DEVELOPMENTAL BIOLOGY

Embryonic

Trio mRNA is abundant and ubiquitous early in embryogenesis; this indicates a substantial maternal contribution. At stages 11-12, Trio message is broadly distributed, with highest levels in invaginating gut (anterior and posterior) and in a repeating pattern within the developing central nervous system (CNS). At stages 13-15, CNS expression is maintained at high levels, while a new pattern of epidermal expression at segment boundaries emerges. This peripheral staining corresponds to the epidermal muscle attachment (EMA) cells, a specialization of the body wall epidermis involved in patterning and maintaining the integrity of muscle attachment sites. EMA cell expression is transient and is undetectable by stage 16, while CNS expression is maintained at a reduced level throughout stage 16. The abundant and sustained expression of Trio RNA in the developing CNS is consistent with a role in axonogenesis (Bateman, 2000).

To examine the temporal and spatial expression of trio during development, in situ analysis of Canton-S wild-type embryos was performed with antisense riboprobes. Extensive accumulation of Trio mRNA was observed in early cleavage stage embryos, indicating maternal contribution of the Trio mRNA. By the cellular blastoderm stage, levels of Trio mRNA are greatly reduced compared with the earlier maternal contribution. As germband extension proceeds, mRNA accumulation in the invaginating mesodermal layer is detected. The first accumulation of Trio mRNA in the developing nervous system is evident during stage 10. Expression in the nervous system persists through approximately stage 15. It appeared that most, if not all, neurons of the CNS express Trio mRNA. In addition, low levels of trio expression are observed throughout the epidermis at these stages. Patches of Trio expression in the lateral epidermis become evident in stage 13 embryos. This accumulation corresponds to muscle attachment sites for the somatic musculature. During the process of dorsal closure, increased levels of Trio mRNA accumulation were observed in leading edge cells that enclose the yolk sack along the dorsal surface. By stage 16, the majority of Trio mRNA accumulation becomes restricted to cells surrounding the developing gut. Throughout embryonic development, Trio expression appears most prominently in cells that are migrating or undergoing dynamic cytoskeletal rearrangements (Liebl, 2000).

To assess the function of Trio, its distribution patterns in Drosophila tissues were examined. Trio protein fused to GST was expressed in bacteria and used to generate rabbit polyclonal and mouse monoclonal (mAb 9.4A) antibodies. In the embryonic CNS, Trio staining is initially detected at stage 12, when axons start extending from the neuronal cell bodies. At stage 13, Trio is detected in the growing axon fascicles running on the longitudinal tracts and on those crossing the midline of the ventral cord. As the CNS develops, the axonal expression becomes more robust in pattern and is detected preferentially in the longitudinal fascicles and weakly in commissural fascicles of stage 16 embryos. These expression patterns suggest that Trio may be involved in axonogenesis that includes axonal extension, fasciculation, or pathway selection. In addition to the neural tissue, Trio is found in the epidermis and strongly in the muscle attachment sites. Trio expression is observed throughout development (Awasaki, 2000).

Larval and adult

In the adult brain, while a large number of cells in the cortex and most neuropil regions are weakly labeled with anti-Trio antibody, strong Trio staining is detected in several groups of neurons, including the MB neurons. A pair of MBs are located in the central brain, and each exhibits a characteristic structure that consists of calyx, peduncle, and five (alpha, alpha', beta, beta' and gamma) lobes. These parts are formed by the neurites emanating from clusters of neurons, Kenyon cells, located in the dorsocaudal cortical regions. All of these neurons extend their processes throughout the peduncle but are classified into three types by their further projection patterns. The individual axons extended from the alpha/beta or alpha'/beta' lobe neurons bifurcate into the alpha and beta, or alpha' and beta', lobes, respectively, and the axons of the gamma lobe neurons project to the gamma lobe after passing through the peduncle. Trio is expressed in a subset of MB neurons and distributed in the cell bodies, calyx, central and lateral peduncles, and alpha', beta', and gamma lobes but not in alpha and beta lobes. To confirm this assignment, MBs were doubly stained with mAb 1D4, which strongly labels the alpha and beta lobes and a part of the peduncle, and weakly labels the gamma lobe but not the alpha' and beta' lobes. These staining patterns were complementary to each other in most MB regions, which confirm the subregional distribution of Trio in MB (Awasaki, 2000).

