pointed

DEVELOPMENTAL BIOLOGY

Embryonic

The first PTD transcripts can be detected during cellular blastoderm stage in two broad stripes in the lateral neurogenic region. As gastrulation [Image] proceeds, P1 transcription is progessively restricted to a row of ectodermal cells, at first three cells wide, then one cell wide. These cells belong to the tracheal anlagen. Additional P1 transcripts are detected in the head region.

From stage 12 through 14, pointed P1 expression is seen in longitudinal glia cells (Klambt, 1993). P2 transcripts are present in the precellular stage at the anterior tip of the embryo, but later, P2 transcripts are located mostly in the mesoderm. At stage 12, P2-driven pointed expression is shut off in the mesoderm and turned on at the midline of the CNS, corresponding to midline glia (Klambt, 1993).

Ectopically expressed pointed P1 forces additional CNS cells to enter the glial-differentiated pathway. Additional glial-like cells are often flanked by cells ectopically expressing neuronal antigen, that is, glial cells are able to induce neuronal antigens in neighboring cells (Klaes, 1994).

The transcription factors encoding genes tailless (tll), atonal (ato), sine oculis (so), eyeless (ey) and eyes absent (eya), and Efgr signaling play a role in establishing the Drosophila embryonic visual system. The embryonic visual system consists of the optic lobe primordium, which, during later larval life, develops into the prominent optic lobe neuropiles, and the larval photoreceptor (Bolwig's organ). Both structures derive from a neurectodermal placode in the embryonic head. Expression of tll is normally confined to the optic lobe primordium, whereas ato appears in a subset of Bolwigís organ cells that are called Bolwigís organ founders. Phenotypic analysis of tll loss- and gain-of-function mutant embryos using specific markers for Bolwigís organ and the optic lobe, reveals that tll functions to drive cells to an optic lobe fate, as opposed to a Bolwigís organ fate. Similar experiments indicate that ato has the opposite effect, namely driving cells to a Bolwigís organ fate. Since tll and ato do not regulate one another, a model is proposed wherein tll expression restricts the ability of cells to respond to signaling arising from ato-expressing Bolwigís organ pioneers. The data further suggest that the Bolwigís organ founder cells produce Spitz (the Drosophila TGFalpha homolog) signal, which is passed to the neighboring secondary Bolwigís organ cells where it activates the Epidermal growth factor receptor signaling cascade and maintains the fate of these secondary cells. The regulators of tll expression in the embryonic visual system remain elusive, no evidence for regulation by the 'early eye genes' so, eya and ey, or by Egfr signaling is found (Daniel, 1999).

Epidermal growth factor receptor is activated in midline regions of the head neurectoderm, in particular in the anlage of the visual system. Moreover, increased and/or ectopic activation of Egfr results in a 'cyclops' phenotype very similar to what has been described for ectopic tll expression. Egfr signaling has been shown to be required in both chordotonal organs and compound eye for the inductive signaling triggered by ato expression. Two questions raised by these observations have been investigated: (1) is Egfr signaling required for tll expression in the optic lobe and (2) is Egfr signaling involved in the recruitment of the secondary (non-atonal-expressing) Bolwigís organ cells? The answer to both these questions is no. tll expression is unaltered when levels of Egfr signaling are manipulated, suggesting that Egfr signaling is not required for tll expression. To investigate the second question, a test was performed for the presence of Egfr-relevant mRNAs or proteins: Rhomboid mRNA, which would be expected to be present only in the signaling cells, and phosphorylated MAPK, Pointed and Argos mRNAs, which would be expected to be expressed in all cells receiving an Egfr-mediated signal. In stage 12 embryos, rho is expressed only in the small group of Bolwigís organ founder cells (the same cells expressing ato). In contrast, activated (phosphorylated) MAPK is present in a larger cluster of cells including the entire Bolwigís organ and part of the adjacent optic lobe. Consistent with this, pnt and aos, both known to be switched on in cells receiving the Spi signal, are expressed at the same stage throughout the entire Bolwigís organ primordium. These gene expression and MAPK activation patterns are consistent with the idea that the Spi signal is activated by rho in the Bolwigís organ founders and passed to the neighboring secondary Bolwigís organ cells where it activates the Egfr signaling cascade. Supporting this view, only 3-4 photoreceptor neurons are found in the Bolwigís organ of embryos lacking rho or spi; furthermore, the size of the posterior lip of the optic lobe is reduced in such embryos. The fact that absence of secondary Bolwigís organ cells in rho or spi mutant embryos can be rescued by blocking cell death in the background of a deficiency that takes out the reaper complex of genes indicates that the Spi signal is not necessary for the specification (recruitment) of secondary Bolwigís organ cells, but rather, for their maintenance (Daniel, 1999).

