Department of Biology, McGill University, 1205 Doctor Penfield Avenue, Montréal QC, H3A 1B1, Canada
* Author for correspondence (e-mail: laura.nilson{at}mcgill.ca)
Accepted 8 March 2005
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SUMMARY |
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Key words: Drosophila, Polarity, gurken, Genetic mosaic
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Introduction |
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Each Drosophila oocyte develops within a cyst of 16 germline
cells, which is surrounded by an epithelium of somatic follicle cells to form
a developmental unit called an egg chamber
(Spradling, 1993). Each
16-cell cyst is derived from a single cell, the cystoblast, through four
synchronized mitoses. Cytokinesis is incomplete during these divisions,
forming a stereotypic pattern of cytoplasmic bridges, called ring canals,
between the cells of the cyst (see Fig.
1A). One of the cells with four ring canals develops as the
oocyte, and comes to occupy the posteriormost position in the cyst, while the
other 15 cells become nurse cells, which supply essential components to the
oocyte through the ring canals. The AP axis of the egg chamber is established
when the oocyte adopts its posterior position within the cyst. The final AP
polarity of the oocyte itself is generated during mid-oogenesis through a
localized signal from the oocyte to the overlying follicle cells. In later
stages, another localized signaling event between the oocyte and follicle
cells defines the DV axis of the egg chamber
(Huynh and St Johnston, 2004
;
Van Buskirk and Schüpbach,
1999
).
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Proper localization of Grk is crucial for the generation of axial polarity.
Mutations in the maelstrom gene alter grk mRNA localization
in early stages and result in defective posterior follicle cell fate
determination (Clegg et al.,
1997). In ovaries overexpressing grk or lacking the
function of squid (sqd) or fs(1)K10, grk is not
restricted dorsally but is instead distributed throughout the anterior cortex
of the oocyte, resulting in expanded induction of dorsal follicle cell fates
(Neuman-Silberberg and Schüpbach,
1993
). The asymmetric localization of grk within the
oocyte requires the association of grk transcripts with hnRNPs and
the activity of the microtubule motor proteins Dynein and Kinesin
(Brendza et al., 2002
;
Duncan and Warrior, 2002
;
Januschke et al., 2002
;
MacDougall et al., 2003
).
However, how these factors function to achieve the unique dorsal anterior
localization of grk is not well understood.
The close association of grk mRNA and protein with the oocyte
nucleus has led to the proposal that anchoring of the message upon export from
the oocyte nucleus mediates or contributes to its localization
(Goodrich et al., 2004;
Norvell et al., 1999
;
Palacios and St Johnston,
2001
; Saunders and Cohen,
1999
). This model has been supported by evidence suggesting that
grk is transcribed primarily or exclusively in the oocyte nucleus,
derived from experiments designed to exclude the possibility of transport from
the nurse cells to the oocyte (Saunders
and Cohen, 1999
). Such a mechanism would require that grk
be a rare gene transcribed in the oocyte nucleus, which is otherwise
transcriptionally quiescent and arrested in meiotic prophase
(King and Burnett, 1959
;
Spradling, 1993
).
Alternatively, grk may be transcribed in the nurse cells and
transported to the oocyte, as are transcripts encoding other spatially
restricted determinants, such as bicoid and oskar
(osk), found at the anterior and posterior poles, respectively
(Johnstone and Lasko, 2001
;
Lipshitz and Smibert, 2000
;
Palacios and St Johnston,
2001
). Production of grk in the nurse cells would require
an alternative model for localization, as a localized synthesis and retention
mechanism would not apply to nurse-cell-derived transcripts. For example,
microtubule-based transport has been shown to function in grk
localization within the oocyte. However, since the existence of a localization
mechanism within the oocyte could equally be proposed to localize
nurse-cell-derived messages or to maintain localization of oocyte-derived
messages, this observation does not address the source of grk
transcripts.
