Centrioles are localized in the apical region of the epithelial cells that formed the eye imaginal

32 Pages·2017·1.28 MB·English

by  Callaini Giuliano

© 2018. Published by The Company of Biologists Ltd.

The developing Drosophila eye: an oncoming model to study
centriole reduction

Maria Giovanna Riparbelli, Veronica Persico, Marco Gottardo, Giuliano Callaini*
Department of Life Sciences, Via A. Moro 2, University of Siena, 53100 Siena, Italy

*Corresponding author: e-mail: [email protected]

Key words: centrosome; centriole elimination; centriole structure; Drosophila.

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JCS Advance Online Article. Posted on 19 January 2018
Summary statement

Centriole elimination in the developing Drosophila eye is preceded by the gradual loss of the
pericentriolar material.

Abstract

In the developing Drosophila eye the centrioles of the differentiating retinal cells are not
surrounded by the microtubule nucleating γ-tubulin suggesting that they are unable to
organize functional microtubule organizing centers. Consistent with this idea Cnn and Spd-2,
involved in γ-tubulin recruitment, and the scaffold protein Plp that plays a role in the
organization of the pericentriolar material are lost in the third larval stage. However, the
centrioles maintain their structural integrity and both the parents accumulate Asl and Ana1.
Although the loading of Asl points to the acquisition of the motherhood condition, the
daughter centrioles failed to recruit Plk4 and do not duplicate. However, it is surprising that
also the mother centrioles that accumulate Plk4 never duplicate. This suggests that the loading
of Plk4 is not sufficient, in this system, to allow centriole duplication. By half of the pupal life
the centriole number decreases and structural defects, ranging from incomplete or lacking B-
tubules, are detected. Asl, Ana1 and Sas-4 are still present, suggesting that the centriole
integrity does not depend on these proteins. t
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Introduction

