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First published online 20 September 2006
doi: 10.1242/dev.02563
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1 Section of Molecular Cell and Developmental Biology, Institute for Cellular
and Molecular Biology, University of Texas, Austin, TX 78712, USA.
2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA
15260, USA.
3 Institute for Neuroscience, University of Texas, Austin, TX 78712, USA.
Author for correspondence (e-mail:
wallingford{at}mail.utexas.edu)
Accepted 3 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin at the apical surfaces of
naïve epithelial cells. Together, our data identify Shroom2 as a central
regulator of RPE pigmentation, and suggest that, despite their diverse
biological roles, Shroom family proteins share a common activity. Finally,
because the locus encoding human SHROOM2 lies within the critical region for
two distinct forms of ocular albinism, it is possible that SHROOM2
mutations may be a contributing factor in these human visual system
disorders.
Key words: Shroom, Shroom2, Retina, RPE, APXL, Melanosome, Ocular albinism
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). RPE pigmentation is a result of the accumulation of
melanosomes-modified lysosomes that contain a dense array of black pigment
granules. Melanosome biogenesis and localization in the RPE requires several
identified proteins including tyrosinase, AP3, members of the BLOC complex,
Rab27a and OA1. Many of the genes encoding these proteins are mutated in human
genetic disorders that affect RPE pigmentation, underscoring their importance
in the process (for reviews, see Marks and
Seabra, 2001; Raposo and
Marks, 2002).
The biogenesis of melanosomes from endosomes is a multistep process
involving a suite of protein components
(Marks and Seabra, 2001;
Raposo and Marks, 2002). Once
formed, mature melanosomes are deployed to the apical surface of the RPE via
both microtubule and actin cytoskeletons
(Barral and Seabra, 2004).
Melanosomes first move to the apical surface along microtubule tracks and are
then thought to be transferred to an apical actin network through which they
are deployed to their final positions
(Burnside et al., 1983;
Gibbs et al., 2004;
King-Smith et al., 1997;
Maniak, 2003;
McNeil et al., 2004;
Troutt and Burnside, 1989).
Indeed, molecules involved in melanosome deployment also underlie some human
visual system disorders. For example, mutations in Rab27a result in Griscelli
syndrome Type I, a disorder in which the movement of melanosomes along actin
filaments is disrupted (Seabra and
Coudrier, 2004). Although microtubules are known to be essential
for the maturation and positioning of endosomes
(Apodaca, 2001;
Bomsel et al., 1990), no
microtubule regulators have yet been identified as essential for melanosome
positioning or biogenesis.
In this paper, we report that Shroom2 (previously called APXL)
(Schiaffino et al., 1995b) is
essential for the proper pigmentation of the RPE. Shroom family proteins are
marked by regions of significant protein sequence similarity between different
family members (Fig. 1A);
however, to date, each member has been found to govern apparently unrelated
biological processes (Hagens et al.,
2006). The most thoroughly studied family member, Shroom3, encodes
an actin-binding protein that is essential for apical constriction, a cell
shape change that is crucial to vertebrate neural tube closure
(Haigo et al., 2003;
Hildebrand, 2005;
Hildebrand and Soriano, 1999).
Shroom1 (previously called APX) is thought to link Na+ channel
activity to the actin cytoskeleton (Prat
et al., 1996; Staub et al.,
1992; Zuckerman et al.,
1999), whereas a third member of the family, Shroom4 (previously
called KIAA1202), has recently been shown to be mutated in humans with
X-linked mental retardation (Hagens et
al., 2005). Recent studies have implicated Shroom2 in regulating
the actin cytoskeleton (Dietz et al.,
2006), but no in vivo developmental studies of Shroom2 have yet
been reported.