Using a novel application of a standard genetic technique to address this question, we generated egg chambers with mosaic germline cysts in which the oocyte lacked the ability to produce grk but the nurse cells retained grk function. This genetic approach provided a stringent functional test for grk production: if grk were produced exclusively in the oocyte nucleus, then mosaic egg chambers with a mutant oocyte would be predicted to exhibit patterning defects. Our results demonstrate that the nurse cells produce functional grk, and that their contribution is sufficient for proper Grk localization within the oocyte and establishment of the oocyte AP and DV axes. While our data do not exclude the oocyte nucleus as a potential additional source of grk transcripts, any such contribution is not required for axis determination. Our findings imply the existence of a mechanism for transport of grk from the nurse cells and its subsequent localization within the oocyte.
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Materials and methods |
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Production of germline mosaic egg chambers
Mosaic ovaries with the nuclear GFP clone marker were produced by
FLP-FRT-mediated mitotic recombination (Xu
and Rubin, 1993) in females of the following two genotypes: y
w P{hsFLP}122; grk2B6 P{neoFRT}40A/P{Ubi-GFPnls}2-1
P{neoFRT}40A and y w P{hsFLP}122; grk2B6
P{neoFRT}40A/P{Ubi-GFPnls}2-1 P{neoFRT}40A;
P{lacW}l(2)mirr6D1. To induce recombination, pupae were
heatshocked in a water bath for 1 hour at 37°C on each of three
consecutive days beginning 2 days after puparium formation. The vials were
maintained at 25°C at all other times, and adult ovaries were harvested 10
days after the first heatshock.
To generate ovaries mosaic for the lacO transgene, recombination was induced as above in females of the following genotype: y w P{hsFLP}122; grk2B6 P{lacO256x}2L P{neoFRT}40A/P{NM}31E P{neoFRT}40A; P{Hsp83-GFP::lacI}. In addition, females were heatshocked at 37°C for 20 minutes, 7 hours before dissection to induce expression of GFP::LacI. To confirm that the number of foci of GFP fluorescence observed in the oocyte nucleus corresponds to the number of copies of the P{lacO256x} transgene present, we induced GFP::LacI expression in non-mosaic females either homozygous or heterozygous for a transgene-containing chromosome. Multiple fluorescent foci are readily detected in all nurse cell nuclei, which are polyploid, but individual foci in the oocyte nucleus are more difficult to detect. In females with two copies of the lacO transgene, two foci were detected in 6/49 stage-8-9 egg chambers, and the remainder exhibited either one focus (8/49) or no foci (35/49). A single focus of GFP fluorescence was detected in the oocyte in 4/23 stage-8-9 egg chambers from females with a single copy of the transgene; importantly, two foci were never observed, indicating that the presence of two foci is diagnostic for homozygosity of the transgene-containing chromosome. Because the inefficiency of detection of the lacO transgene in the oocyte nucleus prevented the unambiguous diagnosis of its absence, we placed the lacO transgene in cis to the grk2B6 allele to allow positive identification of homozygous mutant oocytes by the presence of two foci in the nucleus (see Fig. 1C). Foci were undetectable in nearly all egg chambers after stage 9, precluding the use of this marker for analysis of DV patterning.
Immunohistochemistry
Ovaries were dissected in phosphate-buffered saline (PBS) and fixed at room
temperature for 20 minutes in 5% formaldehyde (EM grade; Polysciences, Inc),
in PBS with 1% NP40, saturated with heptane, then washed three times for 10
minutes in PBS with 0.3% Triton-X100 (PBST). Ovaries were further
permeabilized by incubating at room temperature for 1 hour in PBS with 1%
Triton-X100, then blocked for 1 hour in PBST with 1% bovine serum albumin
(BSA). Ovaries were then incubated overnight at 4°C in PBST with a 1:100
dilution of primary monoclonal antibody [anti-Grk, MAb1D12, or
anti-ß-galactosidase, MAb40-1a, concentrated, Developmental Studies
Hybridoma Bank; anti-Broad Core (BR-C) MAb25E9, supernatant, gift of Greg
Guild], washed three times for 20 minutes in PBST, incubated for 1 hour in
PBST with 1% BSA, then incubated for 90 minutes with goat anti-mouse
AlexaFluor568nm (1:1000, Molecular Probes) in PBST. Samples were
washed three times for 20 minutes in PBST, then incubated for 10 minutes in
PBST with rhodamine-conjugated phalloidin (1:1000, Molecular Probes) and DAPI
(1:1000, Molecular Probes). After manual removal of stage-14 egg chambers,
samples were mounted using the Slowfade Light Antifade Kit (Molecular
Probes).