The centrosome is a structured protein complex that recruits microtubule nucleating proteins
and tubulin (Woodruff et al., 2017) acting as the main microtubule organizing center (MTOC)
of the animal cells (Sanchez and Feldman, 2017). By its ability to nucleate the cytoplasmic
microtubule network, the centrosome plays essential roles in various cellular activities,
including cytoplasmic transport, cell movement, chromosome segregation, primary cilia
formation (Bettencourt-Dias et al., 2011; Arquint et al., 2014; Conduit et al., 2015). The
centrosome is also involved in some aspects of the cell division by organizing the mitotic
spindle and by dictating its orientation through the cell cycle (Roubinet and Cabernard, 2014;
Meraldi, 2016). Therefore, the accurate control of centrosome dynamics is instrumental to
avoid a plethora of cellular defects including ciliopathies, microcephalies, aneuploidy and
cancer (Zyss and Gergely, 2009; Crasta et al., 2012; Vitre and Cleveland, 2012; Chavali et al.,
2014; Godinho and Pellman, 2014).
Centrosome elimination is a common feature during gametogenesis of most organisms
(Manandhar et al., 2005; Mikeladze-Dvali et al., 2012; Pimenta-Marques et al., 2016). Female
germ cells, indeed, lose or inactivate their centrosomes to avoid multipolar spindles at
fertilization. Thus, the assembly of the zygotic centrosome is driven by the sperm provided
centrioles (Schatten, 1994). Centrosome elimination has been also reported in
endoreduplicating intestinal cells of Caenorhabditis elegans (Lu and Roy, 2014) and follicle
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cells of Drosophila melanogaster (Mahowald et al., 1979). The alteration of the centrosome ri
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integrity has been also observed in human post-mitotic cells (Zebrowski et al., 2015) and in u
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Drosophila somatic cyst cells (Riparbelli et al., 2009). m
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Since the organization and integrity of the centrosome depends on a pair of centrioles e
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at its heart (Sluder and Rieder, 1985), understanding centriole dynamics is crucial to decipher e
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centrosome behaviour. We have now a quite detailed knowledge of the process of centriole
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composition, architecture and duplication (Lattao et al., 2017), but there is a poor c
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understanding of how the centrioles were eliminated or inactivated in differentiated cells. ci
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It has been recently reported that the starfish oocytes lose mother centrioles before the
daughters (Borrego-Pinto et al., 2016). In this system the mother centriole retains its
microtubule organizing activity and moves by a dynein dependent process toward the plasma
membrane to be eliminated with the extrusion of the polar body. Thus, the elimination of the
parent centrioles is associated with their different activity. The basal bodies of Caenorhabditis
sensory neurons also degenerate early in neuronal differentiation after the formation of the
ciliary structures (Serwas et al., 2017).
In an attempt to study the mechanisms underlying centriole elimination in post-mitotic
cells, we examined centriole behaviour during the developments of the Drosophila eye. The
adult Drosophila eye is formed by about 750 ommatidial units derived by a complex
differentiation process that requires dramatic cell transformations (Wolff and Ready, 1993;
Carthew, 2007). The eye-antennal imaginal disc of third instar Drosophila larvae is crossed
by the morphogenetic furrow that represents the boundary between the anterior region in
which undifferentiated epithelial cells proliferate and the posterior region that holds the
differentiating rhabdomeric cells (Cagan, 2009; Treisman, 2013). The epithelial cells ahead of
the proliferate randomly, but arrest in G1 as they enter the morphogenetic furrow (Thomas et
al., 1994; Treisman and Heberlein, 1998). The cells posterior to the morphogenetic furrow are
organized into regularly spaced groups, the ommatidial preclusters, within which the
differentiation of the photoreceptors occurs (Tomlinson and Ready, 1987; Wolff and Ready,
1991). The remaining uncommitted cells re-enter the cell cycle and undergo a single round of
cell division within the second mitotic wave, before they are terminally arrested (Wolff and
Ready, 1991; de Nooij and Hariharan, 1995; Firth and Baker, 2005; Escudero and Freeman,
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2007). As the distance from the furrow increases, the ommatidial units became complete with p
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the recruitment of the last three rhabdomeric cells and the differentiation of pigment and lens s
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secreting cone cells (Ready et al., 1976; Wolff and Ready, 1991). Since the post-mitotic cell a
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differentiation occurs progressively, successive developmental stages are present from the d
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anterior to the posterior region of the imaginal disc. Thus, the neuroepithelial cells of the p
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developing Drosophila eye represent a suitable model to study the changes in centriole
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dynamics and organization that accompany terminal cell fate specification. e
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We show here that the centrosomes of the Drosophila retinal cells lose their activity
early during the larval stage, whereas the centrioles start to disappear later by half of the pupal
life. Centriole elimination begins with the gradual disassembly of the microtubule wall,
followed by the disappearance of the cartwheel and the loss of the ninefold symmetry.
Moreover, the centrioles of post-mitotic cells fail to duplicate even if they accumulate Asl and
Plk4.

Results

Neuroepithelial cells lost functional centrosomes
The eye-antennal disc of the Drosophila third instar larva is crossed by a distinct groove, the
morphogenetic furrow, that establishes the anterior and posterior regions of the disc (Fig. 1A).
The apical cytoplasm of the retinal cells in the posterior region showed focal accumulations of
tubulin continuous with longitudinal bundles of microtubules (Fig. 1B). These microtubules
radiated from electron-dense material lining the plasma membrane of short microvillus-like
projections (Fig. 1C). Distinct centrioles were found in the apical cell cytoplasm, but they did
not contact the microtubules nor the peripheral electron-dense material (Fig. 1D). These
observations point to non-conventional microtubule organizing centers like those described in
the cone cells of the Drosophila ommatidia (Mogensen et al., 1993) and suggest that
centrioles of the epithelial cells within the posterior region of the imaginal discs were unable
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to properly recruit centrosomal material. To verify if these centrioles lose their ability to ri
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recruit the main centrosomal proteins during the differentiation process of the ommatidia, we
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first analysed the localization of γ-tubulin, the master protein for microtubule nucleation. γ-
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tubulin was found in the anterior region of the third larval imaginal disc as small spots t
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associated with the centrioles of the interphase cells and as large aggregates at the poles of the c
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mitotic cells (Fig. 1E). Behind the morphogenetic furrow only the cells that undergo a new •
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cell division within the second mitotic wave and a few scattered mitotic cells in the more
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posterior region of the disc displayed distinct accumulations of γ-tubulin at their spindle poles ci
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(Fig. 1E). Although, γ-tubulin did not accumulate to the interphase centrosomes of the ell
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differentiating rhabdomeric cells, a feeble labelling was found at their apical surface (Fig. 1F). of
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This staining did not overlap the centrioles but was presumably associated with the nucleation
sites for the longitudinal microtubule bundles seen at the plasma membrane.
γ-tubulin recruitment at the centrosome mainly depends on the pericentriolar protein
centrosomin (Cnn) (Megraw et al., 1999). Thus, we asked whether the accumulation of this
protein may be reduced during the differentiation of the rhabdomeric cells. We find a weak
Cnn staining on centrioles of the interphase epithelial cells located both in the anterior and
posterior regions of the imaginal disc and within the morphogenetic furrow (Fig. 2A). By
contrast, a strong Cnn accumulation was seen at the poles of the dividing cells in the anterior
proliferating region and at the poles of the spindles within the second mitotic wave (Fig. 2A).
The centrioles of the interommatidial cells were feebly stained, whereas the majority of the
centrioles of the rhabdomeric cells did not accumulate Cnn (Fig. 2A). During the early pupal
stages only a few spots of low intensity were observed (data not shown).
The incorporation of Cnn into the pericentriolar material (PCM) is facilitated in
somatic Drosophila cells by Spd-2 (Conduit et al., 2014a). Thus, we expected a temporal and
spatial colocalization of these proteins during the early phases of the eye development. The
anti-Spd-2 antibody, indeed, mainly recognized the centrioles of the interommatidial cells,
whereas most of the centrioles associated with the apical region of the rhabdomeric cells were
devoid of the Spd-2 protein (Fig. 2B).