In Xenopus embryos lacking Shroom2 function, we find that defects
in RPE pigmentation stem from failures of melanosomes to mature and to
associate with the apical cell surface. Ectopic expression of Shroom2 in
naïve epithelial cells facilitates apical pigment accumulation, and this
activity specifically requires the Rab27a GTPase. Most interestingly, we find
that Shroom2 shares at least one cellular activity with Shroom3: either
protein is sufficient to induce a dramatic apical accumulation of the
microtubule-nucleating protein -tubulin in naïve cells. Together,
our data identify Shroom2 as a central regulator of RPE pigmentation, and
suggest that, despite their diverse biological roles, Shroom family proteins
share at least one common cellular activity.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and embryos were staged according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1994).
Embryos were imaged using a Leica MZ16FA stereoscope and subsequently
processed in Adobe Photoshop 7.0.
In situ hybridization
Whole-mount in situ hybridization was carried out on Xenopus
embryos as described previously (Sive et
al., 2000). Whole-mount embryos were then imaged using a Leica
MZ16FA stereoscope. Subsequently, embryos were prepared for cryosectioning by
fixing in MEMFA overnight at 4°C, washing three times for five minutes in
PBS + 0.1% Tween-20, then cryoprotecting by immersion in 25% sucrose at room
temperature until equilibrated and increased to 40% sucrose overnight at
4°C. Embryos were frozen in TBS tissue freezing medium (Triangle
Biomedical Sciences), sectioned at 12 µm in a cryostat and collected on
gelatin-coated slides. Slides were then mounted either temporarily with 90%
glycerol + 10% 1x PBS, or permanently with DPX (Electron Microscopy
Sciences) and imaged using a Zeiss Axioplan 2 mounted with SPOT digital
camera. Images were subsequently processed using Adobe Photoshop 7.0.
Morphometrics
For cell-surface area measurements, mRNA was injected animally into one
blastomere of a four-cell embryo. Embryos were then cultured until stage 8.
Embryos were then fixed in MEMFA overnight at 4°C, then rinsed three times
for five minutes in PBS + 0.1% Tween-20. Embryos were then imaged using a
Leica MZ16FA stereoscope. Cells were individually traced and surface area
calculated using Image-Pro Plus software. For morphant eye pigmentation
comparison, embryos were injected as described, cultured, fixed, washed and
imaged using a Leica MZ16FA stereoscope. Eyes were then individually traced
and pixel density calculated for each in Image-Pro Plus. For melanosome
morphometrics, TEM images were loaded into Image-Pro Plus software and
analyzed using the roundness and area functions.
Actin visualization
Embryos were fixed overnight at 4°C in MEMFA, then rinsed three times
for five minutes in PBS + 0.1% Tween-20. Embryos were then incubated in 4 U/ml
of Oregon Green 488 phalloidin (Molecular Probes) in PBS + 0.1% Tween-20
overnight at 4°C. Just before visualization on a Zeiss LSM5 PASCAL
confocal microscope, embryos were rinsed twice for 10 minutes in PBS + 0.1%
Tween-20. Images were subsequently processed using Adobe Photoshop 7.0.
Cloning of Shroom2(576-882), truncShroom2 and design of morpholino oligonucleotides
A dominant-negative fragment of mouse Shroom2 was amplified from
CS3+MT-mouse Shroom2 by PCR using Turbo Pfu polymerase (Stratagene) and the
following primer set: 5'-ACTATGGCCAGCCGCACTGTGAGAAC-3' and
5'-CAGTGACCTCTTCTGGAACC-3' (spanning amino acids 576-882). After
addition of 3'A-overhangs post-amplification by Taq polymerase, the PCR
product was cloned into TOPO vector (Invitrogen) and then transferred into
CS2+.
A truncated version of Shroom2, similar to the Shroom2-SPL-MO splice product, was created from CS3+MT-mouse Shroom2 by cutting with SacI inside the ASD2 domain, followed by SalI, blunting with Klenow, cutting the 5' end with BamHI, and ligating into CS107-3STOP at the EcoRV site. Blastula assays were performed as described above.