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Results |
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As a genetic marker for these mosaics, we used a transgene expressing a nuclear form of GFP. This marker was placed in trans to the grk2B6 allele, so that homozygous mutant cells were marked by the lack of GFP (Fig. 1B). Egg chambers with homozygous mutant germline clones were readily recognized by the absence of detectable GFP fluorescence throughout the germline, while mosaic cysts exhibited a combination of nuclei with and without GFP. Though well established as a marker for clonal analysis, we anticipated that the use of nuclear GFP as a genotypic marker in mosaic cysts would be less straightforward. Due to the transport of material from the nurse cells to the developing oocyte through the cytoplasmic bridges connecting the germline cells, any GFP in the oocyte nucleus could include a contribution from the nurse cells. Moreover, it was unclear whether the GFP transgene would be expressed in the transcriptionally inactive oocyte nucleus. To circumvent these issues, we avoided assessing the genotype of the oocyte directly by restricting our analysis to egg chambers in which half the cells were homozygous wild type, as determined by uniformly high levels of GFP, and half were homozygous mutant, as determined by the lack of GFP. Given that the germline mitoses occur in an invariant pattern, mitotic recombination in the first division will always produce precisely eight cells of each genotype. Therefore in egg chambers with eight homozygous wild-type nurse cells, with uniformly high nuclear GFP, and seven grk2B6 homozygous nurse cells, with little or no GFP, we could infer clearly that the oocyte must be the eighth mutant germline cell. In the reciprocal mosaics, with uniformly high nuclear GFP in seven nurse cells and little or none in the remaining eight, we concluded that the oocyte must be the eighth homozygous wild-type cell.
The four consecutive mitoses that generate each germline cyst result in a configuration of germline cells in which the oocyte is directly connected to four of the nurse cells: one is its sister from the first mitosis and the other three are its daughters from the subsequent rounds (see Fig. 1A). In 130/131 examples of germline mosaics with half wild-type and half mutant cells, we noted that three of these four nurse cells shared the deduced oocyte genotype, while the fourth had the opposite genotype. This nearly invariant configuration confirms our assessment of oocyte genotypes and demonstrates that these mosaics are generated primarily by recombination events occurring during the first round of germline mitoses. Multiple recombination events in subsequent mitoses probably generated the single exceptional mosaic.
In parallel, to determine the genotype of the oocyte directly, we generated
germline mosaics using a transgene containing 256 direct repeats of the
lac operator (lacO) as a genetic marker
(Robinett et al., 1996;
Straight et al., 1996
;
Vazquez et al., 2001
). The
presence of the transgene was visualized by the binding of a nuclear
GFP-tagged Lac repressor protein (GFP-LacI), which binds to the lacO
transgene and yields a discrete focus of nuclear fluorescence. In this system,
the integrated lacO transgene itself functions as the genotypic
marker, and is therefore strictly cell-autonomous. In ovaries from females
heterozygous for the lacO transgene, a single focus was present in
the oocyte nucleus, whereas in the nurse cell nuclei, which are highly
polyploid with partially dispersed chromatids, multiple foci were visible. For
generation of germline mosaics, we constructed a chromosome containing the
lacO transgene in cis to the grk2B6
allele, so that homozygous grk mutant cells would be homozygous for
the transgene as well. This configuration allowed us to recognize a homozygous
mutant oocyte directly by the presence of two clear foci of GFP fluorescence
within the oocyte nucleus (Fig.
1C).
Synthesis of grk in the oocyte nucleus is not required for AP axis establishment
We examined whether production of grk in the oocyte nucleus is
required for AP patterning of the egg chamber by observing the position of the
oocyte nucleus. As described above, the movement of the oocyte nucleus from a
central position at the oocyte posterior to an asymmetric location at the
anterior cortex requires the correct polarization of the oocyte microtubule
network, which in turn depends upon induction of posterior follicle cell fates
by Grk signaling in early oogenesis. In the absence of grk function,
this sequence of events is not initiated and the oocyte nucleus remains at the
posterior of the oocyte (Gonzalez-Reyes et
al., 1995; Roth et al.,
1995
).
