. However, cell-autonomous effects of Notch within and posterior to the furrow are independent of Upd. Here, Notch autonomously maintains cells in a proliferative state and blocks photoreceptor differentiation.     

Control of planar cell polarity by interaction of DWnt4 and four-jointed

The Drosophila eye and the wing display specific planar cell polarity. Although Frizzled (Fz) signaling has been implicated in the establishment of ommatidial and wing hair polarity, evidence for the Wnt gene function has been limited. Here we examined the function of a Drosophila homolog of Wnt4 (DWnt4) in the control of planar polarity. We show that DWnt4 mRNA and protein are preferentially expressed in the ventral region of eye disc. DWnt4 mutant eyes show polarity reversals mostly in the ventral domain, consistent with the ventral expression of DWnt4. Ectopic expression of DWnt4 in the dorsoventral (DV) polar margins is insufficient to induce ommatidial polarity but becomes inductive when coexpressed with Four-jointed (Fj). Similarly, DWnt4 and Fj result in synergistic induction of hair polarity toward the source of expression in the wing. Consistent with genetic interaction, we provide evidence for direct interaction of DWnt4 and Fj transmembrane protein. The extracellular domain of Fj is required for direct binding to DWnt4 and for the induction of hair polarity. In contrast to the synergy between DWnt4 and Fj, DWnt4 antagonizes the polarizing effect of Fz. Our results suggest that DWnt4 is involved in ommatidial polarity signaling in the ventral region of the eye and its function is mediated by interacting with Fj.     

The hippo pathway promotes Notch signaling in regulation of cell differentiation, proliferation, and oocyte polarity

Specification of the anterior-posterior axis in Drosophila oocytes requires proper communication between the germ-line cells and the somatically derived follicular epithelial cells. Multiple signaling pathways, including Notch, contribute to oocyte polarity formation by controlling the temporal and spatial pattern of follicle cell differentiation and proliferation. Here we show that the newly identified Hippo tumor-suppressor pathway plays a crucial role in the posterior follicle cells in the regulation of oocyte polarity. Disruption of the Hippo pathway, including major components Hippo, Salvador, and Warts, results in aberrant follicle-cell differentiation and proliferation and dramatic disruption of the oocyte anterior-posterior axis. These phenotypes are related to defective Notch signaling in follicle cells, because misexpression of a constitutively active form of Notch alleviates the oocyte polarity defects. We also find that follicle cells defective in Hippo signaling accumulate the Notch receptor and display defects in endocytosis markers. Our findings suggest that the interaction between Hippo and classic developmental pathways such as Notch is critical to spatial and temporal regulation of differentiation and proliferation and is essential for development of the body axes in Drosophila.     

Left-right asymmetry in the alimentary canal of the Drosophila embryo

In many animal groups, left-right (LR) asymmetry within the body is observed. The left and right sides of the body are generally defined with reference to the anterior-posterior (AP) and dorsal-ventral (DV) axes. In this study, we investigated whether LR asymmetry is solely dependent on the AP and DV polarities in Drosophila embryos. We focused on the proventriculus, a posterior part of the foregut, and the hindgut because LR asymmetry in these body parts is highly stable in normal embryos. In embryos with a fully reversed AP polarity, LR asymmetry in both the proventriculus and the hindgut was re-oriented in relation to the reversed AP polarity. This demonstrates that inversion of AP polarity does not affect LR asymmetry of these tissues, and implies that LR asymmetry is specified in relation to the AP and DV polarities. Our findings were not consistent with the alternative hypothesis that LR asymmetry is predetermined by maternal signals that localize asymmetrically along the LR axis in the oocyte and/or early embryo.