Fgf signaling requires Ras, the Mapk pathway, and Pointed to direct tracheal migration

Branching morphogenesis is a widespread mechanism used to increase the surface area of epithelial organs. Many signaling systems steer development of branched organs, but it is still unclear which cellular processes are regulated by the different pathways. The development of the air sacs of the dorsal thorax of Drosophila was used to study cellular events and their regulation via cell-cell signaling. Two receptor tyrosine kinases play important but distinct roles in air sac outgrowth. Fgf signaling directs cell migration at the tip of the structure, while Egf signaling is instrumental for cell division and cell survival in the growing epithelial structure. Interestingly, Fgf signaling requires Ras, the Mapk pathway, and Pointed to direct migration, suggesting that both cytoskeletal and nuclear events are downstream of receptor activation. Ras and the Mapk pathway are also needed for Egf-regulated cell division/survival, but Pointed is dispensable (Cabernard, 2005).

The air sac of the dorsal thorax grows from a bud that arises during the third larval instar from a wing disc-associated tracheal branch. To illustrate the development of the air sac, a GFP trap line was used that rather ubiquitously expressed membrane bound GFP; tracheal cells were counterstained with an mRFP1-moesin construct under the direct control of the trachea-specific breathless (btl) enhancer. From the early to late third instar stage, a bud-like structure grows out of the transverse connective and spreads on the wing imaginal disc epithelium; this outgrowth corresponds to the primordium of the air sac of the dorsal thorax (Cabernard, 2005).

It has been proposed that the air sac of the dorsal thorax forms de novo from a small group of wing imaginal disc cells, and that the resulting sac subsequently generates a tracheal lumen by an unknown process (Sato, 2002). Since, in the early Drosophila embryo, the lumen arises from an epithelial invagination via cell migration, it was asked whether the cells in the growing air sac are epithelial in nature with a clear apical/basal polarity. For this purpose, a Dα-Catenin-GFP (Dα-Cat-GFP) fusion construct was expressed in the developing air sac and the distribution of GFP from early to late third instar larvae was analyzed. Dα-Cat-GFP labels the adherens junctions (AJs) of epithelial cells. Clearly, the growth of the air sac was accompanied from the early stages onward by an out-bulging of an AJ network, suggesting that most or all of the cells in the growing bud were epithelial in nature, and that a luminal space was generated at the apical side of the epithelial tracheal cells during outgrowth. To confirm this interpretation, use was made of the recent identification of a protein, Piopio (Pio), which is apically secreted into the tracheal lumen in the embryo (Jazwinska, 2003). Indeed, the prospective luminal space in the outgrowing air sac is filled with Pio protein, demonstrating that the air sacs consist of a sac-like epithelial sheet, generating a luminal space as they grow (Cabernard, 2005).

To test whether all cells maintained an apical-basal polarity during air sac budding, single tracheal cells were labelled by using a recently developed assay system that allows for the visualization (and manipulation) of individual tracheal cells in vivo (Ribeiro, 2004). When this scenario was used in the presence of a UAS-Dα-Catenin-GFP chromosome, it was found that, in virtually all cases, such individually labeled air sac cells contacted the lumen and formed AJs with neighboring cells, even when these cells were located at the tip of the outgrowing air sac. The same conclusion was reached when the expression of GFP-moesin was analyzed in single air sac cells; cells at the tip made clear contact with the lumen. Therefore, it is concluded that the air sac is sculpted from an epithelial cell layer, which expands and at the same time generates an apical luminal space filled with secreted proteins (Cabernard, 2005).

It was of interest to better understand how Ras can be used in the same tissue at the same time for different cellular processes. egfr mutant cells can contribute to the tip of the growing air sac, although the clones are relatively small. In the stalk of the air sac, cells lacking Egfr often appeared fragmented, a sign of cell death. Indeed, when egfr mutant cells were sustained with anti-Drice, a marker for apoptotic cells, a strong accumulation of this protein was found. When p35, a viral antiapoptotic protein, was expressed in egfr mutant cell clones, these clones grew to larger sizes and were able to populate the air sac tip at a significantly higher frequency than in the absence of p35. These experiments establish that Egfr is dispensable for migration, and that migration is exclusively triggered by one of the two RTKs, Btl/Fgfr. The experiments also demonstrate that, during the growth phase, Btl/Fgfr signaling is dispensable for cell division; clones can grow to large sizes, although they fail to populate the tip. This same result was obtained with two other components, which are exclusively used by the Fgfr signaling pathway in the air sac (and not the Egfr pathway), namely, Dof and Pointed. Thus, migration and cell division are controlled by two different RTKs, but both RTKs signal via the activation of Ras and the Map kinase pathway to regulate these different cellular outcomes (Cabernard, 2005).

