Axonemal tubules in the distal sperm tail of Wolbachia-infected Drosophila simulans males contain ring-like intraluminal structures that persist after axoneme fragmentation.

Wolbachia are obligate intracellular alphaproteobacteria that enhance their spreading by altering the reproductive mechanisms of several invertebrates. Among the reproductive alterations, Wolbachia also causes cytoplasmic incompatibility that leads to embryo death when infected males are crossed with uninfected females, thus selecting infected females. However, the presence of Wolbachia has important fitness costs and infected Drosophila simulans males produce less sperm than their uninfected counterparts. Such sperm suffer, indeed, of some structural alterations that hinder their proper function. We took advantage of the fact that several sperm have abnormal distal regions of the tail, in which the plasma membrane is broken and the axonemal components splayed, making the ultrastructural aspects clearly observable. We found that axoneme reduction in the distal region of the sperm does not follow a unique pattern as observed in other insects, but occurs by losing accessory tubules or peripheral doublets. The axonemal tubules contain distinct coaxial ring-like structures that are still observed after axoneme fragmentation and form large clusters of several units.

The cilium like region of the Drosophila bifurca spermatocyte: Elongation of a giant axoneme without intraflagellar transport.

The growth of the ciliary axonemes mainly depends on the evolutionary conserved intraflagellar transport (IFT) machinery. However, insect spermatocytes are characterized by cilium-like regions (CLRs) that elongate in the absence of IFT. It is generally believed that the dynamics of these structures relies on the free diffusion of soluble tubulin from the cytoplasm. However, this passive process could allow the elongation of short ciliary axonemes, but it is unclear whether simple diffusion of tubulin molecules can ensure the correct assembly of elongated ciliary structures. To decipher this point we analyzed the assembly of the CLRs held by the primary spermatocytes of Drosophila bifurca. These ciliary structures consist of a very elongated axoneme that grows without IFT and, therefore, could represent a good model in which to evaluate the role played by the free diffusion of soluble tubulin. The observation of wavy microtubules in the axonemal lumen of fully elongated CLRs of D. bifurca may be consistent with the diffusion of tubulin within the axonemal lumen. Progressive consumption of soluble tubulin used for axoneme growth at the apical tip of the CLRs could result in a gradient sufficient to move tubulin from the cytoplasm to the apical end of the forming ciliary structure. When the axoneme reaches its full length, tubulin molecules are not drawn to the tip of the CLRs and accumulate at the base of the axoneme, where its concentration may exceed the threshold need for microtubule polymerization. The presence of γ-TuRCs at the proximal ends of the supernumerary microtubules could enhance their nucleation.

Wolbachia Induces Structural Defects Harmful to Drosophila simulans Riverside Spermiogenesis.

The relationship between cytoplasmic incompatibility and the obligate intracellular alphaproteobacteria Wolbachia has for a long time been reported. Although the molecular mechanisms responsible for this reproductive alteration are beginning to be understood, the effects of Wolbachia on germ cell structure and dynamics have not yet been fully investigated. We report here that the presence of Wolbachia in infected cysts of elongating spermatids is associated with major structural defects that become more evident in mature sperm. We find mitochondrial defects, an improper axoneme structure, reduced sperm numbers, and individualization failures. The large heterogeneous variety of the ultrastructural defects found in elongating spermatids and mature sperm provide the first cytological evidence for the reduced fertility associated with Wolbachia infection in Drosophila simulans males. The observed abnormalities could be the result of the mechanical stress induced by the high bacteria numbers during the process of spermatid elongation, rather than the result of the released factors affecting the proper morphogenesis of the germ cells. Moreover, high Wolbachia densities in male germ cells may not be appropriate for causing cytoplasmic incompatibility as the bacteria are harmful for spermatid differentiation, leading to abnormal sperm that is unlikely to be functional.

Mob4 is essential for spermatogenesis in Drosophila melanogaster.