In egg chambers with germline clones, in which all germline cells were
homozygous for the grk2B6 allele and therefore lacked
grk function, the oocyte nucleus was located at the posterior of the
oocyte in 94/154 cases observed (61%; Fig.
2A). This frequency of mislocalization is consistent with previous
analyses, which report posterior localization of the oocyte nucleus in 31-70%
of egg chambers from homozygous grk mutant females, depending on the
allelic combination (Gonzalez-Reyes et al.,
1995; Roth et al.,
1995
). This defect confirms the effect of the
grk2B6 mutation on grk function and provides an
important internal control for the analysis of egg chambers with mosaic
germlines, which are recovered from the same females. We then analyzed
GFP-marked germline mosaics consisting of eight homozygous wild-type and eight
homozygous mutant cells. In 21/21 control mosaics, in which the oocyte was
homozygous for the wild-type grk allele, the oocyte nucleus was
properly localized at the anterior margin of the oocyte
(Fig. 2B). Strikingly, anterior
localization of the oocyte nucleus was also observed in 22/22 mosaics in which
the oocyte was homozygous for the grk2B6 allele
(Fig. 2C). These observations
indicate that transcription of grk in the oocyte nucleus is not
required for oocyte AP polarity.
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Synthesis of grk in the oocyte nucleus is not required to establish the oocyte DV axis
Coincident with the movement of the oocyte nucleus to the anterior cortex
of the oocyte, grk mRNA and protein also relocalize, remaining
closely associated with the oocyte nucleus and achieving an asymmetric
anterior localization that defines the dorsal side of the oocyte and thus the
DV axis of the egg chamber. While the data presented above indicate that the
production of grk by the nurse cells is sufficient for AP patterning,
this analysis does not address the possibility that, at later stages of
oogenesis, transcription of grk in the oocyte nucleus is required for
DV patterning. Indeed, it has been speculated that grk may be
produced in all germline cells during early stages, for grk-mediated
AP patterning, then produced primarily or exclusively in the oocyte nucleus in
later stages, leading to the dorsally restricted distribution required for DV
patterning (MacDougall et al.,
2003; Norvell et al.,
1999
; Thio et al.,
2000
).
To investigate this possibility, we assessed dorsal follicle cell fate
determination in grk mosaics. As a cell fate marker we used an
enhancer trap inserted in the mirror locus, mirr-lacZ, which
drives expression of a lacZ reporter gene in dorsal anterior follicle
cells in response to Grk-Egfr signaling
(Fig. 4A)
(Jordan et al., 2000;
Zhao et al., 2000
). Egg
chambers with germline clones homozygous for the grk2B6
mutation exhibited no follicle cell expression of mirr-lacZ,
confirming that expression of this reporter is grk-dependent
(Fig. 4B). By contrast, in
germline mosaic egg chambers derived from the same females, expression of the
mirr-lacZ marker was detected in dorsal anterior follicle cells, even
when the oocyte contained no functional copies of the grk gene
(Fig. 4C,D). We found no
significant difference in the number of lacZ-positive follicle cells
between stage-10 germline mosaics with a grk mutant oocyte
(n=12) and germline mosaics with either a wild-type oocyte
(n=7) or grk2B6 heterozygous egg chambers
(n=25), from which germline mosaics are derived. While these data
reveal no gross alterations in DV patterning in the absence of a functional
grk gene in the oocyte nucleus, because the mirr-lacZ
expression pattern is dynamic and the number of positive cells varies even
among stage-10 egg chambers from grk heterozygotes, any subtle
changes in expression within this range would be difficult to detect.
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Border cell migration does not require synthesis of grk in the oocyte
Grk-Egfr signaling is also required to guide the migration of a specialized
subpopulation of follicle cells called border cells. This cluster of cells
delaminates from the anterior follicular epithelium at stage 9 of oogenesis
and migrates posteriorly, between the nurse cells, toward the oocyte. Upon
reaching the anterior margin of the oocyte, the border cells migrate dorsally
and assume a position adjacent to the oocyte nucleus
(Spradling, 1993). Loss of Grk
function in the germline or Egfr function in the border cells themselves leads
to the failure of the dorsal phase of this migration, indicating that Grk
provides a spatial cue that acts through Egfr to guide border cell migration
(Duchek and Rorth, 2001
).