For morpholino oligonucleotide design, genomic sequence for Shroom2 was obtained by BLAST search of trace files from the Joint Genome Institute Xenopus tropicalis genome project. Equivalent regions of X. laevis Shroom2 were amplified by PCR with primers designed against the X. tropicalis sequence. The X. laevis genome sequence was used to design an MO against the 5' splice acceptor site between the ASD1 and ASD2 domains of Shroom2. The sequence of the Shroom2-SPL-MO is 5'-TACTACCATACACACCTTGACAAGT-3'. RT-PCR to check the splicing product was performed with the following primers: Shroom2-spl-up 5'-TGGAATGGAACCGTCTG-3' and Shroom2-spl-dn 5'-GTGTCTGCTTTCCTTCTCAG-3'. The sequence of the control mismatch morpholino (Shroom2-5MM-MO), which was derived from Shroom2-SPL-MO and mismatched at five positions (lowercase), is 5'-TAgTACgATACAgACCTTcACAAcT-3'.
The GenBank Accession Number for Shroom2 in Xenopus tropicalis is: DQ886532.
Lipofection
CS2+mouse Shroom2(576-882) DNA plasmid was transfected into the eye field
of stage 18 embryos using a technique described previously
(Ohnuma et al., 2002).
CS2+memEGFP plasmid DNA was co-transfected to mark the transfected cells. All
plasmids were mixed with DOTAP (Roche) at a ratio of 1 µg of plasmid DNA to
3 µl of DOTAP. Embryos were cultured to stage 42 and fixed overnight at
4°C in MEMFA, then rinsed three times for five minutes in PBS + 0.1%
Tween-20 and imaged using a Leica MZ16FA fluorescence stereoscope. Images were
subsequently processed using Adobe Photoshop 7.0.
Histology and transmission electron microscopy
Embryos were fixed overnight at 4°C in 1% (w/v) paraformaldehyde, 2.5%
glutaraldehyde, 3% sucrose in PBS. They were then briefly washed three times
in PBS and post-fixed in 1% OsO4 for 1 hour at 4°C, after which
they were again briefly washed three times in PBS and dehydrated through a
graded ethanol series (50%, 70%, 80%, 90% and 2 x 100%). Embryos were
further dehydrated twice for 10 minutes in propylene oxide and infiltrated for
1-2 hours in a 50% propylene oxide/50% Epon/Araldite mixture (Polysciences).
They were then incubated overnight in 100% Epon/Araldite resin in Eppendorf
tubes with caps opened to allow for propylene oxide evaporation and resin
infiltration. Embryos were embedded into plastic molds and baked at 60°C
for 2 days. Histological sections 1 µm thick were cut, heat mounted on
glass slides, stained with a 1% Methylene Blue/1% borax solution and imaged on
a Leica DMLB mounted with a Leica DFC320 digital camera. For transmission
electron microscopy, transverse sections were cut through the central retina,
60-80 nm in thickness, and mounted onto slot grids. Sections were stained with
lead citrate and uranyl acetate. Images were obtained on a Philips EM 208
transmission electron microscope (80 kV) via an AMT Advantage HR 1MB digital
camera. Images were subsequently processed using Adobe Photoshop CS.
Antibody staining
Fixed embryos were dehydrated completely in methanol and were bleached in
10% hydrogen peroxide/67% methanol for 3 hours and rehydrated consecutively
with TBS (155 mM NaCl, 10 mM Tris-Cl, pH 7.4). To reduce autofluorescence of
yolk platelets, the embryos were incubated with 100 mM NaBH4 in TBS
for 4 hours at room temperature or overnight at 4°C and rinsed in TBST
(0.1% Triton X-100 in TBS). -Tubulin or myc-tagged mouse Shroom2 was
detected by rabbit polyclonal anti--tubulin (1:200 dilution) or
monoclonal anti-myc (1:1000 dilution, Sigma) antibody diluted in FBS solution
(TBS containing 10% fetal bovine serum and 5% DMSO). Primary antibodies were
detected with Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes), or
Alexa-555 goat anti-mouse IgG (Molecular Probes) diluted 1:250 in FBS
solution. Embryos were cleared in Murray's Clear solution (benzyl
benzoate:benzyl alcohol=2:1).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Hildebrand and Soriano, 1999).