Gamete formation is essential for sexual reproduction in metazoans. Meiosis in males gives rise to spermatids that must differentiate and individualize into mature sperm. In Drosophila melanogaster, individualization of interconnected spermatids requires the formation of individualization complexes that synchronously move along the sperm bundles. Here, we show that Mob4, a member of the Mps-one binder family, is essential for male fertility but has no detectable role in female fertility. We show that Mob4 is required for proper axonemal structure and its loss leads to male sterility associated with defective spermatid individualization and absence of mature sperm in the seminal vesicles. Transmission electron micrographs of developing spermatids following mob4RNAi revealed expansion of the outer axonemal microtubules such that the 9 doublets no longer remained linked to each other and defective mitochondrial organization. Mob4 is a STRIPAK component, and male fertility is similarly impaired upon depletion of the STRIPAK components, Strip and Cka. Expression of the human Mob4 gene rescues all phenotypes of Drosophila mob4 downregulation, indicating that the gene is evolutionarily and functionally conserved. Together, this suggests that Mob4 contributes to the regulation of the microtubule- and actin-cytoskeleton during spermatogenesis through the conserved STRIPAK complex. Our study advances the understanding of male infertility by uncovering the requirement for Mob4 in sperm individualization.

Generation of iPSC-derived human forebrain organoids assembling bilateral eye primordia.

Induced pluripotent stem cell-derived brain organoids enable the developmental complexities of the human brain to be deconstructed. During embryogenesis, optic vesicles (OVs), the eye primordium attached to the forebrain, develop from diencephalon. However, most 3D culturing methods generate either brain or retinal organoids individually. Here we describe a protocol to generate organoids with both forebrain entities, which we call OV-containing brain organoids (OVB organoids). In this protocol, we first induce neural differentiation (days 0-5) and collect neurospheres, which we culture in a neurosphere medium to initiate their patterning and further self-assembly (days 5-10). Then, upon transfer to spinner flasks containing OVB medium (days 10-30), neurospheres develop into forebrain organoids with one or two pigmented dots restricted to one pole, displaying forebrain entities of ventral and dorsal cortical progenitors and preoptic areas. Further long-term culture results in photosensitive OVB organoids constituting complementary cell types of OVs, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections and electrically active neuronal networks. OVB organoids provide a system to help dissect interorgan interactions between the OVs as sensory organs and the brain as a processing unit, and can help model early eye patterning defects, including congenital retinal dystrophy. To conduct the protocol, experience in sterile cell culture and maintenance of human induced pluripotent stem cells is essential; theoretical knowledge of brain development is advantageous. Furthermore, specialized expertise in 3D organoid culture and imaging for the analysis is needed.

Evidence for intraflagellar transport in butterfly spermatocyte cilia.

In the model organism insect Drosophila melanogaster short cilia assemble on spermatocytes that elaborate into 1.8 mm long flagella during spermatid differentiation. A unique feature of these cilia/flagella is their lack of dependence on intraflagellar transport (IFT) for their assembly. Here, we show that in the common butterfly Pieris brassicae, the spermatocyte cilia are exceptionally long: about 40 µm compared to less than 1 µm in Drosophila. By transmission electron microscopy, we show that P. brassicae spermatocytes display several features not found in melanogaster, including compelling evidence of IFT structures and features of motile cilia.

Cell-to-Cell Interactions during Early Drosophila Oogenesis: An Ultrastructural Analysis.

Drosophila oogenesis requires the subsequent growth of distinct egg chambers each containing a group of sixteen germline cells surrounded by a simple epithelium of follicle cells. The oocyte occupies a posterior position within the germ cells, thus giving a distinct asymmetry to the egg chamber. Although this disposition is critical for the formation of the anterior-posterior axis of the embryo, the interplay between somatic and germ cells during the early stages of oogenesis remains an open question. We uncover by stage 2, when the egg chambers leaved the germarium, some unique spatial interactions between the posterior follicle cells and the oocyte. These interactions are restricted to the surface of the oocyte over the centriole cluster that formed during early oogenesis. Moreover, the posterior follicle cells in front of the oocyte display a convoluted apical membrane with extensive contacts, whereas the other follicle cells have a flat apical surface without obvious surface protrusions. In addition, the germ cells located at the posterior end of the egg chamber have very elongated protrusions that come into contact with each other or with facing follicle cells. These observations point to distinct polarization events during early oogenesis supporting previous molecular data of an inherent asymmetry between the anterior and the posterior regions of the egg chambers.

