The expression pattern of Unc5 during Drosophila embryogenesis was determined using in situ hybridization with antisense Unc5 probes and immunohistochemistry using antisera against Unc5 protein. Prior to gastrulation, Unc5 mRNA is strongly expressed in the presumptive mesoderm. Mesodermal expression begins to fade during stages 13-14, persisting only in the cells that form the dorsal vessel. Expression within the CNS begins during late stage 13, shortly after the first axons have extended. Unc5 mRNA can be detected in several dispersed clusters of cells within the CNS, increasing in number as development proceeds. Prominent Unc5 staining is also seen in the peripheral and exit glia, which migrate laterally out of the CNS between stages 14 and 17 (Keleman, 2001).
Anti-Unc5 antisera were generated using a peptide corresponding to the Unc5 amino terminus. Labeling with these antisera reveals accumulation of Unc5 protein on motor axons that exit the CNS ipsilaterally via the segmental nerve root (SN). No staining could be detected on either commissural or longitudinal axons within the CNS, nor on motor axons that exit via the intersegmental nerve (ISN). In the periphery, Unc5 protein can be detected on all branches of the SN. Exit and peripheral glia along both the SN and ISN also express high levels of Unc5 protein. By staining glial cells missing mutants, which lack these glia, it was confirmed that Unc5 protein is expressed on motor axons, not just glia, of the SN, while being undetectable on motor axons of the ISN and its branches (Keleman, 2001).
There is thus a striking complementarity between the expression patterns of Unc5 and the Netrins. Unc5-expressing motor axons avoid midline cells in the CNS and muscles in the periphery that express Netrins. Conversely, commissural axons that are attracted by Netrins do not express Unc5, nor do those motor axons that innervate Netrin-expressing muscles. The finding that Unc5 is expressed on SNa motor axons was particularly satisfying since these axons can be repelled by ectopic expression of NetB, either on all muscles (Mitchell, 1996) or on specific target muscles (Winberg, 1998). These data therefore strongly suggest that repulsion mediated by Netrins and Unc5 helps to guide SNa motor axons out of the CNS and on to their specific muscle targets (Keleman, 2001).
To assess the role of repulsion by Netrins and Unc5 in shaping motor axon pathways, the development of these trajectories was examined in Df(1)NP5 embryos, in which both the NetA and NetB genes are deleted. For this, the general motor axon marker MAb 1D4 and anti-Unc5 were used. No abnormalities were detected in the SNa and SNc projections in these embryos. The lateral migration of peripheral and exit glia, visualized with anti-Repo antibodies, also appears normal in Netrin-deficient embryos. Double-stranded Unc5 RNA was injected into wild-type embryos in an attempt to specifically disrupt Unc5 function by RNA-mediated interference (RNAi). Although this resulted in a strong reduction in Unc5 staining, MAb 1D4 did not reveal any misrouting of SN motor axons in these embryos. Thus, while the expression data suggest a role for Unc5 in repelling SN motor axons out of the CNS and away from Netrin-expressing muscles, the genetic data indicate that repulsion by Netrins is likely to be just one of multiple guidance forces that control these projections (Keleman, 2001).
However, SNa motor axons can be repelled by Netrins. If NetB is ectopically expressed on all muscles using a 24B-GAL4 driver and a UAS-NetB transgene, SNa axons often stall at the edge of the CNS or fasciculate with the ISN (Mitchell, 1996). Does this gain-of-function phenotype depend on Unc5 function? To test this, Unc5 double-stranded RNA was injected into 24B-GAL4/UAS-NetB embryos. In control embryos that were either uninjected or injected with buffer alone, SNa motor axons fail to enter their lateral muscle target region in 54% or 57% of hemisegments, respectively. In contrast, this phenotype is seen in only 14% of hemisegments in Unc5 RNAi embryos. These data establish that SNa motor axons do indeed sense Netrin as a repulsive signal acting through the Unc5 receptor (Keleman, 2001).