In these studies, ectopic expression of Shroom3 in naïve cells resulted
in pigment accumulation, apical constriction, and actin accumulation at the
apical cell surface (Haigo et al.,
2003). Given that the role of Shroom2 during development is
currently unknown, and given its significant protein similarity to Shroom3, we
hypothesized that it might function similarly.
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; Roberts et al.,
1992). Thus, when mRNA is ectopically expressed in these cells,
phenotypic defects can be attributed to direct effects of the exogenous
protein. This cell population has proven to be useful for studying the effects
of misexpressed proteins on epithelial cell behavior
(Chalmers et al., 2005;
Dollar et al., 2005;
Haigo et al., 2003).
When compared with uninjected control cells, expression of ectopic Shroom2
in naïve blastomeres induced a dramatic accumulation of pigment, similar
to that elicited by ectopic Shroom3 (Fig.
1C-E). Interestingly, although expression of Shroom3 resulted in a
significant constriction of apical cell surface area, expression of Shroom2
did not (Fig.
1D,d',E,e',F). Moreover, whereas apical constriction
of Shroom3-expressing cells is associated with robust accumulation of actin at
the apical cell surface (Fig.
1G,H) (see also Haigo et al.,
2003), the accumulation of pigment in Shroom2-expressing cells was
not (Fig. 1I).
Combined, these observations indicate that although Shroom2 is capable of mediating pigment redistribution in epithelial cells, its mechanism of action differs from Shroom3. Shroom2 does not induce apical actin accumulation or apical constriction. That Shroom2 does not possess these abilities seems to suggest that not all Shroom family proteins function in a similar manner.
Expression of dominant-negative Shroom2 disrupts pigmentation of the RPE
In studies of Shroom3 function, a fragment containing the ASD1 domain
(spanning amino acids 754-1108) was shown to act as a dominant-negative;
expression of Shroom3(754-1108) inhibited Shroom3-induced pigment accumulation
and apical actin accumulation in a dose-dependent manner
(Haigo et al., 2003). We asked
if a similar fragment of Shroom2 would likewise behave in a dominant-negative
manner. The corresponding fragment of Shroom2 spans amino acids 576-882, and
indeed, co-expression of Shroom2(576-882) in naïve blastomeres blocked
the activity of ectopic Shroom2 in a dose-dependent manner
(Fig. 2A-D). This observation
indicates that the ASD1 domain of Shroom2 does behave as a dominant-negative,
and suggests that this domain might link the melanosome related functions of
both Shroom3 and Shroom2.
Shroom2 is expressed strongly in the eye of humans
(Schiaffino et al., 1995b),
mice (Dietz et al., 2006) and
Xenopus (Fig. 3). At
the early stages of eye development, Shroom2 mRNA is expressed throughout the
eye field, and with maturation of the eye Shroom2 mRNA expression becomes
specific to the RPE (Fig.
3A,B). Additionally there is Shroom2 mRNA expression in the brain,
ear, somites and kidney (Fig.
3A,a' and data not shown).
Because ectopic Shroom2 induces pigment accumulation in naïve cells
(Fig. 1) and endogenous Shroom2
is expressed in the RPE, we hypothesized that Shroom2 may function in
regulating pigmentation. To test this hypothesis, we took advantage of the
dominant-negative activity of Shroom2(576-882), and to express the construct
specifically in the eye, we employed a lipofection technique developed for
this purpose (Fig. 4A)
(Ohnuma et al., 2002).
Liposomes containing plasmid DNA were injected into the eye field at stage 18,
when embryos are in the early stages of eye evagination, and lipofected
embryos were allowed to develop to tadpole stages (stage 42).
Expression of Shroom2(576-882) by lipofection resulted in a dramatic
disruption of eye pigmentation (Fig.