Since MBs undergo dynamic morphological changes during development, it needed to be clarified how Trio expression is associated with the changes. Trio is distributed in the larval MB, which includes the larval vertical (LV) and medial (LM) lobes, peduncle, calyx, and cell bodies throughout larval life. During the pupal stages, the Trio expression pattern in MB alters as metamorphosis proceeded. Trio staining is found in the vertical and medial lobes at 12 hr after puparium formation (APF), is then confined in the approximate anterior region of the peduncle, with no signals in any lobes at 24 hr APF, and is only detected in the gamma lobe at 48 hr APF. These successive changes in Trio staining correspond to the remodeling processes of the gamma lobe neurons. The gamma lobe neurons are the first neurons generated that form vertical and medial lobes during the larval stages, then undergo degeneration that results in the loss of both lobes around 18 hr APF, and finally regenerate medially to form adult gamma lobes at 24–36 hr APF. Trio is found continuously distributed in the gamma lobe neurons in varying patterns during the larval, pupal, and adult stages. Trio is not observed in the alpha/beta and alpha'/beta' lobe neurons at 24 hr or 48 hr APF (Awasaki, 2000).

Trio is not only expressed in neurons extending neurites at the developing stages, but is also abundantly found in adult brains. This adult expression suggests that Trio functions in some cellular events other than neurite development. To assess the possible Trio function, immunoelectron microscopy was performed to examine the subcellular localization of Trio in adult brains. Trio signals are distributed in a patched pattern in the axons and cell bodies of neurons in the central brain and optic lobes. The patches are, in many cases, associated with clusters of vesicles in the axoplasm. Occasionally, small areas of the plasma membrane close to the patches are stained. This staining, however, may have resulted from diffusion of the dye from the patches. Furthermore, in the lamina neuropil, which exhibits an array of lamina cartridges consisting of synaptic pairs, photoreceptor cells, and lamina neurons, Trio is found in the dendritic terminals of the lamina neurons that contact or intrude into the photoreceptor cells. Occasionally the terminals with the Trio signals form postsynapses, and the signals are largely associated with the plasma membrane in the terminals. No staining is detected in the presynaptic terminals of the photoreceptor cells in adult brains (Awasaki, 2000).

Effects of Mutation or Deletion

Correct pathfinding by Drosophila photoreceptor axons requires recruitment of p21-activated kinase (Pak) to the membrane by the SH2-SH3 adaptor Dock. The guanine nucleotide exchange factor (GEF) Trio has been identified as another essential component in photoreceptor axon guidance. Regulated exchange activity of one of the two Trio GEF domains is critical for accurate pathfinding. This GEF domain activates Rac, which in turn activates Pak. Mutations in trio result in projection defects similar to those observed in both Pak and dock mutants, and trio interacts genetically with Rac, Pak, and dock. These data define a signaling pathway from Trio to Rac to Pak that links guidance receptors to the growth cone cytoskeleton. It is proposed that distinct signals transduced via Trio and Dock act combinatorially to activate Pak in spatially restricted domains within the growth cone, thereby controlling the direction of axon extension (Newsome, 2000).

The development of different axon pathways was assessed in order to examine the trio loss-of-function phenotype during embryonic nervous system development. Embryos from different allelic combinations were collected and stained with the anti-Fasciclin II (Fas II) antibody mAb 1D4, an excellent marker for motor axon pathways. Analysis of motor axon pathfinding revealed defects in the ability of nerve branches to reach their target muscles in trio mutants. The two branches most sensitive to perturbation of small GTPase function, ISNb and SNa, are also most sensitive to the loss of trio activity. These phenotypes were observed in all allelic combinations tested, with some variation in penetrance depending on genetic background. Occasionally, defects in target muscle attachment to the underlying epidermis were observed, which likely reflect a role for trio in EMS cells. To avoid scoring guidance errors that could be caused by the target rather than the growth cone, all segments with abnormal muscle patterning were excluded from the analysis (Bateman, 2000).