Development of the nervous system and establishment of complex neuronal networks require the concerted activity of different signalling events and guidance cues, which include Netrins and their receptors. In Drosophila, two Netrins are expressed during embryogenesis by cells of the ventral midline and serve as attractant or repellent cues for navigating axons. It was asked whether glial cells, which are also motile, are guided by similar cues to axons, and the influence of Netrins and their receptors on glial cell migration was analyzed during embryonic development. In Netrin mutants, two distinct populations of glial cells are affected: longitudinal glia (LG) fail to migrate medially in the early stages of neurogenesis, whereas distinct embryonic peripheral glia (ePG) do not properly migrate laterally into the periphery. It is further shown that early Netrin-dependent guidance of LG requires expression of the receptor Frazzled (Fra) already in the precursor cell. At these early stages, Netrins are not yet expressed by cells of the ventral midline, and evidence is provided for a novel Netrin source within the neurogenic region that includes neuroblasts. Later in development, most ePG transiently express uncoordinated 5 (unc5) during their migratory phase. In unc5 mutants, however, two of these cells in particular exhibit defective migration and stall in, or close to, the central nervous system. Both phenotypes are reversible in cell-specific rescue experiments, indicating that Netrin-mediated signalling via Fra (in LG) or Unc5 (in ePG) is a cell-autonomous effect (von Hilchen, 2010).
Based on the present data, a dual role is postulated for Netrin-mediated signalling in glial cell migration. According to this model, early in neurogenesis, Netrins guide the LGB and its progeny from the lateral edge of the neuroectoderm towards a medial position. At these early stages, Netrins are expressed by cells of the neuroectoderm as well as by NBs, and this Netrin source most likely attracts the LGB via Fra. Ectopic expression of Netrins in the vicinity of the LGB might abolish a possible ventral-to-dorsal gradient and hence (occasionally) results in ectopic clusters. Additionally, attempts were made to express Netrins in the dorsal area of the embryo and thereby attract the LGB and its progeny in the wrong direction. Unfortunately, none of the tested drivers showed Gal4 expression at the appropriate stage and intensity. Further experiments are needed to prove this model (von Hilchen, 2010).
The LGB delaminates from the lateral neuroectoderm close to the sensory organ precursor-derived ePG11 (60%-65% dorsoventral axis, where 0% is the ventral midline). In wild type, it migrates medially while proliferating, whereas it is believed that in Netrin and fra mutants the LGB remains (and proliferates) at its place of birth and does not migrate at all in affected hemisegments. Morphogenetic movements during germ band retraction, mesoderm migration and dorsal expansion of the epidermis complicate this issue. Nevertheless, ectopic clusters mainly remain in close proximity to ePG11. Although ectopic LG have no contact to axons, the lineage develops normally with respect to cell number and marker gene (Msh, the LG-specific marker Naz, Pros) expression. This is contrary to published data on the development and differentiation of LG, which have been postulated to depend on an intimate interaction with longitudinal axons. Nevertheless, LG play an important role in the navigation and fasciculation of longitudinal axons. Accordingly, in hemisegments of Netrin and fra mutants, in which LG are mispositioned from the earliest stages, it was found that the longitudinal axon tracts are thinner, show aberrant projections and fasciculation defects. These neuronal phenotypes were reported previously, but without noticing that the LG are missing in these hemisegments. Similar neuronal phenotypes can be induced by ectopic expression of unc5 in glial cells (repo>unc5), which only affects LG and shifts them away from the midline to more lateral positions or even into the PNS. In some hemisegments with ectopic LG clusters, longitudinal tracts show a weaker phenotype. In these cases, other glial cells (from within the same hemisegment or an adjacent hemisegments) fill the gaps on top of the longitudinal axons and hence seem to compensate for the loss of LG (von Hilchen, 2010).
From these data, it is concluded that the longitudinal axon phenotypes observed in Netrin and fra mutants are, at least partially, a secondary effect of the lack of LG in corresponding neuromeres. Additionally (and somewhat confusingly), these neuronal phenotypes in fra mutants can be partially rescued by elav-Gal4 and Mz605-Gal4, but neither rescues the LG phenotype. Further experiments with other Gal4 drivers that allow a more restricted spatio-temporal expression of UAS-fra might help to resolve this issue (von Hilchen, 2010).
