hibris is regulated by Notch and Ras in a Toll10b mutant background. This regulation was confirmed in vivo in wild-type embryos. hbs expression was examined in Notch and Ras loss-of-function embryos and embryos overexpressing activated forms of Notch and Ras in the mesoderm. A dominant negative Ras construct activates hbs expression in the somatic mesoderm. Zygotic null Notch embryos show lower hbs transcription. Conversely, an activated form of Notch upregulates hbs in the mesoderm, while an activated form of Ras almost completely inhibits hbs expression. These results argue that, upon stimulation, Notch activates hbs, while Ras acts as a negative signal, and predicts that hbs expression in the somatic mesoderm would be restricted to fusion-competent cells (Notch dependent) and excluded from founder cells (Ras dependent). It is not known whether this regulation is direct, that is, Notch or Ras effectors act directly on the hbs promoter, or indirect, that is, Notch/Ras converts cell fate, which in turn would lead to hbs upregulation/downregulation by some other effector (Artero, 2001).
hbs expression in the mesectoderm and developing CNS midline partially overlaps with the expression of the transcription factor single minded (sim). In sim embryos, the mesectodermal progeny survive but fail to differentiate or migrate to appropriate locations. In sim mutant embryos, hbs expression is abolished at the CNS midline. When sim was misexpressed in all neuroblasts with the sca-GAL4 driver, the domain of hbs expression at the CNS midline was expanded. Yet when sim was misexpressed in all post-mitotic neurons using elav-GAL4 drivers hbs expression was unaltered (Dworak, 2001).
Notch is pivotal in the development of multiple tissue types. At the developing ventral midline, Notch activity is essential for establishing sim expression. Consequently, in NXK11 mutant embryos hbs expression is also lost at the CNS midline at stage 12 and onwards, after depletion of the Notch maternal contribution. In addition, Notch is crucial for the development of fusion competent myoblasts. In NXK11 mutant embryos, where myoblasts are transformed to founder cells, hbs expression is absent in the mesoderm. An examination was made to determine whether hbs is downstream of two mesoderm-specific transcription factors, bap and mef2. hbs expression in visceral mesoderm is greatly decreased in bap mutant embryos, but unaffected in the mef2 mutant embryos (Dworak, 2001).
Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophila embryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. This approach was validated by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Strong evidence is presented showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo (Solano, 2003).
the expression of 14 genes was studied that are localized close to the genomic DNA fragments isolated in the library and tested previously for their Engrailed-specific binding ability. The results are shown for four genes (frizzled2, hibris, branchless, frazzled) that are representative of the different pathways where engrailed seems to be involved. frizzled 2 expression is activated in the presence of (VP16-En) and repressed in the presence of En. This suggests that engrailed might act as a repressor on fz2 expression. hibris is expressed along the wing margin and in the presumptive region of wing vein L3 and L4 in wild type. This expression is slightly activated in the presence of (VP16-En), but strongly repressed when En is overexpressed, suggesting that hbs expression is regulated by engrailed in vivo. branchless is essentially expressed in a dorsal/posterior territory surrounding the wing pouch in wild type. In the presence of (VP16-En), several additional patches of bnl expression are detected within the wing pouch, whereas no activation of bnl is observed after wild type En overexpression. As expected, because MS1096 drives Gal4 expression only in the wing pouch, endogenous bnl expression outside the wing pouch is not affected, showing the specificity of the experiment. Finally, frazzled is slightly expressed in wild-type wing disc. This expression is activated when (VP16-En) is overexpressed, and repressed upon En overexpression (Solano, 2003).
Proteins belonging to the Ig superfamily are frequently implicated in cell-cell adhesion. The ability of Hbs, Sns, Kirre, IrreC-Rst and Sidestep (Side) to bind homotypically was tested with the S2 cell aggregation assay. As a negative control, S2 cells were transfected with RmHa3 vector, and as a positive control, S2 cells were transfected with Fasciclin II-RmHa3. Homotypic aggregation was observed for Fasciclin II and Kirre. To test for heterotypic interactions, the S2 cells were labeled with either DiI (red) or DiO (green), and the aggregates were examined using confocal microscopy. When Fasciclin II-transfected cells (red) were mixed with RmHa3-transfected cells (green), all aggregates formed contained only red cells. Similarly, when Kirre-transfected cells (red) were mixed with RmHa3-transfected cells (green), aggregates were again all comprised of only red cells. When Kirre-transfected cells (red) were mixed with Hbs- or Sns-transfected cells (green), the resultant aggregates all had both red and green cells, but when Kirre-transfected cells (red) were mixed with RmaHa3- or Irrec-transfected (green) cells, all the resultant aggregates contained only red fluorescent cells. This is the first evidence suggesting that Nephrin proteins interact heterophilically in trans with other potentially non-Nephrin extracellular partners (Dworak, 2001).
