In an attempt to identify gene targets of ash2, an expression analysis was performed by using cDNA microarrays. Genes involved in cell cycle, cell proliferation, and cell adhesion are among these targets, and some of them are validated by functional and expression studies. Genes involved in cell adhesion and/or development of the neural system (i.e., FasII, mfas, Ama, Lac, and shg) are two of the main classes regulated by ash2. Even though trithorax proteins act by modulating chromatin structure at particular chromosomal locations, evidence of physical aggregation of ash2-regulated genes has not been found. This work represents the first microarray analysis of a trithorax-group gene (Beltran, 2003).
Lac shows closest similarity to members of the Ig superfamily belonging to the neuronal Ig cell adhesion molecule (IgCAM) class, which includes vertebrate L1, NCAM, TAG1, Contactin and IgLONs, and Drosophila Neuroglian, Wrapper and Klingon. Members of this family have been shown to engage in homophilic and/or heterophilic interactions to mediate cell adhesion, raising the possibility that the molecular mechanism by which Lac contributes to tracheal development could be based on its adhesive properties. In order to establish whether Lac also works as a homophilic cell adhesion molecule, its activity was tested in a bead aggregation assay. In this assay, chimaeric molecules consisting of the protein of interest and the Fc fragment of human IgG are coupled to polystyrene beads, and assayed for their ability to form aggregates. Whereas control beads do not aggregate, Lac-Fc coated beads form aggregates, showing that Lac can work as a homophilic cell adhesion molecule. This in vitro assay confirms the adhesion properties of the Lac protein suggested by the cell detachment phenotype observed in mutant tracheae (Llimargas, 2004).
Grasshopper Lachesin is expressed on the surfaces of differentiating neuronal cells from the onset of neurogenesis in both the central and peripheral nervous systems. Lachesin expression begins in some cells of the neurogenic ectoderm immediately after engrailed expression begins in the posterior cells of each future segment. All neurogenic cells express Lachesin early, but only those cells that become neuroblasts continue to express Lachesin. Ectodermal cells in the neurogenic region that adopt non-neuronal fates lose Lachesin at the time that they diverge from a potentially neurogenic pathway. Neuroblasts, ganglion mother cells and neurons all express Lachesin early in their lives, but expression becomes restricted to a subset of neurons as development progresses. Sensory neurons express Lachesin as they delaminate from the body wall ectoderm. Lachesin is also present on growing axons of the CNS and PNS and becomes restricted to a subset of axons later in development. This expression is unique among known insect neurogenic genes and suggests a role for Lachesin in early neuronal differentiation and axon outgrowth (Karlstrom, 1993).
The gene coding for the Drosophila Lac cell surface protein is expressed in a dynamic pattern including the developing trachea. Lac mRNA is first detected in the cellularized blastoderm, where it is excluded from the ventral side. Its expression persists in the early ectoderm, uniformly until completion of gastrulation, and with a peculiar segmental pattern consisting of an alternate strong-weak stripe in each segment from around stage 10, which progressively fades away and disappears by stage 15. Strong expression is detected in specific tissues such as the trachea (from early stage 13), hindgut, foregut and nervous system. In the nervous system Lac is detected in subsets of neurons starting from stage 11, and, later, in subsets of glia too, where it has been reported to be a target of glial cells missing (Egger, 2002; Freeman, 2003; Llimargas, 2004 and references therein).
