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.

CPAP promotes timely cilium disassembly to maintain neural progenitor pool.

A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms ofNPCs maintenance remain unclear. Here, we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, andOFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control.

The developing Drosophila eye - a new model to study centriole reduction.

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, which are involved in γ-tubulin recruitment, and the scaffold protein Plp, which plays a role in the organization of the pericentriolar material, are lost in the third-instar larval stage. However, the centrioles maintain their structural integrity, and both the parent centrioles accumulate Asl and Ana1. Although the loading of Asl points to the acquisition of the motherhood condition, the daughter centrioles fail to recruit Plk4 and do not duplicate. However, it is surprising that the mother centrioles that accumulate Plk4 also never duplicate. This suggests that the loading of Plk4 is not sufficient, in this system, to allow centriole duplication. By halfway through pupal life, the centriole number decreases and structural defects, ranging from being 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.

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.

The "transition zone" of the cilium-like regions in the Drosophila spermatocytes and the role of the C-tubule in axoneme assembly.

The fruit-fly Drosophila melanogaster harbours different types of ciliary structures: ciliary projections associated with neurons of type I and cilium-like regions (CLRs) found during male gametogenesis. The latter deserve particular attention since they are morphologically similar to vertebrate primary cilia and transform into the sperm axonemes during spermiogenesis. Although, all the centrioles are able to organize the CLRs, we found that the mother centriole docks first to the plasma membrane suggesting a new intrinsic functional asymmetry between the parent centrioles. We also show that the CLRs lack the Y-links that connect the axoneme doublets with the plasma membrane in conventional primary cilia. Moreover, the C-tubules, that are lacking in the axoneme of the primary cilia, persisted along the CLRs albeit modified into longitudinal blades. Remarkably, mutant flies in which the CLRs are devoid of the C-tubules or their number is reduced lack sperm axonemes or have incomplete axonemes. Therefore, the C-tubules are dispensable for the assembly of the CLRs but are essential for sperm axoneme elongation and maintenance in Drosophila.

Does Unc-GFP uncover ciliary structures in the rhabdomeric eye of Drosophila?

The uncoordinated (unc) gene product, a potential ortholog of mammalian orofaciodigital syndrome 1 (Ofd1), is involved in the assembly of the ciliary axoneme in Drosophila and it is, therefore, constrained to cell types that have ciliary structures, namely type 1 sensory neurons and male germ cells. Here, we show that evenly spaced Unc-GFP spots are present in the eye imaginal discs of third-instar larvae. These spots are restricted to the R8 photoreceptor cell of each ommatidium in association with mother centrioles. This finding is unexpected because the Drosophila eye is of the rhabdomeric type and would be expected to lack ciliary structures.

The Drosophila centriole - conversion of doublets into triplets within the stem cell niche.

We report, here, that two distinct centriole lineages exist in Drosophila: somatic centrioles usually composed by microtubule doublets and germ line centrioles characterized by triplets. Remarkably, the transition from doublets to triplets in the testis occurs within the stem cell niche with the formation of the C-tubule. We demonstrated that the old mother centriole, which stays in the apical cytoplasm of the male germline stem cells (GSCs), is invariably composed of triplets, whereas its daughter is always built of mixed doublets and triplets. This difference represents the first documentation of a structural asymmetry between mother and daughter centrioles in Drosophila GSCs and might reflect a correlation between the architecture of parent centrioles and their ability to recruit centrosomal proteins. We also found that the old mother centriole is linked to the cell membrane by distinct projections that might play an important role in keeping its apical position during centrosome separation.

Procentriole assembly without centriole disengagement - a paradox of male gametogenesis.

Disengagement of parent centrioles represents the licensing process to restrict centriole duplication exactly once during the cell cycle. However, we provide compelling evidence that this general rule is overridden in insect gametogenesis, when distinct procentrioles are generated during prophase of the first meiosis while parent centrioles are still engaged. Moreover, the number of procentrioles increases during the following meiotic divisions, and up to four procentrioles were found at the base of each mother centriole. However, procentrioles fail to organize a complete set of A-tubules and are thus unable to function as a template for centriole formation. Such a system, in which procentrioles form but halt growth, represents a unique model to analyze the process of cartwheel assembly and procentriole formation.