Cell adhesion is essential for morphogenesis; however, the mechanisms by which cell adhesion coordinates precisely regulate morphogenesis are poorly understood. This study analyzes the morphogenetic processes that organize the interommatidial precursor cells (IPCs) of the Drosophila pupal eye. The Drosophila immunoglobulin superfamily members Hibris and Roughest are essential for IPC morphogenesis in the eye. The two loci are expressed in complementary cell types, and Hibris and Roughest proteins bind directly in vivo. Primary pigment cells employ Hibris to function as organizers in this process; IPCs minimize contacts with neighboring IPCs and utilize Roughest to maximize contacts with primaries. In addition, evidence is provided that interactions between Hibris and Roughest promote junction formation and that levels of Roughest in individual cells determine their capacity for competition. These results demonstrate that preferential adhesion mediated by heterophilic interacting cell-adhesion molecules can create a precise pattern by minimizing surface free energy (Bao, 2005).
To properly organize the ommatidia into a precise pattern, the interommatidial precursor cells (IPCs) undergo dynamic cell rearrangements between 18 and 42 hr after puparium formation (APF). These cells will eventually differentiate as secondary and tertiary pigment cells (2ºs, 3ºs) and mechanosensory bristles. Emergence of the interommatidial lattice was further analyzed with an antibody to the β-catenin ortholog Armadillo (Arm), a core component of the adherens junction. Based on this work, IPC and ommatidial patterning was classified into four stages (hours are based on the approximate center of the eye field), which are briefly described (Bao, 2005).
(1) Initial cell sorting (18-24 hr APF). Initially, IPCs are scattered between ommatidia with a relaxed apical profile. As development progresses, two cells emerge from the IPC pool to enwrap the cone cells and become 1ºs; the remaining IPCs simultaneously line up in single file to contact 1ºs from adjacent ommatidia. Concurrently, some cells are removed by apoptosis (Bao, 2005).
(2) Emergence of 3ºs (24-27 hr APF). Typically, three cells are initially positioned equally at a vertex. One cell reaches past the other two to contact a third 1º ; this cell will then physically “invade” the vertex and mature as a 3º (Bao, 2005).
(3) Selection of 2º s (27-36 hr APF). Cells that fail to become 3ºs either become 2ºs or are removed by programmed cell death. During this final cell-fate decision, cell-cell adhesion becomes visibly polarized as IPCs form detectable junctional contacts with 1º s but not with other IPCs. In addition, a 'scalloping' of membrane profiles is observed as 1ºs push between IPCs, further confirming that the adhesion between 1ºs and IPCs is greater than between neighboring IPCs. By 36 hr APF, the hexagonal pattern is essentially complete: it is composed of a single 2º at each side and a 3º or bristle organule at each vertex (Bao, 2005).
(4) Maturation (36-42 hr APF). Visible adherens junctions return to the interfaces between IPCs (now 2ºs and 3ºs). Contacts are now smoothed as the scalloping caused by invasive 1º contacts is now relaxed (Bao, 2005).
One particularly striking feature of this morphogenetic process is the dynamic nature of the cell junctions, which were visualized with the junctional protein Arm. For example, the level of Arm in the cone cells was constant but the levels of Arm in the IPCs decreased: this was seen by comparing the levels of Arm in the two cell groups. This drop in Arm levels is followed by its complete loss between IPCs after 3ºs emerge and eventual reemergence at the final maturation stage to levels similar to cone cells. Thus, junctions appear to be diminished during the period of maximal cell rearrangement, suggesting that IPCs are free to move during these stages (Bao, 2005).