4C). In addition to this patchy hypopigmentation, eyes with
Shroom2(576-882)-expressing lens cells appeared to have an abnormal lens
(Fig. 4B,C). Previous
experiments demonstrate that co-lipofected plasmids are almost uniformly
co-expressed (Ohnuma et al.,
2002), so we lineage-traced our lipofections with co-lipofected
GFP. In all cases, hypopigmented regions of Shroom2(576-882)-expressing eyes
displayed high levels of GFP fluorescence
(Fig. 4c'). Using the
same method, expression of GFP alone had no effect on eye morphology or
pigmentation (Fig.
4B,b').
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Injection of Shroom2-SPL-MO into two animal blastomeres at the four-cell stage (Fig. 5B) resulted in severe defects in RPE pigmentation when assayed at stages 35 and 39 (Fig. 5C-e') and compared with stage-matched controls. From our Shroom2-SPL-MO sequence, we designed a mismatch control morpholino with five mismatched base pairs (Shroom2-5MM-MO). This MO is unable to bind to the target site of our Shroom2-SPL-MO and therefore cannot disrupt splicing. Our Shroom2-5MM-MO control morphants had no defects in pigmentation (Fig. 5D,d',F) indicating that our Shroom2-SPL-MO knockdown phenotype is specific and not an artifact of morpholino toxicity. Our findings that either mosaic dominant-negative Shroom2(576-882) expression or Shroom2 morpholino knock-down results in significant defects in RPE pigmentation and indicate that Shroom2 functions at some level in the regulation of this process.
Proper RPE development is known to be necessary for several aspects of
vertebrate eye development and most notably for retinal lamination (e.g.
Raymond and Jackson, 1995;
Jensen et al., 2001). Given
the striking RPE hypopigmentation in both dominant-negative Shroom2(576-882)
mosaic embryos and in Shroom2-SPL-MO morphants, we assessed overall eye
development in the absence of Shroom2 function. Shroom2-SPL-MO morphant eyes
were often smaller in size but contained all ocular structures. In severely
affected Shroom2-SPL-MO morphant eyes the lens was smaller and malformed, and
the cornea appeared thicker; additionally, cell shapes in these tissues were
sometimes abnormal (Fig. 6A,D;
data not shown). When viewed histologically, the most severely affected
Shroom2-SPL-MO morphants often showed prominent retinal lamination defects
when compared with stage-matched controls and Shroom2-SPL-MO morphants showed
increased numbers of pyknotic nuclei in their retinas
(Fig. 6A,D). Less severely
affected Shroom2-SPL-MO morphant retinas displayed significantly better
retinal lamination, although pigmentation defects were still obvious
(Fig. 6B,E).
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Melanosome localization and biogenesis is defective in Shroom2 morphants
TEM analysis also demonstrated that although melanosomes were clearly
present in Shroom2-SPL-MO morphants, their distribution was not limited to the
apical surface, as is the case in control RPE
(Fig. 6C,F). This failure of
melanosome positioning is suggestive of a defect in some aspect of RPE
polarization. Such a failure of RPE polarization could explain the retinal
lamination defects observed in Shroom2-SPL-MO morphants
(Fig. 6), as defective
polarization of the RPE has been shown to have non-cell autonomous effects on
lamination of the retina (Jensen et al.,
2001; Jensen and Westerfield,
2004).
In addition to positioning defects, melanosome morphology was also disrupted in Shroom2-SPL-MO morphants (Fig. 6C,F). We were curious about the nature of these defects and so melanosome structure was further imaged with high magnification TEM (Fig. 7). A representative control RPE region is shown in Fig. 7A, in which melanosomes of various sizes are present, most of which show the dense black pigment accumulation and smooth-edged morphology typical of RPE melanosomes. By contrast, two representative regions of Shroom2-SPL-MO morphant RPE are shown in Fig. 7B. Overall melanosome number in Shroom2-SPL-MO morphants is similar to that in controls; however, the pigment density within these melanosome is in many instances decreased and their morphology is severely abnormal. Numerous small vesicular structures are obvious and in many cases these vesicles appear not to have coalesced into a single unit. This `bunch of grapes' morphology may reflect a failure of endosomes to fuse as the melanosome develops.