To ensure that the observed defects in ISNb guidance result from the loss of trio, embryos carrying trio^BX4, a precise excision of the P[1372/3] insertion that restores the locus, were examined. These embryos display wild-type levels of ISNb stop short (2.8%), demonstrating that the phenotype is insertion dependent. The defects observed in SNa development were similar to those of the ISNb phenotype. In trio mutants, SNa sometimes fails to extend either its lateral branch, to contact muscles 5 and 8, or its vertical branch, to contact longitudinal muscles 21-24. Occasionally, SNa fails to reach its target domain altogether; instead it stalls beneath the ventral target domain of ISNb. Although not highly penetrant, these SNa defects were seen in all allelic combinations examined but not in wild-type or precise excision controls. Consistent with defects observed in motoneurons, analysis of axon trajectories in the CNS reveal an inability of axons to pathfind correctly. During the early development of the CNS, longitudinal axons are required to cross segment boundaries and extend into neighboring segments, such that by the late stages of embryonic development (stage 17), distinct 1D4-positive fascicles form continuous pathways along the length of the CNS. In mutant embryos lacking trio function, defects are observed in the formation of these pathways. The most dramatic and persistent disruption was seen in the lateralmost Fas II-positive longitudinal pathway, where breaks and/or inappropriate direction of these interneuronal axons are often observed. In trio^1372/3/trio^1372/3 embryos, 20.6% of stage 17 A2–A8 hemisegments fail to connect to the neighboring segment, compared with only 1.2% (n = 320) in wild-type embryos. Similar defects are seen in multiple allelic combinations (Bateman, 2000).

Mutations in trio cause specific defects in the formation of multiple embryonic axon pathways, implying a role for trio activity in developing axons. However, because trio expression is not restricted to neurons, it is possible that its activity is required elsewhere and that the axonal phenotypes observed represent functions outside the nervous system. To exclude this possibility, axon pathway formation was examined in mutant embryos while simultaneously expressing a wild-type trio construct in postmitotic neurons using the GAL4 driver elaV-GAL4. These embryos show a marked reduction in pathfinding errors in both the CNS and the motor nervous system, indicating that the axonal defects in trio mutants result from a lack of trio function in neurons (Bateman, 2000).

Dosage-sensitive genetic interactions between trio and Abl have been documented. A number of observations support the interaction of Abl and Trio in a common regulatory network. First, the dosage-sensitive genetic interactions between trio and Abl are reciprocal, as assayed by either viability or CNS architecture. Heterozygous mutations in trio worsen the Abl mutant phenotype, while heterozygous mutations in Abl worsen trio mutant phenotypes. A background of compromised signaling (Abl¹/Abl⁴, Df(3L)FpaI/trio^M89, or trio^P0368/10/trio^M89) is enhanced by reduced activity of another member of this network (Liebl, 2000).

As further evidence for the involvement of Abl and Trio in a common signaling network, the Abl and trio homozygous mutant phenotypes show a synergistic interaction. Neither the Abl mutant background nor the trio mutant background have dramatic phenotypic consequences on CNS architecture. Phenotypes similar to those reported here have been observed in a variety of trio mutant combinations (Awasaki, 2000; Bateman, 2000). However, combining these two backgrounds to generate trio, Abl homozygous mutant embryos results in dramatic disruption of the CNS scaffolding. Taken alone, this synthetic enhancement may represent common or independent signaling pathways involving Abl and Trio. However, combined with the dosage-sensitive interactions between Abl and trio observed, it is likely that these molecules are involved in overlapping or interdependent networks. Similar synergistic effects between Abl and fax, and Abl and dab, have been reported. In addition to the reciprocal genetic enhancement between Abl and trio, a null allele of fax (fax^M7) greatly worsens the trio hypomorphic mutant's viability, while a dab null allele weakly modifies this background (Liebl, 2000).

The ena gene was identified through its ability to suppress the Abl mutant phenotype. Since reductions in ena compensate for the absence of Abl, it has been hypothesized that a precise balance between Abl and Enabled activity is required for viability. Similar to the genetic interaction between Abl and ena, heterozygous mutations in ena can partially alleviate the trio mutant phenotype. One interpretation of this interaction is that a balance between trio and ena is required, and Trio may possess a biochemical function that is antagonistic to Enabled's. Since neural enriched isoforms of Mena, the murine homolog of Enabled, are believed to be involved in filopodia formation to extend the growth cone, a potential antagonistic role for Trio is the retraction of growth cones. Drosophila Trio's second DH domain stimulates the formation of stress fibers in REF-52 cells (Newsome, 2000). In neurons, the formation of similar actin-myosin contractile filaments leads to neurite retraction. Therefore, a balance between the biochemical activities of Enabled and Drosophila Trio may be required for a proper balance between extension and retraction of the growth cone in response to attractive and repulsive pathfinding cues (Liebl, 2000).