Using laser ablation studies, it has been demonstrated that 1ºs are centrally important for the process of organizing IPCs into a correctly patterned interommatidial lattice. However, the mechanism by which one cell can provide such remarkably precise patterning information to a larger collection of uncommitted cells has not been not clear. The dynamic interactions between Hibris and Roughest provide such a mechanism (Bao, 2005).
The 'differential adhesion hypothesis' (DAH) proposes that sorting-out and segregation of cell populations are driven by differences in the intensities of cell adhesions. Given motile and cohesive cell populations, DAH predicts that weakly cohesive cells will tend to be displaced by more strongly cohesive ones; this process can direct cells to segregate away from unlike cell populations, and it can control tissue spreading during, for example, germ layer maturation in the embryo. DAH has been supported by several observations. For example, quantitative differences in the level of cadherin expression can lead two cell populations to be mutually immiscible: less cohesive cells will envelope more cohesive ones, creating a 'sphere within a sphere' configuration. Recently, the importance of differential adhesion for patterning developing tissue has been demonstrated in the pupal retina. Cone cells segregate from other cells and assemble into a simple pattern by minimizing surface area, as do soap bubbles. This assembly is mediated at least in part by E- and N-cadherins, and manipulating cadherin levels within the cone cells or their neighbors can alter the final cone cell pattern. These experiments illustrate that differential adhesion caused by differences in cadherin expression can mediate morphogenesis and pattern formation (Bao, 2005).
The current data indicate that IPC patterning follows a mechanism that shows unique aspects when compared with these classical DAH experiments. First, manipulating E-cadherin levels does not alter the morphogenesis or arrangement of IPCs. Even when two neighboring IPCs have higher levels of E-cadherin, adhesion between these two IPCs or their final patterning is not affected. More critically, IPCs do not aggregate together or segregate away from their neighbors. Rather, they separate away from each other to minimize IPC:IPC contacts, and aggregate with ommatidial cores to maximize 1º :IPC contacts. That is, the data indicate that IPCs have a preference for adherence to 1ºs. This preference can be seen most clearly at 27 hr APF: the junctions between IPCs and 1ºs are strong and elaborate; the junctions between IPCs are indistinct, and 1ºs are seen to push between IPCs to maximize contact and create a scalloping effect. The result is the precise aggregation of two different cell populations (Bao, 2005).
Why do IPCs sort away from other IPCs and preferentially adhere to 1ºs? The data indicate that interactions between Hibris and Roughest provide the mechanism. The immunoglobulin-class proteins Roughest and Hibris are utilized by IPCs and 1ºs, respectively, to form heterophilic interactions. Several lines of evidence support this view: (1) both Hibris and Roughest are required for proper interommatidial lattice assembly; (2) hibris is expressed in 1ºs as well as in cone cells and roughest is expressed in IPCs at the time of IPC rearrangement in the eye; (3) expression of ectopic Hibris in either the 1º or IPC is sufficient to relocalize Roughest protein -- conversely, downregulation of Hibris in 1ºs leads to decreased levels of Roughest protein at the 1º :IPC interface; (4) Hibris and Roughest are capable of directly binding each other when isolated in tissue culture experiments (Bao, 2005).
After 1ºs are specified and start to express Hibris, levels of Roughest protein decrease between IPCs and increase in the borders between IPCs and 1ºs; for example, at 30 hr APF, Roughest protein is undetectable between IPCs. Furthermore, ectopic Hibris in 1ºs is sufficient to attract still more Roughest protein toward the 1º :IPC border; by contrast, ectopic Roughest in 1ºs does not attract additional Roughest. It is concluded that although Roughest can show homophilic interactions in S2 cells, it strongly prefers heterophilic interactions with Hibris in situ (Bao, 2005).
Ubiquitous Hibris expression greatly increases the levels of cell-junction proteins between IPCs. Similarly, individual IPCs that received ectopic Hibris form E-cadherin-rich borders with neighboring IPCs that are sharp, straight, and significantly enlarged (Bao, 2005).
