Ptpmeg protein expression was examined and it was found that Ptpmeg was enriched along fiber tracts in the brain at all stages examined from third instar into adulthood, including the periods when MB axons begin to exhibit defects. As expected, ptpmeg1/Df(3L)ED201 mutants exhibited little or no Ptpmeg expression. Consistent with mosaic analyses indicating that ptpmeg did not act in the MB neurons, Ptpmeg expression was not detected on MB axons, However, Ptpmeg was expressed by many neurons in the central brain and the developing visual system. In the embryonic CNS, Ptpmeg expression was also detected in midline glial cells, indicating that expression of Ptpmeg was not entirely restricted to neurons (Whited, 2007).
Although Ptpmeg was expressed on neuronal processes, it was largely excluded from synapse-rich neuropil regions in the central brain. The subcellular localization of Ptpmeg in the central brain was examined in greater detail using a set of highly polarized neurons in the ellipsoid body (EB) that strongly express Ptpmeg. EB neurons are located in two clusters, one in each brain hemisphere. Each EB neuron extends a neurite which branches to form a dendritic tuft and an axon ring. Ptpmeg was concentrated on the cell bodies and proximal neurites of EB neurons. Ptpmeg expression was also detected in EB dendritic regions, but it did not extend into the axon terminals. Even when overexpressed using an EB-specific promoter (EB1-Gal4), Ptpmeg could not be detected in the axon ring. Rather, Ptpmeg accumulated to increased levels in the EB cell bodies (particularly near the cell surface) and on the proximal neurites and the dendrites. These data demonstrate that Ptpmeg can localize to discrete regions within a neuron (Whited, 2007).
To study the function of ptpmeg, a four base-pair insertion was introduced into the locus using homologous recombination-mediated gene replacement, creating ptpmeg1. The 4 bp insertion in ptpmeg1 is predicted to introduce a translational frameshift, truncating Ptpmeg within the PDZ domain. As predicted, ptpmeg1 mutants did not express full-length Ptpmeg protein. In addition to ptpmeg1, two additional disruptions of the ptpmeg locus were obtained from publicly available collections. ptpmeg2 (pGATB-NP4498) contains a transposable element insertion upstream of the Ptpmeg open reading frame and expresses reduced levels of Ptpmeg protein. Genetic data below suggests ptpmeg2 is a weak loss-of-function allele. In addition, Df(3L)ED201, contains an ~224 kb chromosomal deletion that disrupts the ptpmeg locus and is predicted to delete ~43 additional protein coding genes (Whited, 2007).
Homozygous ptpmeg1 adults were viable and fertile, but often became trapped alive in their food when cultured under normal conditions. This phenotype was rescued by expression of a Ptpmeg cDNA in the nervous system using elav-Gal4, raising the possibility of nervous system disruptions in ptpmeg mutants.Connectivity patterns in the adult brain were examined and significant disruptions of mushroom body (MB) axon projections were observed(Whited, 2007).
The MBs of the adult fly are a higher order brain structure involved in
multiple behaviors including olfactory memory and sleep. MB neurons each
extend an axon that bifurcates to send one branch dorsally and one branch
medially. Each α'/ß' neuron extends one axon branch dorsally, into the
α' lobe, and one branch medially, into the ß' lobe.
Similarly, each α/ß neuron extends one axon branch dorsally, into
the α lobe, and one branch medially, into the ß lobe. In
ptpmeg mutant adults, the dorsally projecting MB lobes were often
reduced in thickness and/or length. Meanwhile, the medially projecting MB lobes were often overextended, with the medial lobes of one hemisphere reaching the midline and
sometimes fusing with the medial lobes from the contralateral hemisphere. By contrast, the cell body and dendritic regions of MB neurons appeared normal in ptpmeg mutants. Thus, ptpmeg is important for MB axon branch development (Whited, 2007).
In addition to exhibiting α lobe defects, ptpmeg mutants
also had disrupted ß lobes. In wild-type animals and in
ptpmeg1 heterozygotes, the ß lobe terminated before
reaching the midline of the brain. In ptpmeg1 mutants, the ß lobes often touched the midline and in some cases completely fused with the contralateral ß lobe. Similar to the α lobe defects, the ß lobe defects were rescued by the expression of wild-type ptpmeg in neurons. Thus, ptpmeg regulates both α and ß lobe patterning. Analysis of ß lobe
development in Df(3L)ED201 animals was not included in this analysis
of ptpmeg function, as Df(3L)ED201 caused dominant ß
lobe defects that were not rescued by ptpmeg expression, suggesting
that disruption of genes in addition to ptpmeg contributed to
Df(3L)ED201-derived ß lobe defects (Whited, 2007).