To determine whether synthesis of grk in the oocyte nucleus is
required for border cell guidance, we examined the position of the border cell
cluster in germline mosaic egg chambers of the appropriate stage. Border cells
were dorsally localized along the anterior margin of the oocyte in 12/14
germline mosaics with a wild-type oocyte, comparable to previous observations
of wild-type egg chambers (Duchek and
Rorth, 2001). In germline mosaics with a grk mutant
oocyte, the border cell cluster was dorsally localized in 14/15 cases. These
data indicate that synthesis of functional grk transcripts in the
oocyte nucleus is not required to guide border cell migration.
Production of grk in the oocyte nucleus is not required for localization of Grk protein
The observation that AP and DV patterning occur normally in germline
mosaics with a homozygous grk mutant oocyte suggests that the Grk
signal is properly localized in these egg chambers. To test this prediction,
we visualized the localization of the Grk protein in mosaic egg chambers using
a monoclonal antibody. No specific immunoreactivity was observed in 14/14
homozygous mutant germline clones, confirming the specificity of the antibody
(data not shown). In germline mosaics, the localization of Grk in those with a
homozygous mutant oocyte (Fig.
6B,C) was indistinguishable from that of those with a wild-type
oocyte (Fig. 6A). This
observation indicates that normal Grk localization is achieved even when the
transcript is derived exclusively from the nurse cells and indicates that
production of wild-type grk transcripts in the oocyte is not required
to generate a spatially restricted Grk signal.
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Discussion |
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Does the oocyte nucleus produce grk?
Evidence for transcription of grk in the oocyte nucleus has been
reported previously (Saunders and Cohen,
1999). After treatment of females with colchicine to disrupt
microtubule-based transport from the nurse cells to the oocyte, osk
transcripts were retained in the nurse cells while grk transcripts
were detected exclusively in the oocyte, consistent with grk
transcription in the oocyte nucleus. However, it is also possible that there
is a fundamental difference between grk and osk in the
timing or mode of transport. For example, blocking transport of grk
to the oocyte could require a level of microtubule disruption equivalent to
that which would disrupt oocyte specification
(Koch and Spitzer, 1983
),
potentially confounding analysis by this method. Alternatively, transport may
occur via a microtubule-independent mechanism, or along microtubules that are
stable and therefore insensitive to disruption by treatment with colchicine,
which affects only dynamic microtubules. Such a population of stable
microtubules, present at least in the early stages of oogenesis, has recently
been described (Roper and Brown,
2004
).
In a complementary approach, transcripts from a reporter construct under
the control of the grk promoter were observed to accumulate
exclusively in the oocyte, consistent with activity of this promoter
exclusively in the oocyte nucleus
(Saunders and Cohen, 1999).
However, subsequent mapping of grk transcripts suggested that the
grk transcription start site is located farther upstream than
previously recognized (Thio et al.,
2000
), indicating this construct probably contained elements
present within the grk 5' UTR. While there is no evidence that
this sequence is relevant to mRNA transport or localization, strong
conclusions about grk promoter activity cannot be drawn.
Our genetic approach demonstrates that the nurse cells provide grk to the oocyte. However, because it is difficult to detect subtle or late changes in patterning, our data do not exclude a contribution of grk by the oocyte nucleus. Given the lack of obvious defects in mosaics with a grk mutant oocyte, and the greater synthetic capacity of the polyploid nurse cells, it seems likely that any grk contribution by the oocyte nucleus would be minor. Nevertheless, the important conclusion of this work is the clear demonstration that the nurse cells transcribe grk, and that these transcripts are sufficient for normal Grk localization and the establishment of the oocyte AP and DV axes. These observations imply the existence of a mechanism for their transport from the nurse cells and subsequent dorsal anterior localization within the oocyte.