How does Ras control cell migration in the tip and cell division in the remaining air sac? To start to address these questions, whether high levels of constitutive active Ras were compatible with directional cell migration was tested and RasV12 was expressed in wild-type tissue in small cell clones. Interestingly, such clones expanded considerably and grew to large sizes in the center of the air sac or in the stalk, resulting in bulgy outgrowths; however, clones expressing RasV12 never contributed to the tip of the air sac. This finding suggests that unrestricted levels of Ras in a cell perturb its capacity to read out the migratory cues (presumably the Bnl/Fgf ligand); wild-type cells were apparently much better in taking up the leading position. In line with this interpretation, expression of an activated version of Btl (Torso-Btl/Fgfr) also resulted in bulky outgrowths. In addition, cells expressing the chimeric Btl receptor never populated the tip. Quite in contrast, activated Egfr (Egfr fused to a lambda dimerization site) was not able to perturb air sac guidance, but it also triggered higher division rates in clones, generating bulgy outgrowths (Cabernard, 2005).

To test whether single cells expressing activated receptor constructs changed their behavior with regard to cytoskeletal dynamics, the expression of either the activated version of Fgfr or Egfr was induced in early third instar stages and the behavior of such cells was analyzed with live imaging of cultured discs. Cells in the stalk of the air sac expressing activated Fgfr showed extremely dynamic cytoskeletal activity and formed large lamelipodia extending away from the air sac, similar to cells at the tip. Quite in contrast, cells expressing activated Egfr did not show increased lamelipodia formation, and their basal side remained relatively inactive (Cabernard, 2005).

Since the expression of constitutive active versions of the two different RTKs during air sac growth had different effects, whether the endogenous receptors activated the Ras/Mapk pathway to different levels in wild-type air sacs was investigated. In order to monitor the strength of Mapk signaling, an antibody recognizing the double-phosphorylated form of Erk, dpErk were used. Indeed, high levels of dpErk was detected in the nucleus of tip cells; lower dpErk levels were found in the cells in the center of the air sac, and dpErk was mostly cytoplasmic (Cabernard, 2005).

From all of the above-mentioned data, it is concluded that air sac development makes use of two distinct RTKs to control directed organ extension via cell migration (Fgfr) and organ growth via cell division (Egfr). This study carefully analyzed air sac outgrowth from early to late stages, by using a number of different markers labeling either membranes or AJs of individual air sac cells, or the apical luminal compartment. It was found that the thoracic air sac is modeled out of the existing tracheal epithelium, and that a luminal space is generated by the migration of a few cells away from the cuticle of the existing tracheal branch; the luminal space is then expanded by increasing the cell number in the sac-like epithelial structure via cell division. During this process, all cells remain within the epithelium and only round up when they divide. Even those cells that send out filopodia and lamelipodia and migrate in the direction of Bnl/Fgf remain embedded within the epithelium, contact the lumen, and form AJs with their neighbors. Thus, the directed outgrowth of the thoracic air sac during larval development is very similar to the budding of tracheal branches in the early embryo, in that epithelial cells form extensions from the basal side, ultimately resulting in cell movement toward the Fgf ligand. In contrast, during tubule formation of MDCK cells in culture, cells initially depolarize and migrate to form chain-like structures before they repolarize and form the luminal cavity; tubulogenesis is thus accompanied with partial epithelial-to-mesenchymal as well as mesenchymal-to-epithelial transitions. The tube-forming process has been subdivided into different stages such as cyst, extension, chain, cord, and tubule. In the case of the MDCK model system, growth factors have been proposed to trigger branching by inducing a dedifferentiation that allows the monolayer to be remodeled via cell extension and chain formation. Similar to the MDCK system, it was found in Drosophila that growth factor signaling induces the formation of cellular extensions, the first sign of outgrowth. Also, in both systems, cell division is an integral part of the process, but it occurs randomly throughout the structure and not locally at the point of outgrowth. However, two different RTKs are used in the air sac to control extension (migration) and cell division, and chain and chord stages are not observed. It thus appears that both similarities and differences exist between these different cellular systems (Cabernard, 2005).

It has already been reported that cells divide during air sac formation. The cell division rates have been semiquantified and it was found that the elongating structure does not grow preferentially at the tip. The genetic analysis demonstrates that the Egfr is essential for cells to divide and survive efficiently in the air sac. Egfr signals via Ras and the Mapk pathway, but it does not require the Pnt transcription factor to regulate cell division. It is not yet known which ligand activates Egfr, and whether expression of this ligand is induced at early stages of development by Fgf signaling. As shown before (Sato, 2002), the complete lack of Fgfr signaling results in the absence of air sacs; Fgf signaling might thus be used at the onset of the budding process to initiate or trigger cell division, but it is clearly dispensable in later stages. Since cells in the tracheal branch, which gives rise to the air sac primordium, also divide in the absence of Fgf signaling, it is possible that the role of Fgf signaling consists in generating an outgrowth via directed cell movement, triggering cell division indirectly (Cabernard, 2005).