In Shroom2-SPL-MO morphants, melanosome aggregates were nearly 90% larger
in area than their control counterparts
(Fig. 7C, P<0.001),
again consistent with a defect in melanosome maturation
(Cortese et al., 2005). We
found the `bunch of grapes' morphology to be intriguing, and we sought to
quantify this defect with morphometric software. We analyzed melanosome shape
using a `roundness' function, which compares the predicted circumference based
on radius to the actual circumference of an object. This function therefore
quantifies the regularity of the surface of an object. A score of 1.00
indicates a perfect circle. We found that melanosomes in wild-type animals had
a mean roundness of 1.36, owing to their normally elongate shape
(Fig. 7A,D). By contrast,
melanosomes in Shroom2-SPL-MO morphants had an average roundness of over 1.7,
consistent with their highly irregular surfaces
(Fig. 7B,D). This difference
was statistically significant (P<0.001). Combined, these results
indicate that Shroom2 is not only necessary for RPE pigmentation and the
apical localization of RPE melanosomes, but also for melanosome
biogenesis.
Rab27a is required for Shroom2-dependent melanosome distribution
We next sought to probe the mechanisms by which Shroom2 may function to
govern RPE pigmentation. Using ectopic expression in naïve
Xenopus blastomeres (see Fig.
2F), we have previously demonstrated that Shroom3-mediated pigment
accumulation and apical constriction require the activity of the Rap1 GTPase
(Haigo et al., 2003) (P.D.F.,
unpublished). We therefore tested for an interaction between Shroom2 and Rap1.
Expression of high doses of dominant-negative Rap1-N17 mutant
(Kitayama et al., 1990) had no
effect on the ability of Shroom2 to elicit pigment accumulation in naïve
blastomeres (Fig. 2E).
Expression of high doses of Rap1GAP
(Rubinfeld et al., 1991),
which also inhibits Rap1 activity, likewise failed to inhibit Shroom2 activity
when co-expressed in Xenopus blastomeres
(Fig. 2E).
We next tested for interaction between Shroom2 and Rab27. This small GTPase
is a crucial mediator of actin-mediated melanosome movement in the eye
(Futter et al., 2004;
Gibbs et al., 2004).
Rab27a-null (Ashen) mice have melanosome distribution
defects in the RPE, where the melanosomes cannot move beyond the adherens
junction to the apical processes (Futter
et al., 2004). Given the hypopigmentation and melanosome
distribution defects in Shroom2-SPL-MO morphants, Rab27a is a good candidate
for mediating Shroom2 activity. For these experiments, we used
dominant-negative mutations of Rab27a identified in humans with Griscelli
syndrome, a disease characterized in part by albinism
(Bahadoran et al., 2003;
Menasche et al., 2003). The
Rab27a-L130P missense mutation is unable to connect melanosomes to myosin and
the actin cytoskeleton (Bahadoran et al.,
2003), and both Rab27a-L130P and Rab27a-A152P mutations
significantly interfere with the GTP and GDP binding activity of Rab27a
(Menasche et al., 2003).
Indeed, co-expression of either dominant-negative Rab27a-L130P or Rab27a-A152P
inhibited the ability of Shroom2 to trigger pigment concentration in
naïve cells (Fig. 2F).
These observations place Rab27a downstream of Shroom2 in the control of
melanosome distribution in epithelial cells and, furthermore, suggest that
Shroom family members use distinct downstream effector GTPases in regulating
cellular processes.
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-tubulin in naïve epithelial cells).
Rab27 is thought to act at the level of the actin cytoskeleton, linking
melanosomes to myosin motors that allow them to move along the apical actin
network and into their final positions below the apical surface
(Futter et al., 2004;
Gibbs et al., 2004;
Seabra and Coudrier, 2004).
Our findings that Shroom2 expression does not elicit significant actin
accumulation (Fig. 1I) and that
it acts upstream of Rab27 (Fig.