Centrioles disappeared during eye development
Since the behaviour of the centrosome depends on the centrioles at its heart we analysed their
dynamic during the eye development. We traced centrioles by the localization of the
conserved centriole-specific core protein Sas-4 that provides a link between the cartwheel and
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the microtubule wall (Hsu et al., 2008; Tang et al., 2011) and may represent a bona fide p
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marker of centrioles in the developing eye. s
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Distinct centriole pairs were found behind the morphogenetic furrow on the narrow a
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apical regions of the cells that formed the ommatidial preclusters and within the d
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undifferentiated interommatidial cells (Fig. 3A). Each rhabdomeric cell also displayed two p
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apical centrioles. Since the ommatidia consist of eight rhabdomeric cells, we found evenly
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spaced groups of eight couples of centrioles within the posterior region of the disc (Fig. 3A). e
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Surprisingly the Sas-4 signal had a different intensity within the centrioles of the same e
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pair (Fig. 3B). To decipher if this signal was a property of only one parent centriole, we ell
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counterstained the eye imaginal discs with an antibody against centrobin (Cnb) that
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specifically recognized the daughter centrioles. Centrobin stain overlapped with the centrioles al
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that had lower Sas-4 intensity (Fig. 3B) that may be, therefore, believed as daughters. The
lower Sas-4 accumulation at the daughter centrioles was also observed when the parent
moved away (Fig. 3B).
The eye imaginal disc dramatically changed shape during the transition to the pupal
stage becoming a thin disc-like epithelium in which the ommatidia are separated by a matrix
of unpatterned interommatidial cells arranged in double or triple rows. At about 25 h APF
(after puparium formation) the interommatidial cells sort into single rows disposed in a
precise hexagonal pattern (Fig. 3C). At this developmental stage, the centrioles of the
rhabdomeric cells were barely detectable because the apical surface of these cells had
retracted below the cone cells. By contrast, the centrioles of the interommatidial, cone and
primary pigment cells may be easily identified because they lie at approximately the same
focal plane at the surface of the ommatidial units. Each cell had one distinct centriole pair at
this stage of development. Therefore, apical views of the retina show groups of 12 centrioles,
8 from cone cells and 4 from primary pigment cells, surrounded by the centriole pairs of
several interommatidial cells (Fig. 3C). At about 45 h APF the interommatidial cells flattened
and distinct mechano-sensory bristles were visible at the anterior vertex of each ommatidium
(Fig. 3D). The majority of the interommatidial, cone and pigment cells showed single Sas-4
spots (Fig. 3D). By 65 h APF only 2-3 spots of Sas-4 were found in the apical region of the
ommatidial units (Fig. 3E). The quantification of the centrioles as defined by Sas-4 staining
confirmed the progressive reduction of their number from 25 to 65 h APF (Fig. 3F,G).