To determine whether ptpmeg acts in the MB neurons, marked clones of homozygous mutant ptpmeg1 neurons were generated in
otherwise heterozygous animals using the MARCM system. Mutant
clones of varying sizes were generated, including small clones containing one
to 10 mutant α/ß cells and medium clones containing
~10 to 50 mutant α/ß cells, but in no cases were MB axon defects observed. Larger MB-restricted clones were generated in which
nearly all α/ß neurons along with some α'/ß' and γ neurons were mutant, but MB axon branches still appeared normal. Clones containing substantial amounts of mutant brain tissue outside the MBs did confer phenotypes, but did not permit the identification of the critical cell populations in which ptpmeg was required. These data suggest that Ptpmeg acts in neurons to control MB axon patterning, but does not act in the MB neurons themselves (Whited, 2007).
The MB axon defects observed in ptpmeg mutant adults could arise
in at least two different ways. In the first scenario, ptpmeg mutants
could be defective in the initial pathfinding or elaboration of MB axon
branches. Alternatively, ptpmeg MB axons might initially pathfind and
elaborate normally, but become progressively abnormal at later times. To
distinguish these possibilities, MB axon development was examined in
ptpmeg mutants, initially focusing on the α/ß neurons,
which are born early during pupariation. By 18
hours post-pupal formation (PPF), α/ß dorsal and medial axon
branches can be detected and by 48 hours PPF their branching is well
established. In ptpmeg1 animals, dorsal branches appeared
normal at both 18 hours PPF and 48 hours PPF, whereas medial lobe branches were normal in all hemispheres at 18 hours and in 33 of 36 hemispheres at 48 hours. The large increase in MB defects observed between 48 hours PPF and adult - from 0% to ~55% of
dorsal lobes defective and from ~10% to ~80% of medial lobes defective
- indicates that α/ß axon branching defects are detected only after
the α/ß axon projections are well-established. This suggests that
ptpmeg is not essential for branching or pathfinding of α/ß axons, but is rather required for these branches to be maintained into the adult (Whited, 2007).
The onset of MB axon projection defects were followed in the dorsal
lobes of ptpmeg1/Df(3L)ED201 animals, using a marker that
labels all subsets of MB neurons throughout development. In early third
instar larvae, the MB lobes of wild type and ptpmeg mutants were
indistinguishable. As larval MB lobes are composed of largely of γ axons with some
α'/ß' axons, the initial extension of these axons thus
appeared normal. Between third instar and 18 hours PPF in wild-type animals, branches from additional α'/ß' neurons and from α/ß
neurons enter the dorsal MB region. However, the overall innervation of dorsal
MB regions temporarily decreases due to the pruning of the dorsal branches of
γ neurons. Since the dorsal lobes of 18 hours PPF wild-type and ptpmeg mutants were indistinguishable, this stage of development also appears to proceed normally in ptpmeg mutants. The dorsally projecting MB lobes thicken during pupation as additional α/ß neurons send branches into this region. At 24 hours PPF, the dorsal lobes of ptpmeg mutant MBs remained essentially
indistinguishable from wild type, as only 1 of 24 hemispheres exhibited defects. At later times, however, MB defects became common: ~15%-20% of dorsal MB lobes exhibited defects at 36 hours and 48 hours PPF (4 of 24 and 5 of 27 hemispheres defective, respectively), increasing to nearly 50% by the first day of adulthood (Whited, 2007).
The morphology of the dorsal lobes in ptpmeg mutants was also
informative. Not only did the loss of ptpmeg cause a preferential
reduction in the distal region of dorsal lobes, dots of axonal material were
frequently observed near the regions where dorsal lobes were reduced. Together
these data are consistent with the loss of ptpmeg causing axon
retraction in the dorsal lobe (Whited, 2007).
Taken together, the ptpmeg1 and
ptpmeg1/Df(3L)ED201 time-course data provide a consistent
picture in which MB axon elaboration is initially normal, but becomes aberrant
over time. As the majority of defects are detected only after the initial
elaboration of MB axons is completed, these data suggest that ptpmeg
is required for a later stage in MB development. The finding that the onset of
dorsal lobe defects is slightly earlier in ptpmeg1/Df(3L)ED201 animals than in ptpmeg1/ptpmeg1 animals raises the possibility
that ptpmeg1 might not be a null allele. Such residual
ptpmeg activity could also explain the partial penetrance of MB
defects observed here. Alternatively, partial penetrance could reflect the
ability of Ptpmeg-independent pathways to maintain apparently normal patterns
of MB axon branches in some hemispheres (Whited, 2007).