Human brain organoids assemble functionally integrated bilateral optic vesicles.

During embryogenesis, optic vesicles develop from the diencephalon via a multistep process of organogenesis. Using induced pluripotent stem cell (iPSC)-derived human brain organoids, we attempted to simplify the complexities and demonstrate formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day 30, brain organoids attempt to assemble optic vesicles, which develop progressively as visible structures within 60 days. These optic vesicle-containing brain organoids (OVB-organoids) constitute a developing optic vesicle's cellular components, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections, and electrically active neuronal networks. OVB-organoids also display synapsin-1, CTIP-positive myelinated cortical neurons, and microglia. Interestingly, various light intensities could trigger photosensitive activity of OVB-organoids, and light sensitivities could be reset after transient photobleaching. Thus, brain organoids have the intrinsic ability to self-organize forebrain-associated primitive sensory structures in a topographically restricted manner and can allow interorgan interaction studies within a single organoid.

Early Drosophila Oogenesis: A Tale of Centriolar Asymmetry.

Among the morphological processes that characterize the early stages of Drosophila oogenesis, the dynamic of the centrioles deserves particular attention. We re-examined the architecture and the distribution of the centrioles within the germarium and early stages of the vitellarium. We found that most of the germ cell centrioles diverge from the canonical model and display notable variations in size. Moreover, duplication events were frequently observed within the germarium in the absence of DNA replication. Finally, we report the presence of an unusually long centriole that is first detected in the cystoblast and is always associated with the developing oocyte. This centriole is directly inherited after the asymmetric division of the germline stem cells and persists during the process of oocyte selection, thus already representing a marker for oocyte identification at the beginning of its formation and during the ensuing developmental stages.

Sas-4 Colocalizes with the Ciliary Rootlets of the Drosophila Sensory Organs.

The Drosophila eye displays peculiar sensory organs of unknown function, the mechanosensory bristles, that are intercalated among the adjacent ommatidia. Like the other Drosophila sensory organs, the mechanosensory bristles consist of a bipolar neuron and two tandemly aligned centrioles, the distal of which nucleates the ciliary axoneme and represents the starting point of the ciliary rootlets. We report here that the centriole associated protein Sas-4 colocalizes with the short ciliary rootlets of the mechanosensory bristles and with the elongated rootlets of chordotonal and olfactory neurons. This finding suggests an unexpected cytoplasmic localization of Sas-4 protein and points to a new underscored role for this protein. Moreover, we observed that the sheath cells associated with the sensory neurons also display two tandemly aligned centrioles but lacks ciliary axonemes, suggesting that the dendrites of the sensory neurons are dispensable for the assembly of aligned centrioles and rootlets.

The Microtubule Cytoskeleton during the Early Drosophila Spermiogenesis.

Sperm elongation and nuclear shaping in Drosophila largely depends on the microtubule cytoskeleton that in early spermatids has centrosomal and non-centrosomal origins. We report here an additional γ-tubulin focus localized on the anterior pole of the nucleus in correspondence of the apical end of the perinuclear microtubules that run within the dense complex. The perinuclear microtubules are nucleated by the pericentriolar material, or centriole adjunct, that surrounds the basal body and are retained to play a major role in nuclear shaping. However, we found that both the perinuclear microtubules and the dense complex are present in spermatids lacking centrioles. Therefore, the basal body or the centriole adjunct seem to be dispensable for the organization and assembly of these structures. These observations shed light on a novel localization of γ-tubulin and open a new scenario on the distribution of the microtubules and the organization of the dense complex during early Drosophila spermiogenesis.

Tissue specific requirement of Drosophila Rcd4 for centriole duplication and ciliogenesis.