It is not envisioned that the stimulation of stress fiber formation leading to neurite retraction is the only possible biochemical function of Trio. In fibroblasts, human Trio activates Rac1 with its first DH domain, inducing the formation of lamellipodia and membrane ruffles (Bellanger, 1998; Seipel, 1999). Similar biochemical activity reported for the first DH domain of Drosophila Trio (Newsome, 2000) would presumably lead to lamellipodia/filopodia formation analogous to the activity of Enabled in the growth cone. The elucidation of the biochemical interrelationships between Abl, Ena, Fax, and Trio suggested by their genetic interactions awaits detailed analyses (Liebl, 2000).

In addition to a role for trio during development of the CNS, observations of trio expression in leading edge cells during dorsal closure and an uninflated, blistered wing phenotype in the trio hypomorphic background suggest additional roles for trio during development. Dominant-negative Rho subfamily constructs can disrupt the actin cytoskeleton in leading edge cells, with subsequent effects on dorsal closure. Intense trio expression in leading edge cells may indicate that Trio plays a role in this process. Expressing dominant-negative Cdc42 proteins in wing discs can produce wing blisters similar to those observed in the trio hypomorphic background. Mutations in inflated, a Drosophila integrin, produce similar wing blisters, as well. Since the mammalian c-Abl kinase is activated in response to integrin-mediated cell adhesion, and Abl is expressed in wing imaginal disc epithelial cells, the blistered wing phenotype seen in trio hypomorphic animals potentially presents a modifiable phenotype with which to explore additional aspects of Trio signaling networks (Liebl, 2000).

The trio^E4.1/Df(3L)FpaI embryos appear normal as a whole structure, and the gross morphology of the CNS also look similar to wild type. When the embryos are stained with mAb 1D4, however, defects in the axon patterning in the CNS are found. mAb 1D4 stains three longitudinal fascicles at each lateral side of the ventral nervous system in wild-type embryos at stage 17. In trio^E4.1/Df(3L)FpaI embryos, however, the stained fascicles are arranged in an abnormal pattern. The outermost fascicles are very discontinuous and fused to the adjacent inner fascicles. This phenotype is more pronounced in the mutant first instar larvae, in which the outermost fascicles are thin and frequently disrupted, producing gaps. Portions of the axons turn vertically to the inner fascicles, showing orthogonal patterns. Axons in other fascicles also exhibit irregular arrangements. These abnormal mAb 1D4 staining patterns are similarly observed in trio^E4.1 homozygous embryos and larvae, indicating that trio^E4.1 is a functionally strong or null allele for these axonal phenotypes. To trace the axon pathways more accurately in the mutants, embryos expressing Tau-Myc protein under the control of the lim3 promoter in a small number of neurons were further examined. lim3 is expressed in a subset of interneurons and motor neurons, including the RP motor neurons in embryos and first instar larvae. The Tau-positive axons extending medially from a lateral cluster of neurons turn vertically to navigate on the longitudinal tract and form a fascicle with the axons extended from other segments in the wild-type larva. In the trio^E4.1/Df(3L)FpaI larvae, these axons do not faithfully extend on the longitudinal tracts and exhibit a wavy pattern or frequently turn along three-dimensional axes to follow the wrong tracts. These misrouting phenotypes demonstrate that Trio has an essential role for axon patterning in the embryonic and larval CNS (Awasaki, 2000).

In contrast to the clear defects in the mutant CNS, mild aberrations have been observed in the motor axons projecting to the body wall muscles. ISNb, a motor axon fascicle, innervates the ventrolateral muscles 6, 7, 12, and 13 in a stereotypical fashion. In mutant embryos, while most ISNb fascicles correctly extend toward the target muscles, 10% of ISNb exhibit a stall or fusion phenotype; 7% of the fascicles stall on muscle 7 or prior to entering the muscle territory after exiting the CNS, and 3% of the fascicles fuse to another fascicle ISNd and stall shortly (Awasaki, 2000).