2F) suggest a potential role for Shroom2 at the level of
microtubules. This notion was intriguing in light of our recent finding that
Shroom3, a protein closely related to Shroom2, coordinates microtubule
assembly in epithelial cells by governing the localization of the
microtubule-nucleating component -tubulin (C. J. Lee, H. M. Scherr and
J.B.W., unpublished).
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-tubulin localization. Again, using naïve blastomeres
(Fig. 1B), we expressed either
Shroom2 or Shroom3 by mRNA injection, and assessed -tubulin
distribution by immunohistochemistry. We found that expression of either
protein resulted in a dramatic accumulation of -tubulin at the apical
cell surface (Fig.
8A-C,a'-c'). Despite the differences in apical
constriction and actin accumulation (Fig.
1F-I), Shroom2 and Shroom3 were equally effective in driving the
re-localization of -tubulin. The accumulated -tubulin overlapped
entirely with regions of pigment accumulation
(Fig.
8A-C,a'-c').
Our Shroom2 construct is myc-tagged and immunohistochemistry revealed that
the effect of Shroom2 on -tubulin was cell-autonomous
(Fig. 8D-F). Moreover, we found
that Shroom2 protein, when expressed in naïve blastomeres, did not
co-localize with the accumulated -tubulin. Rather, the Shroom2 signal
decorated the cell surface immediately apical to the accumulated
-tubulin (Fig.
8D-F).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin accumulation at the apical epithelial cell surface (Figs
2,
8). When combined, these
results position Shroom2 at the interface of the actin- and microtubule-based
cytoskeletons, and strongly suggest that the regulation of both of these is
required for proper melanosome development and localization in the RPE.
Shroom2, RPE pigmentation and eye development
Melanosome biogenesis is a complex process that has only recently begun to
be elucidated in vivo. Melanosomes derive from the endosomal network of the
cell and go through several distinct stages of maturation to form the mature
melanin-containing organelle (reviewed by
Raposo and Marks, 2002).
Shroom2 morphants show obvious RPE defects where melanosomes are larger than
normal and appear to be composed of small vesicular bodies that have not
coalesced into a uniform melanosome (Fig.
6). It is not clear at what step of the maturation process
melanosome biogenesis is disrupted in these morphants, but our analysis of
Shroom2 function makes it possible to propose an explanation.
We find that Shroom2 acts upstream of Rab27a and that Shroom2 is sufficient
to drive apical accumulation of -tubulin, a crucial component of
microtubule-nucleating complexes
(Gunawardane et al., 2000;
Job et al., 2003;
Stearns et al., 1991). Given
the known functions of -tubulin and its apical localization in RPE
cells (Rizzolo and Joshi,
1993), we suggest that the principle role for Shroom2 is to
regulate microtubule assembly in the RPE by governing the localization of
-tubulin. Such a function for Shroom2 is consistent with the observed
melanosome phenotype, as an intact microtubule cytoskeleton is known to be
necessary for proper maturation and fusion of endosomes in epithelial cells
(Bomsel et al., 1990;
Clarke et al., 2002;
Gruenberg et al., 1989). In
addition to morphological defects, melanosomes in Shroom2 morphants also fail
to localize properly to the apical surface of the RPE
(Fig. 5). It is thought that
apical deployment of melanosomes first involves movement on the microtubule
cytoskeleton and then transfer to the actin cytoskeleton for final deployment
(Maniak, 2003). As such, a
role for Shroom2 in microtubule organization could also explain the failure of
melanosomes to accumulate apically in morphants.
Loss of Shroom2 function also results in defects in retinal lamination and
in anterior eye development (Fig.
5). We can envision several possible scenarios to account for the
observed lamination defects. Shroom2 could play a direct role in the retina by
regulating the survival of retinal cells, by influencing cell fate
specification events during the early phases of retinal development or by
dictating the final laminar position of differentiating retinal neurons.