Rcd4 is a poorly characterized Drosophila centriole component whose mammalian counterpart, PPP1R35, is suggested to function in centriole elongation and conversion to centrosomes. Here, we show that rcd4 mutants exhibit fewer centrioles, aberrant mitoses, and reduced basal bodies in sensory organs. Rcd4 interacts with the C-terminal part of Ana3, which loads onto the procentriole during interphase, ahead of Rcd4 and before mitosis. Accordingly, depletion of Ana3 prevents Rcd4 recruitment but not vice versa. We find that neither Ana3 nor Rcd4 participates directly in the mitotic conversion of centrioles to centrosomes, but both are required to load Ana1, which is essential for such conversion. Whereas ana3 mutants are male sterile, reflecting a requirement for Ana3 for centriole development in the male germ line, rcd4 mutants are fertile and have male germ line centrioles of normal length. Thus, Rcd4 is essential in somatic cells but is not absolutely required in spermatogenesis, indicating tissue-specific roles in centriole and basal body formation.

Centrioles and Ciliary Structures during Male Gametogenesis in Hexapoda: Discovery of New Models.

Centrioles are-widely conserved barrel-shaped organelles present in most organisms. They are indirectly involved in the organization of the cytoplasmic microtubules both in interphase and during the cell division by recruiting the molecules needed for microtubule nucleation. Moreover, the centrioles are required to assemble cilia and flagella by the direct elongation of their microtubule wall. Due to the importance of the cytoplasmic microtubules in several aspects of the cell life, any defect in centriole structure can lead to cell abnormalities that in humans may result in significant diseases. Many aspects of the centriole dynamics and function have been clarified in the last years, but little attention has been paid to the exceptions in centriole structure that occasionally appeared within the animal kingdom. Here, we focused our attention on non-canonical aspects of centriole architecture within the Hexapoda. The Hexapoda is one of the major animal groups and represents a good laboratory in which to examine the evolution and the organization of the centrioles. Although these findings represent obvious exceptions to the established rules of centriole organization, they may contribute to advance our understanding of the formation and the function of these organelles.

The Singularity of the Drosophila Male Germ Cell Centriole: The Asymmetric Distribution of Sas4 and Sas6.

Drosophila spermatocytes have giant centrioles that display unique properties. Both the parent centrioles maintain a distinct cartwheel and nucleate a cilium-like region that persists during the meiotic divisions and organizes a structured sperm axoneme. Moreover, the parent centrioles are morphologically undistinguishable, unlike vertebrate cells in which mother and daughter centrioles have distinct structural features. However, our immunofluorescence analysis of the parent centrioles in mature primary spermatocytes revealed an asymmetric accumulation of the typical Sas4 and Sas6 proteins. Notably, the fluorescence intensity of Sas4 and Sas6 at the daughter centrioles is greater than the intensity found at the mother ones. In contrast, the centrioles of wing imaginal disc cells display an opposite condition in which the loading of Sas4 and Sas6 at the mother centrioles is greater. These data underlie a subtle asymmetry among the parent centrioles and point to a cell type diversity of the localization of the Sas4 and Sas6 proteins.

Drosophila Doublefault protein coordinates multiple events during male meiosis by controlling mRNA translation.

During the extended prophase of Drosophila gametogenesis, spermatocytes undergo robust gene transcription and store many transcripts in the cytoplasm in a repressed state, until translational activation of select mRNAs in later steps of spermatogenesis. Here, we characterize the Drosophila Doublefault (Dbf) protein as a C2H2 zinc-finger protein, primarily expressed in testes, that is required for normal meiotic division and spermiogenesis. Loss of Dbf causes premature centriole disengagement and affects spindle structure, chromosome segregation and cytokinesis. We show that Dbf interacts with the RNA-binding protein Syncrip/hnRNPQ, a key regulator of localized translation in Drosophila We propose that the pleiotropic effects of dbf loss-of-function mutants are associated with the requirement of dbf function for translation of specific transcripts in spermatocytes. In agreement with this hypothesis, Dbf protein binds cyclin B mRNA and is essential for translation of cyclin B in mature spermatocytes.