Since Trio is strongly expressed in MB throughout development, the effects of trio mutations on the MB structure were examined. mAb 1D4 labels MB of the wandering third instar larvae, which include the peduncles and the LV and LM lobes but not the cell bodies of the MB neurons and calyx. The core regions of the peduncle, LV, and LM are clearly unstained. These staining patterns indicate that mAb 1D4 is a useful marker for visualizing the whole neurite structure of the larval MB. In both trio^P0368/10/Df(3L)FpaI and trio^P0368/10/trio^E4.1 larvae, the LV and LM lobes are found to be abnormally developed: thin or small lobes in the position of the LV lobe (15 of 18 MBs in trio^P0368/10/trio^E4.1); short lobes in the position of the LM lobe (15 of 18 in trio^P0368/10/trio^E4.1). In most cases, the peduncles appeared normal, with the core region remaining unstained, whereas the core regions of LV and LM are ambiguous when compared with wild type. The degree of defects and altered morphology vary among the individual mutant MBs. These observations suggest that the MB neurons in the trio mutant extend their axons normally in the peduncle but often fail to project further along the lobe-forming tracts (Awasaki, 2000).

In the adult MB, mAb 1D4 labels the alpha and beta lobes strongly, and the gamma lobe weakly. In trio^P0368/10/Df(3L)FpaI and trio^P0368/10/trio^E4.1 flies, shortened or deformed lobes exhibiting weak mAb 1D4 staining are found in the position of the medial lobes, while the peduncles are formed in an apparently normal shape. Based on the position and staining intensity, the abnormal lobes possibly arise from the gamma lobe neurons, in which Trio is continuously expressed during their differentiation in wild type. Moreover, in place of the alpha and beta lobes, strangely shaped lobes with strong staining are found at the anteriormost region of the peduncle, and sometimes unexpectedly close to the calyx. Since no expression of Trio is observed in the alpha/beta lobe neurons at any stages in wild type, the aberrant formation of the alpha and beta lobes is likely caused by an indirect consequence of the defects in the preexisting larval lobes that the alpha/beta lobe axons later follow (Awasaki, 2000).

Since Trio expression is not confined to the MB neurons, it is uncertain whether the defects in MB are caused by the loss of Trio function in the MB neurons or are a secondary effect resulting from structural alterations in the adjacent brain regions involved in MB development. To discriminate between these possibilities, clonal analyses were performed using the MARCM system, with which only mutant clones can be labeled. Clones of the MB neuroblasts were induced in first instar larvae and analyzed in wandering third instar larvae. The wild-type MB neurons extend their axons through the peduncle and bifurcate into the LV and LM lobes. Mutant MB clones exhibit an abnormal axonal pattern. The axons emanating from the trio^E4.1 clones appear to navigate normally through the peduncle to the approximate region of bifurcation, but the axons found in the two lobes are sparsely distributed, with an apparent reduction in the overall fluorescent intensity of the lobes. The fluorescent intensity of larva-specific spur-shaped lateral (LSL) projection remains high. These observations indicate that the axons in the mutant clones are either stalled on the tracts or misrouted to LSL. In addition, the trio neuroblast clones interestingly exhibits a bundle of neurites overextended from the calyx, a major dendritic cluster of MBs. It remains to be revealed, however, whether the neurites have the properties of axons or dendrites. Taken together, these phenotypes are primarily caused by an alteration in the intrinsic nature of the mutant clones. Thus, it is concluded that Trio plays an essential role in the development of MBs through controlling the directional extension of the axons and/or dendrites (Awasaki, 2000).

Rac GTPases regulate the actin cytoskeleton to control changes in cell shape. To date, the analysis of Rac function during development has relied heavily on the use of dominant mutant isoforms. Here, loss-of-function mutations have been used to show that the three Drosophila Rac genes, Rac1, Rac2 and Mtl, have overlapping functions in the control of epithelial morphogenesis, myoblast fusion, and axon growth and guidance. They are not required for the establishment of planar cell polarity, as had been suggested on the basis of studies using dominant mutant isoforms. The guanine nucleotide exchange factor, Trio, is essential for Rac function in axon growth and guidance, but not for epithelial morphogenesis or myoblast fusion. Different Rac activators thus act in different developmental processes. The specific cellular response to Rac activation may be determined more by the upstream activator than the specific Rac protein involved (Hakeda-Suzuki, 2002).