The antibody raised against Lac was used to determine the subcellular localization of the protein. Protein distribution essentially recapitulates mRNA expression, although the protein appears to accumulate in the ectodermal derivatives until the end of embryogenesis, when Lac expression has already disappeared from most tissues. Interestingly, the Lac protein is not homogeneously distributed on the cell surface, but rather it accumulates in a lateral region. To precisely determine in which compartment Lac is localized, double-labelling experiments were carried out. Drosophila epithelial cells contain an adhesive belt, the ZA (zonula adherens), which encircles the whole cell just below the apical surface, and in which E-Cadherin and its associated proteins are found. Apical to ZA, a subapical region (SAR) is observed, where different protein complexes localize, such as the Crb complex. Septate Junctions (SJ) are found basal to the ZA, and are also composed of different protein complexes. Lac is found adjacent, and lateral, to the SAR protein Crb. It is also more basally located than the cytoplasmic protein Armadillo (Arm), which accumulates in the ZA via association with E-Cadherin. The lack of co-localization between Lac and Crb or Arm is especially conspicuous in the large columnar cells of the hindgut. By contrast, Lac co-localizes with Coracle (Cor; Cora FlyBase), Neurexin IV (Nrx), Disclarge (Dlg) and Fasciclin 3 (Fas3), well-known components of the SJs. In particular, Lac completely co-localizes with Cora and Fas III, and partially with Nrx and Dlg; staining for these markers appears to be more enhanced in the most apical region of the apicolateral membrane corresponding to the region of the SJs (Llimargas, 2004).
To more finely determine the localization of Lac protein along the apicolateral membrane, embryos were analyzed by immunoelectron microscopy. In order to preserve the structure and antigenicity of the sample, electron microscopy analysis was carried out following cryofixation and freeze-substitution techniques. The results show that Lac distribution is restricted to the membrane region, where the septae are observed, further confirming that Lac is associated with SJs (Llimargas, 2004).
Interestingly, Lac is expressed in the same tissues in which SJs have been reported. However, SJ formation occurs midway through embryogenesis, which is later than the onset of Lac expression. As is the case for Dlg, Scrib and Lgl, it is proposed that Lac is gradually integrated or recruited into the SJs while or once they are formed (Llimargas, 2004).
To assess the role of the Lac protein in tracheal morphogenesis the BG1462 line, which has a P-element inserted into the 5'UTR of the Lac gene, was analyzed. BG1462 is late embryonic lethal, both when homozygous and over two deficiencies that uncover the Lac gene. In addition, its lethality can be reverted by precise excision of the P-element. Thus, BG1462 has been renamed as Lac1 (Llimargas, 2004).
The Lac2 mutation was generated by imprecise excision of BG1462. Lac2 is a deletion that removes the Lac ATG without affecting nearby genes. Comparison of the tracheal phenotypes in homozygous conditions, and over deficiencies that uncover the gene, indicate that both Lac1 and Lac2 behave as strong loss-of-function alleles or null alleles. In situ hybridization with a Lac probe showed no, or very low, signal in homozygous Lac1 and Lac2 embryos. Immunostaining with a specific anti-Lac antibody produced a similar result: no protein expression was detected in homozygous Lac2 embryos, although residual levels of signal were detected in Lac1 homozygous embryos in tissues other than the trachea. Consistent with these results, Lac1 and Lac2 mutants display the same tracheal phenotype (Llimargas, 2004).
The early events of tracheal development, such as guidance and primary branching, occur normally in Lac mutant embryos. In addition, branch fusion and extension of terminal branches also show a normal pattern, indicating that Lac is not required for these processes. However, from stage 15, Lac1 and Lac2 mutants start to display several defects that become more apparent by stage 16. In particular, most branches become more sinuous or convoluted than in the wild-type; this is especially conspicuous in the dorsal trunk. The lumen shows an uneven appearance, with expansions and constrictions along the tubes, and in addition an abnormal accumulation of lumen components is detected when monitoring the lumenal antigen 2A12. Numerous lumenal breaks and discontinuities are observed, mainly in the dorsal and lateral branches. Moreover, tracheal tubes do not inflate at the end of embryogenesis indicating that they do not become functional (Llimargas, 2004).
The specific tracheal requirement of Lac activity was assessed by inducing Lac expression in the tracheal cells of otherwise Lac1 mutants, and it was confirmed that these embryos display a normal tracheal system (Llimargas, 2004).