The midbody interactome reveals unexpected roles for PP1 phosphatases in cytokinesis.

The midbody is an organelle assembled at the intercellular bridge between the two daughter cells at the end of mitosis. It controls the final separation of the daughter cells and has been involved in cell fate, polarity, tissue organization, and cilium and lumen formation. Here, we report the characterization of the intricate midbody protein-protein interaction network (interactome), which identifies many previously unknown interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome revealed that PP1ß-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex. This de-phosphorylation antagonizes Aurora B kinase to modify the functions and interactions of centralspindlin in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins.

The Microtubule-Depolymerizing Kinesin-13 Klp10A Is Enriched in the Transition Zone of the Ciliary Structures of Drosophila melanogaster.

The precursor of the flagellar axoneme is already present in the primary spermatocytes of Drosophila melanogaster. During spermatogenesis each primary spermatocyte shows a centriole pair that moves to the cell membrane and organizes an axoneme-based structure, the cilium-like region (CLR). The CLRs persist through the meiotic divisions and are inherited by young spermatids. During spermatid differentiation the ciliary caps elongate giving rise to the sperm axoneme. Mutations in Klp10A, a kinesin-13 of Drosophila, results in defects of centriole/CLR organization in spermatocytes and of ciliary cap assembly in elongating spermatids. Reduced Klp10A expression also results in strong structural defects of sensory type I neurons. We show, here, that this protein displays a peculiar localization during male gametogenesis. The Klp10A signal is first detected at the distal ends of the centrioles when they dock to the plasma membrane of young primary spermatocytes. At the onset of the first meiotic prometaphase, when the CLRs reach their full size, Klp10A is enriched in a distinct narrow area at the distal end of the centrioles and persists in elongating spermatids at the base of the ciliary cap. We conclude that Klp10A could be a core component of the ciliary transition zone in Drosophila.

The male stem cell niche of Drosophila melanogaster: Interactions between the germline stem cells and the hub.

The Drosophila male stem cell niche is a well characterized structure in which a small cluster of somatic cells send self-renewal signals to neighbouring germ cells. Although the molecular information involved in the stem cell fate have been identified, much less is understand on the mechanisms driving their short-range specific release. Our ultrastructural analysis reveals distinct protrusions of the stem cell plasma membrane that interdigitate with membrane protrusions of the facing hub cells. Some of these protrusions are very elongated and extend into the hub and could correspond to the Mt-Nanotubes. Therefore, the interface between the stem cells and the hub appears more complex than previously reported and the membrane protrusions of the stem cells might represent specialized surface areas involved in the niche-stem cell communication. We also noticed the presence of clathrin-coated vesicles in the germline plasma membrane that might be also involved in delivering information from the hub.

Plk1/Polo Phosphorylates Sas-4 at the Onset of Mitosis for an Efficient Recruitment of Pericentriolar Material to Centrosomes.

Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4's localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and γ-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and γ-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion.

A transient microtubule-based structure uncovers a new intrinsic asymmetry between the mother centrioles in the early Drosophila spermatocytes.

Parent centrioles are characterized in most organisms by individual morphological traits and have distinct asymmetries that provide different functional properties. By contrast, mother and daughter centrioles are morphologically undistinguishable during Drosophila male gametogenesis. Here we report the presence of previously unrecognized microtubule-based structures that extend into the peripheral cytoplasm of the Drosophila polar spermatocytes at the onset of the first meiosis and are positive for the typical centriolar protein Sas-4 and for the kinesin-like protein Klp10A. These structures have a short lifespan and are no longer found in early apolar spermatocytes. Remarkably, each polar spermatocyte holds only one microtubule-based structure that is associated with one of the sister centriole pairs and specifically with the mother centriole. These findings reveal an inherent asymmetry between the parent centrioles at the onset of male meiosis and also uncover unexpected functional properties between the mother centrioles of the same cells.