Centrioles of the pupal eye lost the scaffold protein Plp, but maintained the core
proteins Asl and Ana1
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It has recently suggested that centriole elimination in Drosophila somatic cells and female p
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germ cell line could be a consequence of the loss of different components of the PCM s
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(Pimenta-Marques et al., 2016). This prompted us to verify if also the centriole disappearance a
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during later stages of eye development was associated with the loss of distinct centriolar d
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constituents that play main roles in centriole/centrosome biogenesis. Since, we have found p
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that γ-tubulin is the first PCM component to be lost, and then Cnn and Spd-2, we asked
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whether the dynamics of the main core centriole proteins involved in PCM recruitment and e
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organization might be also affected during eye development. e
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A distinct role in PCM organization is played by the coiled-coil Pericentrin-like ell
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protein (Plp) that is radially arranged around the centriole wall and organizes a distinct
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scaffold before the PCM was recruited (Mennella et al., 2012). Moreover, the direct al
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interaction between Plp and Cnn is required for normal centrosome organization and activity
during interphase and mitosis of the Drosophila syncytial embryo (Lerit et al., 2015; Richens
et al., 2015). We then sought to analyse the distribution of this peripheral centriolar
component during the eye development. Plp was associated with all the centrioles of the
rhabdomeric cells in the larval eye imaginal disc (Figs 1E, 4A), but start to disappear early in
the pupal stage. By 25 h APF, the anti-Plp antibody recognized 7-8 small spots within the
apical surface of each ommatidium out of 12 recognized at this stage by the Sas-4 antibody
(Fig. 4A). At 45 h APF there were usually 6 Sas-4 spots in the apical region of each
ommatidium but only2-3 of them maintained a detectable Plp signal (Fig. 4A).
The assembly of the PCM around the centrioles requires the products of the genes
Asterless (Asl) and Anastral spindle 1 (Ana1) (Lattao et al., 2017). The recruitment of Spd-2
seems, indeed, to be initially supported by Asl (Conduit et al., 2014a), although additional
observations suggested that Drosophila centrioles lacking Asl may efficiently recruit PCM
(Galletta et al., 2014). Asl, in turn, is recruited and maintained at the centriole by Ana1 (Fu et
al., 2016; Saurya et al., 2016). These proteins extend from the inner centriole to the outermost
part of it and are usually recruited at the daughter centrioles when they acquire motherhood
(Fu and Glover, 2012). Both Ana1 (Fig. 4B) and Asl (Fig. 4C) were found within the
centrioles of the rhabdomeric cells during the larval stage. When the apical surface of the
photoreceptor cells turned by 90° at the beginning of pupal life, the staining for Asl and Ana1
was barely detectable. By contrast, the centrioles of the cone and primary pigment cells
displayed strong Ana1 and Asl signals (Fig. 4B,C). Thus, the apical surface of each
ommatidium at 25 h APF displayed a cluster of 12 centrioles surrounded by the centriole pairs
of the interommatidial cells (Fig. 4B,C). By 45 h APF, we find 6-7 spots of Ana1 or Asl
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within each ommatidial units (Fig. 4B,C). At 65 h APF the spots reduced to 2-3 (Fig. 4B,C). p
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Daughter centrioles failed to recruit Plk4 a
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To understand how long the centrioles maintain their duplication properties we look at the d
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localization of Plk4, a protein kinase at the head of the centriole duplication process p
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(Bettencourt-Dias et al., 2005; Habedanck et al., 2005). Plk4 has a distinct cell cycle
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dependent localization on the centrioles of the anterior region of the larval imaginal disc e
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where the undifferentiated cells proliferated randomly. At the beginning of interphase Plk4 e
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was associated with only one centriole of each pair (Fig. 5A). This centriole expressed more ell
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Sas-4 and was identified as the mother on the basis of the lack of centrobin (Fig. 3B). As
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interphase progressed the daughter centrioles gradually accumulated Plk4 and reached the al
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same fluorescence intensity as the mothers. Both the parent centrioles soon displayed a small
daughter lacking Plk4. The tight centriole pairs then migrated to the opposite poles of the cells
during prometaphase/metaphase (Fig. 5A) and disengaged at anaphase (Fig. 5A). At the end
of telophase the parent centrioles were widely separated and each sister cells inherited a pair
of centrioles with only the mother expressing Plk4 (Fig. 5A). EM analysis of the anterior
region of the larval eye imaginal disc confirmed the presence of interphase cells with