A group of mutants, previously reported by Beitel and Krasnow, display a tracheal phenotype similar to that of Lac (Beitel, 2000). One of these mutations, bulb, maps to the same region as the Lac gene. The following observations suggest that bulb is a mutation of the Lac gene. First, the bulb mutation fails to complement both Lac1 and Lac2: bulb/Lac1 embryos show the same kind of tracheal phenotype as bulb and Lac1 homozygotes, and they do not survive. Second, although in bulb embryos Lac is expressed in several tissues in a similar way to the wild type, no Lac expression is detected in tracheal cells. Thus, bulb appears to be a regulatory mutation of Lac (Llimargas, 2004).
To analyze in more detail the nature of the Lac mutant defects, a tauGFP construct was used to visualize the tracheal cells. The convoluted shape of the tracheal tubes, mainly the dorsal trunk, is a consequence of them having lengthened too much. However, this extra growth does not appear to derive from an increase in the number of tracheal cells, but rather from an increase in the length of the cells. For example, the same number of cells was scored in the dorsal trunk between metameres 7 and 8 (18±2 cells on average in this interval in the wild-type, n=14; and 18.3±1.7 in Lac1, n=16), but the total length of the branch in this interval in Lac mutants is 110% of that of wild-type embryos (n=10 intervals measured for Lac1 and for wild-type embryos). Accordingly no extra tracheal cell proliferation is detected in Lac mutants, as assessed by the absence of alpha-phosphorylated histone H3 staining. Similar observations were reported for bulb (Beitel, 2000). Moreover, no enlargement of the nuclei of the mutant cells was detected as compared with the wild type. These results reveal that Lac plays a role in regulating organ size, probably by affecting the shape of the cells (Llimargas, 2004).
Many lumenal breaks are the result of an anomalous behavior of the tracheal cells, which detach from one another in some of the tracheal branches. Cell detachments that break the tracheal tubes are observed, particularly in those branches where cells elongate more and are thought to be subjected to stronger pulling forces. Indeed, these are the cells where a lessening in cell adhesion should be more readily detected. This phenotype points to a role for the Lac protein in cell adhesion (Llimargas, 2004).
In contrast, the tracheal cells of Lac mutants retain a normal epithelial polarity, as judged by the distribution of an apical marker, such as Crumbs (Crb), and a normal polarization of the cytoskeleton, as judged by scoring the minus end of the microtubules with a nodGFP transgene (Llimargas, 2004).
SJs have been proposed to play a prominent role in the formation of trans-epithelial diffusion barriers. Since subcellular localization experiments showed that Lac is a component of the SJs, whether Lac is also involved in the formation of such a barrier in the tracheal tubes was tested. A dye permeability assay was performed by injecting a 10 kDa rodhamine-labeled dextran into the hemocoel of Lac mutant embryos at the end of embryogenesis. In wild-type embryos, the dextran does not show any diffusion into the tracheal lumen, even more than one hour after injection. However, in Lac mutant embryos, the dye diffuses very quickly and completely fills the tracheal lumen, showing that these mutants are unable to establish or maintain the tracheal diffusion barrier. In addition, the dye was found to be internalized in the salivary glands, indicating that they are also affected. In agreement with this, necrotic tissue was observed in the salivary gland region of Lac mutant embryos at later stages. Lac mutants display defects in the accumulation of tracheal lumen components such as 2A12. The fact that Lac is necessary to maintain the trans-epithelial diffusion barrier suggests that the improper accumulation of lumen components in Lac mutants could be due to leakage of the tracheal tubes rather than to a defect in secretion (Llimargas, 2004).
Localization or stabilization of some (but not all) SJs proteins has been found to be dependent upon that of other components. For example, Cora has a very faint staining and a diffuse localization in Nrx mutants, whereas localization of Dlg or Fas3 shows no obvious defect in those same mutants. To understand how Lac relates to other SJs proteins their mutual requirement for localization was examined (Llimargas, 2004).