The second population of glial cells that is guided by Netrin-mediated signalling comprises ePG. Nine ePG migrate from the ventral nerve cord into the PNS of each abdominal hemisegment, but it is ePG6 and ePG8 in particular, both progeny of NB2-5, that show a stalling phenotype in NetABδ, NetBδ and unc58 mutants. It was shown by rescue experiments and analysis of Netrin single mutants that only NetB provided by cells of the ventral midline repels ePG6 and ePG8 via the Unc5 receptor. Although both Netrins are expressed by the ventral midline, they clearly do not share a redundant function for ePG guidance. A similar observation has been reported for unc5-expressing motoaxons, which respond differently to each Netrin. To date, the nature of these differences between the two Netrins in combination with Unc5 remains unresolved. In NetA?-NetB™, only NetB is expressed, but is tethered to the membrane of the cell. In these embryos, no ePG stalling was observed, further supporting the notion that only NetB is required for ePG migration and indicating that this signalling is at short range. But why do all ventrally derived ePG express unc5 mRNA transiently during their migratory phase? Further work will be needed to clarify why NetB-Unc5 signalling is selectively required for normal migration of ePG6 and ePG8 (von Hilchen, 2010).
It is widely accepted that embryonic glial cells use neurons or neuronal processes as the substrate for migration. It was shown previously that most migrating ePG follow certain axonal projections. Could such neuron-glia contact be sufficient for proper guidance? The questions would then be (1) how do glial cells actually identify their respective neuronal projections and (2) how is directionality of migration given? Four-dimensional analysis of ePG migration, however, has revealed that ePG6 and ePG8 do not necessarily follow axons, but may also use other glial cells as substrate. These two cells leave the CNS later than other ePG, they are the only ePG that can overtake other cells, and they may migrate on top of ePG rather than along peripheral nerves. Is this possible lack of axonal association (and perhaps adhesion) the reason why these cells need an additional guidance system? (von Hilchen, 2010).
The initial migration of the LGB and its early progeny cannot occur along axons because at these early stages axonal projections are not yet established. As discussed, in most cases the LG phenotype affects the entire LGB lineage and hence is an early guidance defect. So it might well be that early LGB guidance is dependent on Netrin-Fra signalling without any neuronal contribution. After the first division of the LGB, neuronal projections are established, Net-Fra attraction is no longer required and fra expression is switched off. The results of the fra rescue experiments show that at least the timing of fra expression is crucial: gcm-Gal4-induced fra expression can rescue the LG phenotype, whereas a slightly later expression driven by repo-Gal4 cannot. The expressivity of the LG phenotype in NetAB? mutants is only 30%. How are 70% of the LGB 'rescued'? A second, as yet unknown, signalling mechanism could guide the LGB medially, either by ventral attraction or dorsal repulsion. Similar redundancies have been reported, e.g. for border cell migration in the ovary or germ cell migration in early embryogenesis (von Hilchen, 2010).
In addition to the selectivity of the phenotypes in different populations of glial cells, as discussed above, another interesting observation comes from the rescue experiments in unc58 and fra3/fra4 mutants. Control experiments were performed to test whether pan-glial expression of either receptor affects glial cell migration. Glial expression of UAS-fra in an otherwise wild-type background does not alter the glial pattern in the CNS or PNS. Since unc5 is expressed normally in these experiments, repulsion of ePG6 and ePG8 into the periphery occurs as normal. By contrast, pan-glial expression of unc5 is able to shift LG to a more lateral position in the CNS and LG can even leave the CNS and lie in the periphery, whereas all other glial cells are properly positioned. Why do only certain glial cells react upon Netrin-mediated signalling? More precisely, what conveys the ability for LG to 'read' Netrin-mediated signalling? In addition to the receptors, cells might require downstream molecules that could be differentially expressed and hence provide competence to react to Netrins. Several such molecules have been described for both vertebrates and invertebrates. Loss-of-function mutants for Drosophila homologues of these possible downstream molecules were analyzed, but none showed phenotypes comparable to those of fra3/fra4 or unc58. Previous data demonstrate a function for the small GTPases Rac1 and Rho1 in ePG migration in Drosophila, and it was recently shown that they can act downstream of Unc5 signalling in vertebrates. A dominant-negative form of Rho1 was expressed using cas-Gal4 in an otherwise wild-type background and stalling of ePG6 and ePG8 (cas>Rho1N19) was obtained. Expression of a constitutively active form of Rho1 in ePG6 and ePG8 in an unc5 mutant background (unc58; cas>Rho1V14), however, did not restore their stalling phenotype. Although the possiblity cannot be ruled out that ectopic expression of such constructs leads to artificial phenotypes, these data indicate that Rho1 is not downstream of Unc5, but rather acts in parallel. Further experiments will be needed to unravel the signalling complex of Unc5 and Fra/Dcc, and glial cell migration in the Drosophila embryo might serve as a powerful model system for this purpose (von Hilchen, 2010).
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date revised: 15 February 2011
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