Having demonstrated a requirement for ptpmeg in the maintenance of
MB axon branches, it was asked whether ptpmeg was exclusively required
for later stages of development or whether ptpmeg might be needed for
the initial pathfinding of other axons in the brain. This
question was addressed by examining the role of ptpmeg in the formation of the
ellipsoid body (EB). EB axons normally grow to reach the midline and then extend ventrally to form a closed ring, which is completed by 48 hours PPF. In
ptpmeg1 mutants, EB axons reached the midline, but their
extension toward ventral regions halted prematurely, leaving an omega-shaped
EB open along its ventral aspect. EB axon defects persisted into the adult;
ptpmeg1 mutant adults displayed a ventral cleft in the EB
ring. ptpmeg1/Df(3L)ED201 adults showed similar
defects. In contrast to the MB, which was established normally in
ptpmeg mutants but became increasingly aberrant over time, the EB
appeared never to form normally and the axonal projections defects in the
ptpmeg mutant EB did not become more severe at later time points. Taken together these data suggest that ptpmeg is critical for the initial pathfinding of EB axons. The EB projection defects appeared restricted to axons; the dendritic tufts and cell bodies of EB neurons appeared normal in ptpmeg mutants (Whited, 2007).
The identity of the cells in which ptpmeg acts to control EB axon
patterning was examined through tissue-specific rescue and genetic mosaic
experiments. Expression of a wild-type Ptpmeg cDNA in neurons using
Elav-GAL4 rescued the EB defects of ptpmeg mutants, indicating that ptpmeg was required in neurons to correctly pattern the EB axonal ring. To determine if ptpmeg was required within the EB neurons, marked clones of
homozygous mutant ptpmeg1 EB neurons were examined in
otherwise heterozygous animals using the MARCM system. Animals containing
ptpmeg1 mutant EB neurons were analyzed, including animals in which essentially all EB neurons were mutant. In no case were defects observed in EB axon projections. Furthermore, EB-specific expression of a wild-type Ptpmeg cDNA using EB1-Gal4 failed to rescue the EB defect. Thus, whereas
EB neurons express Ptpmeg, they do not require ptpmeg to control the
trajectories of their axons, suggesting that ptpmeg acts in other
neurons to control EB axonal projections (Whited, 2007).
The Ptpmeg subfamily of tyrosine phosphatases is characterized by the
presence of FERM, PDZ and PTP domains, and the
requirements for these domains in brain development were examined. The role of
the FERM domain on ptpmeg function was examined using a naturally occurring splice variant that encodes a Ptpmeg without the FERM domain. When expressed in neurons, the ΔFERM variant of Ptpmeg strongly rescued the EB defect. In the MBs, the ΔFERM variant rescued the ß lobe overextension phenotype of ptpmeg1, and any differences between ΔFERM and wild-type ptpmeg transgene rescue of the ß lobe defect were not of statistical significance. By contrast,
the ΔFERM variant did not significantly rescue the α lobe
reduction of ptpmeg1, and there was a
highly significant difference between ΔFERM and wild-type transgenes for
α lobe rescue. These data suggest that the FERM
domain is important for Ptpmeg's role in α lobe maintenance, but not
essential for ß lobe maintenance and EB pathfinding (Whited, 2007).
The function of the Ptpmeg PDZ domain was examined by mutating residues in
the GLGF motif that forms part of the substrate-binding pocket of other PDZ
domains. As a GF to AA mutation in the GLGF motif of the PDZ domain protein Enigma
disrupts its ability to bind ligand, these amino acids were mutated in Ptpmeg, creating
Ptpmeg[G494A,F495A]. Ptpmeg[G494A,F495A] rescued the EB defects of ptpmeg1 mutants as effectively as a wild-type Ptpmeg cDNA. Ptpmeg[G494A,F495A] also rescued both the MB α and ß lobe defects. However, the ability of Ptpmeg[G494A,F495A] to rescue the MB defects was reduced compared to wild-type
Ptpmeg for both the α lobe and ß lobe, suggesting that the PDZ domain contributes to the effectiveness of Ptpmeg in maintenance of the MBs (Whited, 2007).