The expression of several SJs proteins in Lac2 mutants was analyzed. No differences were detected in the levels of expression of these proteins in the tissues analyzed. However, some changes were detected in the subcellular localization of the SJs proteins tested, as is the case for Cora, Dlg, Scrib, Nrx, Fas3 and Nrg. These proteins are no longer tightly localized to the SJs region, but rather are spread into more basolateral positions, although they are still found in the membrane and an apically concentrated distribution is still observed. These differences are particularly conspicuous in the salivary glands. The salivary glands consist of large columnar cells, where the SJs are restricted to a relatively small region in the lateral membrane, making it easier to observe a mislocalization of the SJs proteins. By contrast, these defects are not so easily detectable in other tissues, including the trachea, because of the wider distribution of the SJs on the lateral cell surface. These results show that Lac plays a role in the proper recruitment or accumulation of several SJs proteins into the complexes (Llimargas, 2004).
To determine the requirement of known SJs proteins in Lac localization, Lac distribution was assayed in cora and zygotic lgl mutants. No defects were detected in the levels of Lac protein, but subtle changes were detected in the localization of Lac, which was not so sharply enhanced at the most apical part of the apicolateral membrane. Again, this mild change in Lac localization was more obvious in the salivary glands than in other tissues. These results indicate that the impairment of SJ integrity affects Lac accumulation (Llimargas, 2004).
All together, these results suggest Lac as a new component of the SJs. Therefore, it was reasoned that SJs could be more generally involved in ensuring proper cell length and cell adhesion during tracheal morphology, and thus mutants for genes encoding other SJ proteins were examined. In particular, cora and Nrx mutant embryos were examined: they both display tracheal phenotypes very similar to those of Lac mutant embryos. In particular, both mutants show overgrown tubes with unusual expansions, defects in the accumulation of lumen antigens and lumen breaks. These results demonstrate that there is a general role for SJ in tracheal morphogenesis, and that Lac, as a new component, contributes to this function (Llimargas, 2004).
Reference names in red indicate recommended papers.
Beitel, G. J. and Krasnow, M. A. (2000). Genetic control of epithelial tube size in the Drosophila tracheal system. Development 127: 3271-3282. 10887083
Beltran, S., et al. (2003). Transcriptional network controlled by the trithorax-group gene ash2 in Drosophila melanogaster. Proc. Natl. Acad. Sci. 100(6): 3293-8. 12626737
Egger, B., Leemans, R., Loop, T., Kammermeier, L., Fan, Y., Radimerski, T., Strahm, M. C., Certa, U. and Reichert, H. (2002). Gliogenesis in Drosophila: genome-wide analysis of downstream genes of glial cells missing in the embryonic nervous system. Development 129: 3295-3309. 12091301
Freeman, M. R., Delrow, J., Kim, J., Johnson, E. and Doe, C. Q. (2003). Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function. Neuron 38: 567-580. 12765609
Hemphala, J., Uv, A., Cantera, R., Bray, S. and Samakovlis, C. (2003). Grainy head controls apical membrane growth and tube elongation in response to Branchless/FGF signalling. Development 130: 249-258. 12466193
Karlstrom, R. O., Wilder, L. P. and Bastiani, M. J. (1993). Lachesin: an immunoglobulin superfamily protein whose expression correlates with neurogenesis in grasshopper embryos. Development 118(2): 509-522. 8223276
Llimargas, M., et al. (2004). Lachesin is a component of a septate junction-based mechanism that controls tube size and epithelial integrity in the Drosophila tracheal system. Development 131: 181-190. 14681183
Paul, S. M., Ternet, M., Salvaterra, P. M. and Beitel, G. J. (2003). The Na+/K+ ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal system. Development 130: 4963-4974. 12930776
date revised: 20 November 2004
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