Shroom2 is expressed in the retinal neuroepithelium prior to neurogenesis
(Fig. 3), making each of these
scenarios formally possible. Alternatively, the observed lamination defects in
Shroom2 morphants could be an indirect consequence of loss of Shroom2 function
in the RPE. The RPE plays an important role in regulating retinal lamination
(e.g. Raymond and Jackson,
1995). In fact, disruption of normal cell polarity specifically in
the RPE is sufficient to induce lamination defects in the zebrafish retina
(Jensen et al., 2001;
Jensen and Westerfield, 2004).
These defects could reflect a role for the Shroom2 protein in regulating the
polarity of RPE cells. Finally, ocular albinos display macular hypoplasias
where retinal morphogenesis is impaired in areas of the retina that are
apposed to hypopigmented RPE (Jeffery,
1997), so our observed neural retina defects could be a result of
observed defects in melanosome biogenesis in the RPE. Further studies will be
required to elucidate the molecular basis for the lamination defects and the
anterior segment defects in Shroom2 morphants and to determine if they result
from direct roles of Shroom2 in this process.
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-tubulin localization and microtubule assembly (C. J. Lee, H. M. Scherr
and J.B.W., unpublished). These data may suggest that microtubule organization
is a key function of Shroom family proteins in general. However, we show here
that -tubulin and Shroom2 do not co-localize apically
(Fig. 8D-F). It is relevant
then to point out that while Shroom2 has minimal effect on actin when
overexpressed (Fig. 1I), the
other family members, Shroom1, Shroom3 and Shroom4, are each associated with
the actin cytoskeleton (Dietz et al.,
2006; Hagens et al.,
2005; Haigo et al.,
2003; Hildebrand,
2005; Zuckerman et al.,
1999). As such, it is possible be that Shroom2 itself is localized
apically by association with actin, and in turn drives the apical localization
of -tubulin. One intriguing possibility is that a general function for
Shroom family proteins is to link the actin and microtubule cytoskeletons.
Shroom2 and ocular albinism in humans
RPE pigmentation deficiencies in humans with Type I X-linked ocular
albinism result in a loss of visual acuity stemming from defective melanosome
biogenesis and hypopigmentation of the RPE
(Shen et al., 2001). Mutations
in the OA1 gene are thought to underlie X-linked ocular albinism, but
mutations in this gene have not been identified in all individuals
(Bassi et al., 1995;
Schiaffino et al., 1995a;
Tijmes et al., 1998).
Furthermore, a second distinct human pigmentation disorder for which no
causative mutations have been identified, ocular albinism with sensorineural
deafness (OASD), also maps to the same region of the X-chromosome
(Tak et al., 2004;
Winship et al., 1993). The
locus encoding human Shroom2 lies within the crucial region for these
albinism-associated disorders, and the Shroom2 gene is immediately adjacent to
OA1 on the human X-chromosome (Schiaffino
et al., 1995b), as it is in Xenopus tropicalis (not
shown). We have demonstrated herein that Shroom2 loss of function results in
striking RPE pathologies that include hypopigmentation stemming from
melanosome biogenesis and localization defects, similar to those observed in
individuals with ocular albinism and mouse models (e.g.
Cortese et al., 2005;
Tak et al., 2004). That the
locus encoding human Shroom2 lies within the genomic region associated with
two distinct forms of ocular albinism suggests that Shroom2 mutations may
possibly be a contributing factor in these human visual system disorders.
These data suggest that further studies are needed in humans with ocular
albinism, and that a detailed analysis of the Shroom2 locus should be
undertaken in individuals with OASD or ocular albinism type I in which OA1
mutations are not present.
|
ACKNOWLEDGMENTS |
|---|
-tubulin antibody. This work was supported by grants
from the Knights Templar Eye Foundation and American Health Assistance
Foundation Macular Degeneration Research Program (#M2006-024) to J.M.G.; by a
grant from NIH (1RO1GM74104) to J.B.W.; and by a Career Award in the
Biomedical Sciences from the Burroughs Wellcome Fund to J.B.W. |
Footnotes |
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