Implications for grk mRNA localization
We have been unable to visualize the distribution of grk mRNA in
germline mosaics, due to the incompatibility of GFP fluorescence with
conditions required for in-situ hybridization and the inconsistent levels of
signal obtained with available anti-GFP antibodies. However, it seems likely
that the normal distribution of Grk in mosaics with a mutant oocyte reflects
proper localization of grk mRNA. The alternative possibility, that
grk mRNA is mislocalized in mosaics with a grk mutant oocyte
but yields a properly localized protein, would imply that synthesis of
wild-type grk transcripts in the oocyte nucleus is required to
localize nurse-cell-derived transcripts. Although there is evidence for
translational regulation of unlocalized grk, which would account for
the normal Grk distribution observed in such mosaics
(Norvell et al., 1999), it is
unclear how localization of grk transcripts from the nurse cells
would depend on additional grk production in the oocyte nucleus.
Although we cannot exclude such a model, due to its complexity it seems less
likely.
The site of grk transcription has important implications for the
consideration of potential localization mechanisms. While the localization of
the grk mRNA between the oocyte nucleus and adjacent cortex has led
to the proposal that this distribution arises from grk transcription
in the oocyte nucleus and local anchoring of grk transcripts
(Goodrich et al., 2004;
Norvell et al., 1999
;
Palacios and St Johnston,
2001
; Saunders and Cohen,
1999
), such a model would not address the proper localization and
patterning function of grk contributed by the nurse cells. As
grk encodes a secreted protein, it has also been suggested that a
dorsal anterior concentration of exocytic pathway components within the oocyte
could contribute to its localization. A careful analysis of transitional
endoplasmic reticulum and Golgi compartments, however, reveals a uniform
distribution and indicates that polarized Grk distribution is driven by the
localization of its mRNA (Herpers and
Rabouille, 2004
).
The mechanism of transport of grk transcripts from the nurse cells
to the oocyte is unknown. However, within the oocyte, proper dorsal anterior
localization of grk mRNA requires the heterogeneous nuclear
ribonuclear protein (hnRNP) proteins Sqd (also known as Hrp40) and Hrb27C
(also known as Hrp48), which bind to the 3' UTR of the grk mRNA
(Goodrich et al., 2004;
Neuman-Silberberg and Schüpbach,
1993
; Norvell et al.,
1999
). These hnRNPs form a complex with the nascent grk
transcript, then recruit cytoplasmic proteins to the grk RNP complex
upon its export from the nucleus to regulate grk localization and
translation in the cytoplasm (Goodrich et
al., 2004
; Norvell et al.,
1999
). Although this model proposes that the interaction of Sqd
and Hrb27C with the grk transcript occurs in the oocyte nucleus
(Goodrich et al., 2004
;
Norvell et al., 1999
), it
seems plausible to suggest that these complexes assemble in the nurse cells to
regulate grk localization within the oocyte. Localization of
grk mRNA within the oocyte also requires transport on microtubules,
because disruption of the minus-end-directed microtubule motor cytoplasmic
Dynein results in defects in grk localization
(Brendza et al., 2002
;
Duncan and Warrior, 2002
;
Januschke et al., 2002
;
MacDougall et al., 2003
). It
is unclear, however, whether these factors are required for transport of
grk into the oocyte, as the most obvious consequence of loss of their
function is mislocalization of grk mRNA to the oocyte anterior
margin.
Taken together with previous work, our data favor a model in which
grk transcripts are assembled in the nurse cell nuclei into hnRNP
particles containing Sqd and Hrb27C, followed by recruitment upon nuclear
export of cytoplasmic factors regulating localization and translation. These
grk-containing complexes would ultimately associate with microtubule
motors, resulting in minus-end-directed transport along microtubules emanating
from the dorsal anterior region; indeed there is evidence for a scaffold of
microtubules around the oocyte nucleus
(Clark et al., 1997;
MacDougall et al., 2003
;
Theurkauf et al., 1992
).
Remaining to be resolved, however, is a mechanism that would distinguish
microtubule-based localization of grk from that of anteriorly
localized messages that are not dorsally restricted. Although grk
could interact with specific trans-acting factors in a distinct hnRNP
particle, a potential sorting mechanism would nevertheless require differences
in the dorsally oriented microtubules to allow recognition by motors carrying
grk-containing particles. While modifications of tubulin itself or
the association with distinct microtubule-associating proteins could
distinguish microtubule networks
(Westermann and Weber, 2003
),
the mechanism underlying the sorting of grk from other anterior
transcripts remains to be determined.
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ACKNOWLEDGMENTS |
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