Endogenous Rac GTPases thus function in morphogenesis of the epidermis, mesoderm, and nervous system. Are they regulated by the same or different upstream activators in each of these tissues? The guanine nucleotide exchange factor Trio activates Rac1, Rac2 and Mtl in vitro, and loss of trio function in the visual system results in projection errors of photoreceptor axons similar to those observed in Rac triple mutants. Axon guidance errors and occasional stalling defects also occur in embryos lacking zygotic trio function. Axon stalling becomes severe in both the CNS and PNS if the maternal trio function is also eliminated. As with the Rac proteins, low levels of Trio activity are sufficient but essential for axon growth. This critical requirement for Trio in axon growth is particularly striking, given that the Drosophila genome encodes at least 22 other Rho family GTPase exchange factors, several of which are also expressed in the developing nervous system (Hakeda-Suzuki, 2002).

In the embryonic nervous system and adult visual system, loss of trio function thus results in defects remarkably similar to those observed upon loss of Rac function, consistent with the idea that Trio and Rac proteins act in a common pathway in vivo. An epistasis experiment was performed to test this. Overexpression of the Trio GEF1 domain using the eye-specific GMR promoter results in a severely disrupted eye morphology and highly aberrant photoreceptor axon projections. If Trio signals through Rac proteins in vivo, then these defects should be dependent on Rac function. This is indeed the case. Both the eye morphology and axon projection defects are almost completely suppressed in animals homozygous for loss-of-function mutations in either Rac1 or Rac2. Mtl alone does not suppress this trio gain-of-function phenotype. The Rac1;Rac2;Mtl triple mutant phenotype is completely epistatic to the trio gain-of-function phenotype. These data demonstrate that Trio GEF1 does indeed act through Rac proteins in vivo, and further suggest that Rac1 and Rac2 are its preferred substrates. The trio loss-of-function phenotype is however much more severe than the Rac1;Rac2 double mutant phenotype, suggesting that endogenous Trio may also activate Mtl, at least when Rac1 and Rac2 are lacking (Hakeda-Suzuki, 2002).

Having identified Trio as the primary activator of Rac proteins during axon growth, whether Trio is required for any of the other Rac functions was investigated. Dorsal closure occurs normally in embryos lacking both maternal and zygotic trio function. Myoblast fusion also appears complete in these embryos, but myotubes often fail to attach themselves correctly to the epidermis. Thus, although it is expressed in both the epidermis and mesoderm, Trio is not required for either dorsal closure or myoblast fusion (Hakeda-Suzuki, 2002).

Thus endogenous Rac proteins control cell-sheet spreading, cell fusion, and axon growth and guidance, and they also regulate axon branching. Each of these processes involves its own characteristic restructuring of the cytoskeleton, and hence is likely to be mediated by a different set of Rac effectors. What determines which of these effector pathways will be stimulated when Rac proteins are activated? One possibility would be that distinct Rac proteins have distinct effectors. This may well be the case for myoblast fusion, which can be mediated by Rac1 or Rac2, but not Mtl. However, in most cases Rac1, Rac2 and Mtl have largely overlapping functions, indicating that they also share a common set of effectors. A similar pattern of overlapping functions in diverse processes has also recently been reported for the three C. elegans Rac genes. In general, the cellular response is therefore unlikely to be dictated by the specific Rac protein involved. These results suggest an alternative possibility. Trio, despite its widespread expression, is required for only a limited subset of Rac functions. This suggests that the set of effectors a Rac protein engages, and hence the cellular response it induces, might also depend on how or where it has been activated. Trio, for example, might activate Rac proteins to a level, for a duration, or in a subcellular location, that allows it to stimulate only those effector pathways that control motility and guidance. Exploring the basis for specificity in Rac function is an important task for the future (Hakeda-Suzuki, 2002).

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date revised: 15 January 2006

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