The evidence indicates that 1ºs and IPCs prefer to adhere to each other based on their expression of Hibris and Roughest, respectively. One principle of thermodynamics states that the binding of two adherent molecules will lead to a reduction of free energy within the system, provided the equilibrium constant of association (ka) is greater than the equilibrium constant of dissociation (kd). The essential role of Hibris and Roughest in IPC morphogenesis prompts making an assumption: among the various molecules being displayed in the surfaces of 1ºs and IPCs, Hibris and Roughest play a major role in determining the flow of free energy. Roughest has a higher affinity for Hibris than itself, and therefore heterophilic binding between Roughest and Hibris leads to a greater reduction in free energy. As a result, contacts between IPCs and 1ºs contribute to a reduction of free energy and are favored, while contacts between IPCs and IPCs do not contribute to reduction of free energy and are disfavored (Bao, 2005).
Other features of the developing pupal eye provide important components to this patterning process. After 1ºs are specified, they establish cell junctions with each other and with cone cells. These cone cell/1º units are not free to move within the epithelial plane and form a functional patterning unit. Therefore, 1ºs function as the organizers in this context. In contrast, IPCs have reduced levels of junctional proteins and are free to move within the epithelium. Numerous filopodia from IPCs observed by SEM studies also point to their potential for high motility. Taken together, these data suggest that IPC morphogenesis follows a preferential adhesion model: IPCs exhibit preferential adhesion to 1ºs; 1ºs function as organizers for IPC morphogenesis, and IPC:1º contacts are free energy favored while IPC:IPC contacts are disfavored (Bao, 2005).
The ommatidial clusters are poorly organized until 18 hr APF, when the morphogenetic movements of the IPCs begin to organize clusters into a hexagonal array. Preferential adhesion of IPCs to 1ºs yields two major outcomes. (1) IPCs compete to adhere directly to the limited, Hibris-rich surface presented by the 1ºs. High motility of IPCs permits this competition to proceed and achieve a favored configuration. (2) Preferential adhesion can also lead to the removal of cells that fail to contact a 1º . Specifically, IPCs that adhere to 1ºs have an increased chance to survive since the Hibris:Roughest interactions provide a greater opportunity to establish a stable junction. By the same token, those cells that do not have access to 1ºs are disadvantaged and are commonly dropped from the apical surface; these cells are likely to be eventually removed by programmed cell death. As a result, each stage proceeds with a progressive reduction of the IPC:IPC contacting surfaces and an increase in IPC:1º contacting surfaces (Bao, 2005).
At the onset of IPC morphogenesis (18 hr APF), the average size of IPC:IPC contacts is not significantly different from the size of IPC:1º contacts. During the time cells in multiple layers are sorted into single file after the initial cell-sorting stage (24 hr APF), IPC:IPC contacts are significantly reduced. After emergence of 3ºs, this reduction in IPC:IPC contacts is particularly dramatic. The IPC:1º contacts are increased by a scalloped profile, a further demonstration that IPC:IPC contacts are disfavored. To complete this pattern, therefore, IPC:IPC contacts are further minimized by reducing the number of candidate 2ºs to one cell between each 3º and bristle. Thus, IPC morphogenesis reveals a mechanism by which pattern is determined through minimizing disfavored cell-cell contacts and maximizing preferred cell-cell contacts (Bao, 2005).
Finally, it is interesting to note how 2ºs are selected. After emergence of 3ºs, two IPCs are commonly found between a 3º and bristle. In many ways, these two IPCs are equal: each contacts two 1ºs and each establishes equally strong cell junctions; each forms a scalloped contour with two neighboring 1ºs, and each is exposed to the same molecular cues. However, evidence is provided that these two cells have a low affinity for each other, a situation that is not favored by minimum free energy principles. One cell will be removed. How is this cell chosen? Clues came from manipulating levels of Roughest, which altered each cell's capacity for competition. Artificially high levels of Roughest rendered a cell a supercompetitor: the targeted cell even replaced two cells to become both a 2º and a 3º. Presumably, high levels of Roughest promote a higher level of cell junctions, which makes a cell more competitive and determines the survivor. Conversely, low levels of Roughest put the targeted cell at a disadvantage during this competition. Therefore, during the selection of a 2º , differing levels of Roughest expressed by each cell may determine its fate: survival or death (Bao, 2005).
Neph1/Nephrin family members are required for the development of a wide array of tissues including axonal pathfinding and myoblast fusion in Drosophila and formation of the slit diaphragm in the developing mammalian kidney. The role observed for preferential adhesion in IPC morphogenesis and patterning in the Drosophila eye leads to the interesting possibility that similar mechanisms are utilized broadly in pattern formation (Bao, 2005).
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