Interestingly, a recent study addressing the role of GDNF/Ret signaling in kidney branching morphogenesis in vivo has shown that ret mutant cells (which are unable to respond to GDNF) can contribute to the primary outgrowth of the ureteric bud, but are excluded from the ampulla that forms at its tip. Apparently, a Ret-dependent proliferation of tip cells under the influence of GDNF controls branch outgrowth. This study found that in Drosophila, in the developing air sac, cells lacking Fgfr are also excluded from the tip. However, evidence is provided that Fgf signaling is translated into directed migration in the leading structure and not into a local increase in cell proliferation. The isolation and cultivation of wing imaginal discs allows for using 4D imaging to document cell behavior during air sac growth. It was found that numerous tip cells extend long filopodia and lamelipodia, similar to the findings reported earlier (Sato, 2002). Tip cells not only produce extensions, but they indeed change their respective position with time, and move forward over the substrate in the direction of the filopodia/lamelipodia. Thus, tip cells are clearly motile and migrate in the direction of Bnl/Fgf. Cell clones incapable of responding to different families of ligands were produced and marked and were examined with regard to their capacity to populate the air sac tip. Among the receptors analyzed, only Btl/Fgfr was strictly required for cells to populate the leading tip of the air sac. Considering the observation that cells in the tip actively migrate, that Btl/Fgfr signaling is required for tracheal cell migration in the embryo, that tracheal cells migrate to ectopic sources of Bnl/Fgf in the embryo and the larva (Sato, 2002), and that cells form numerous filopodia and lamelipodia upon constitutive activation of the Fgf signaling pathway, it is concluded that Fgf steers cell migration in the tip of the air sac and leads to its directional outgrowth on the surface of the wing imaginal disc. The demonstration that the MARCM system can be used to analyze gene function with regard to cell migration in the developing air sac prompted an investigation of the role of Ras and the Mapk pathway in Fgf-directed cell movement (Cabernard, 2005).

Using the MARCM system, it was found that Ras activation is essential for cells to migrate at the tip of the air sac. The requirement for Cnk and Ksr strongly suggests that one important branch downstream of Ras in the control of cell migration is the Mapk pathway. This interpretation is supported by the somewhat surprising finding that the transcription factor Pnt is also strictly required for cell migration. In the Drosophila embryo, genes regulated by Fgf signaling at the transcriptional level and essential for migration have not been identified so far; although both pnt itself and blistered/DSrf are targets of Fgf signaling with important functions in tracheal morphogenesis, they are not required for migration. One possible target of Fgf signaling in the dorsal air sac cells might be the btl/fgfr gene itself. Attempts were made to rescue the pointed defects in air sac development by supplementing a btl transgene under the control of UAS sequences. It was found that even when Btl/Fgfr is provided by the transgenes, pnt mutant clones do not reach the tip. A second gene that might have been a transcriptional target of Pointed is dof; however, it was found that Dof protein is still present in pnt mutant clones (Cabernard, 2005).

The results demonstrate that the outgrowth of the dorsal air sac along the underlying wing imaginal disc is controlled by Btl/Fgfr and Egfr. Fgf signaling is required for directional outgrowth via cell migration, and Egf signaling is required for organ size increase sustaining cell division/cell survival. Both signals use the Ras/Mapk pathway to elicit their cellular responses. To what extent these two pathways regulate different downstream targets is not known at present. However, this study shows that Pointed is only required downstream of Fgf signaling in the control of directed cell migration, and not downstream of Egf signaling in the control of cell division/survival. Since the activation of the Map kinase pathway is much stronger in the cells at the tip as compared to the cells in the central portion or in the stalk of the air sac (according to the levels of dpErk), it is thought that the local availability of the Bnl/Fgf ligand results in a local signaling peak. Egf signaling in more central and proximal cells does not result in a strong activation of the Map kinase pathway, yet this activation is apparently sufficient to control cell division and survival. The independent regulation of cell migration and cell division by two different RTKs might be even more important in later stages of dorsal air sac development, when the growing tip is yet farther away from the main body of the air sac. It will be interesting to find whether other growing branched tissues use similar mechanisms to uncouple directional expansion and size increase (Cabernard, 2005).

Larval

In the differentiation of photoreceptors in the eye imaginal disc, pointed is activated by MAP Kinase through the sevenless pathway. The signal for the Sevenless receptor is Bride of sevenless.