duplicated centrioles (Fig. 5B). The short procentrioles formed by nine A-tubules and some
growing B-tubules were orthogonal to the proximal end of the mother centrioles that were in
turn formed by nine doublet microtubules (Fig. 5B).
No sign of centriole duplication was seen behind the morphogenetic furrow, except
within the second mitotic wave area. The retinal cells of the posterior region of the imaginal
disc displayed a centrioles pair in which only the mother showed a distinct Plk4 accumulation
(Fig. 5C). However, the mother centrioles did not support procentriole assembly despite they
accumulated Plk4. At 25 h APF each retinal cell expressed two Sas-4 spots, but only one Plk4
spot was present (Fig. 5D). As the eye development proceeded the number of centrioles
expressing Plk4 further reduced. The Plk4 labelling was barely detectable by 45 APF when
each cone and pigment cell displayed a single Sas-4 spot (data not shown).
EM analysis revealed distinct procentrioles closely associated with the mothers only
just posterior the morphogenetic furrow where the cells start to divide to enter the second
mitotic wave (data not shown). Behind this tight proliferative area each cell had only two
centrioles that lost their orthogonal configuration and became disorientated (Fig. 5E). One
centriole of the pair was shorter and look like a procentriole (Fig. 5E). This centriole was
composed by a distinct cartwheel and single A-tubules. Only occasionally one or two B-
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tubule was observed. Therefore, in differentiating rhabdomeric cells, the procentrioles did not p
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complete their doublet content and failed to elongate properly. Despite the several cells s
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examined by serial sections in the posterior region of the larval imaginal disc (n=47), never a
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the parent centrioles were seen in association with a newly formed daughter. The different d
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length of the parent centrioles during interphase is unusual, since sister cells inherit at the end p
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of mitosis two disengaged and equally sized centrioles. Serial sections of late telophase cells
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displayed a pair of different sized centrioles at their poles (Fig. 5F) pointing to the failure of e
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centriole elongation during the previous interphase. e
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Centrioles of post-mitotic cells lost their structural integrity
The mother centrioles within the anterior and the posterior regions of the larval imaginal disc
were built by nine doublets of microtubules and a central cartwheel (Fig. 6A). This
organization persisted through the early pupal life. By 45 h APF all the centrioles scored
(n=27) still maintained a central cartwheel, but often they displayed distinct defects of the
microtubule wall. These defects were more evident in cross sections and ranged from
incomplete or lacking B-tubules (Fig. 6B,C). We excluded that these centrioles were remnant
procentrioles that failed to grow. The assembly of the B-tubule occurs, indeed, during
centriole elongation by arc-like projections starting from the external side of the A-tubule,
whereas the disassembly of the B-tubule occurred at the opposite side (Fig. 6B) or at the
middle region of the same tubule (Fig. 6C). The majority of the centrioles scored during later
stages of eye development (75%, n =34) lost their cartwheel and the centriole wall was often
built by eight singlet-doublet tubules (Fig. 6D). Thus, the diameter of these centrioles
(148±2.3 nm; n=9) was reduced compared to the diameter of the centrioles that maintained
the ninefold symmetry (165±1.9 nm: n=21).

Discussion

Recent findings suggest that the centrioles may be modified during development, yet
maintaining their function. In the Caenorhabditis embryo, indeed, the centrioles remodel to
nucleate the axoneme of sensory neurons (Nechipurenko et al., 2017) and during Drosophila
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spermiogenesis the sperm basal body is modified both in protein composition and in
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ultrastructure (Khire et al., 2016). However, the centrioles do not longer persist at the base of s
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the mature sensory cilia in Caenorhabditis suggesting that they are dispensable for cilia m
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maturation and maintenance (Serwas et al., 2017). Similarly, the centrioles of the e
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differentiating Drosophila ommatidia gradually lose their structural organization and then e
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disappear. It is unclear why it would be advantageous to remove the centrioles from the A
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developing Drosophila eye. Perhaps, centriole elimination could prevent the assembly of too
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many centrosomes and unnecessary mitotic spindles that may impair cell differentiation by
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imposing abnormal divisions. It has been, indeed, proposed that centrosome inactivation in ell
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differentiated cells may function as a barrier restricting cell cycle re-entry (Wong et al., 2015). f
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