The importance of catalytic activity for ptpmeg
function was examined by creating three forms of Ptpmeg in which residues crucial for
phosphatase function were mutated. Both Ptpmeg[C877S] and Ptpmeg[Y650F,D787A]
contained mutations that disrupt catalysis, whereas
Ptpmeg[R883M] contained a mutation predicted to disrupt substrate binding. Whereas
expression of a wild-type Ptpmeg cDNA in neurons completely rescued the EB
axon defects of ptpmeg mutants, none of the three phosphatase domain
mutants significantly rescued EB defects. Similarly, none of the phosphatase mutants rescued either the α lobe or ß lobe defects in the MB. In no case did expression of a mutant form of Ptpmeg cause a dominant EB axon or MB axon phenotype in an otherwise normal animal. Control experiments demonstrated that each mutant protein was expressed at a level comparable to wild-type transgenic protein as detected by western blot. These results demonstrate that the phosphatase activity of Ptpmeg is crucial for all of the ptpmeg functions observed in this study, including EB axon pathfinding and the stabilization of MB axon branching, where Ptpmeg inhibits retraction of dorsal lobe branches and prevents overextension of medial lobe branches (Whited, 2007).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Ptpmeg
Bauler, T. J., et al. (2007). Normal TCR signal transduction in mice that lack catalytically active PTPN3 protein tyrosine phosphatase. J. Immunol. 178(6): 3680-7. Medline abstract: 17339465
Gu, M. and Majerus, P. W. (1996a). The properties of the protein tyrosine phosphatase PTPMEG. J. Biol. Chem. 271(44): 27751-9. Medline abstract: 8910369
Gu, M., Meng, K. and Majerus, P. W. (1996b). The effect of overexpression of the protein tyrosine phosphatase PTPMEG on cell growth and on colony formation in soft agar in COS-7 cells. Proc. Natl. Acad. Sci. 93(23): 12980-5. Medline abstract: 8917530
Hironaka, K., Umemori, H., Tezuka, T., Mishina, M. and Yamamoto, T. (2000). The protein-tyrosine phosphatase PTPMEG interacts with glutamate receptor delta 2 and epsilon subunits. J. Biol. Chem. 275: 16167-16173. Medline abstract: 10748123
Hsu, E. C., et al. (2007). Suppression of hepatitis B viral gene expression by protein-tyrosine phosphatase PTPN3. J. Biomed. Sci. [Epub ahead of print]. Medline abstract: 17588219
Jing, M., et al. (2007). Degradation of tyrosine phosphatase PTPN3 (PTPH1) by association with oncogenic human papillomavirus E6 proteins. J. Virol. 81(5): 2231-9. Medline abstract: 17166906
Park, K. W., et al. (2000). Molecular cloning and characterization of a protein tyrosine phosphatase enriched in testis, a putative murine homologue of human PTPMEG. Gene 257(1): 45-55. Medline abstract: 11054567
Sahin, M., Slaugenhaupt, S. A., Gusella, J. F. and Hockfield, S. (1995). Expression of PTPH1, a rat protein tyrosine phosphatase, is restricted to the derivatives of a specific diencephalic segment. Proc. Natl. Acad. Sci. USA 92: 7859-7863. Medline abstract: 7644504
Takeuchi, K., Kawashima, A., Nagafuchi, A. and Tsukita, S. (1994). Structural diversity of band 4.1 superfamily members. J. Cell Sci. 107: 1921-1928. Medline abstract: 7983158
Takeuchi, T., Miyazaki, T., Watanabe, M., Mori, H., Sakimura, K. and Mishina, M. (2005). Control of synaptic connection by glutamate receptor delta2 in the adult cerebellum. J. Neurosci. 25: 2146-2156. Medline abstract: 15728855
Uchida, Y., Ogata, M., Mori, Y., Oh-hora, M., Hatano, N. and Hamaoka, T. (2002). Localization of PTP-FERM in nerve processes through its FERM domain. Biochem. Biophys. Res. Commun. 292: 13-19. Medline abstract: 11890665
Wang, Z., et al. (2004). Mutational analysis of the tyrosine phosphatome in colorectal cancers. Science 304: 1164-1166. Medline abstract: 15155950
Whited, J. L., Robichaux, M. B., Yang, J. C. and Garrity, P. A. (2007). Ptpmeg is required for the proper establishment and maintenance of axon projections in the central brain of Drosophila. Development 134(1): 43-53. Medline abstract: 17138662
date revised: 8 July 2007
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