Asterless is a scaffold for the onset of centriole assembly.

Centrioles are found in the centrosome core and, as basal bodies, at the base of cilia and flagella. Centriole assembly and duplication is controlled by Polo-like-kinase 4 (Plk4): these processes fail if Plk4 is downregulated and are promoted by Plk4 overexpression. Here we show that the centriolar protein Asterless (Asl; human orthologue CEP152) provides a conserved molecular platform, the amino terminus of which interacts with the cryptic Polo box of Plk4 whereas the carboxy terminus interacts with the centriolar protein Sas-4 (CPAP in humans). Drosophila Asl and human CEP152 are required for the centrosomal loading of Plk4 in Drosophila and CPAP in human cells, respectively. Depletion of Asl or CEP152 caused failure of centrosome duplication; their overexpression led to de novo centriole formation in Drosophila eggs, duplication of free centrosomes in Drosophila embryos, and centrosome amplification in cultured Drosophila and human cells. Overexpression of a Plk4-binding-deficient mutant of Asl prevented centriole duplication in cultured cells and embryos. However, this mutant protein was able to promote microtubule organizing centre (MTOC) formation in both embryos and oocytes. Such MTOCs had pericentriolar material and the centriolar protein Sas-4, but no centrioles at their core. Formation of such acentriolar MTOCs could be phenocopied by overexpression of Sas-4 in oocytes or embryos. Our findings identify independent functions for Asl as a scaffold for Plk4 and Sas-4 that facilitates self-assembly and duplication of the centriole and organization of pericentriolar material.

Citron kinase controls a molecular network required for midbody formation in cytokinesis.

Cytokinesis partitions cytoplasmic and genomic materials at the end of cell division. Failure in this process causes polyploidy, which in turn can generate chromosomal instability, a hallmark of many cancers. Successful cytokinesis requires cooperative interaction between contractile ring and central spindle components, but how this cooperation is established is poorly understood. Here we show that Sticky (Sti), the Drosophila ortholog of the contractile ring component Citron kinase (CIT-K), interacts directly with two kinesins, Nebbish [the fly counterpart of human kinesin family member 14 (KIF14)] and Pavarotti [the Drosophila ortholog of human mitotic kinesin-like protein 1 (MKLP1)], and that in turn these kinesins interact with each other and with another central spindle protein, Fascetto [the fly ortholog of protein regulator of cytokinesis 1 (PRC1)]. Sti recruits Nebbish to the cleavage furrow, and both proteins are required for midbody formation and proper localization of Pavarotti and Fascetto. These functions require Sti kinase activity, indicating that Sti plays both structural and regulatory roles in midbody formation. Finally, we show that CIT-K's role in midbody formation is conserved in human cells. Our findings indicate that CIT-K is likely to act at the top of the midbody-formation hierarchy by connecting and regulating a molecular network of contractile ring components and microtubule-associated proteins.

The cilium-like region of the Drosophila spermatocyte: an emerging flagellum?

Primary cilia and flagella are distinct structures with different functions in eukaryotic cells. Despite the fact that they share similar basic organization and architecture, a direct developmental continuity among them has not been reported until now. The primary cilium is a dynamic structure that typically assembles and disassembles during mitotic cell cycles, whereas the sperm axoneme is nucleated by the centriole inherited by the differentiating spermatid at the end of meiosis. Fruit flies display a remarkable exception to this general rule. Drosophila spermatocytes have an unusual axoneme-based structure reminiscent of primary cilia (the cilium-like region, or CLR). This structure persists through the meiotic divisions when it is internalized with the centriole to organize the centrosome and is finally inherited by young spermatids. Examination of elongating spermatids by transmission electron microscopy (EM) and cold regrowth experiments suggests that the motile axoneme derives directly from the elongation and remodelling of the immotile CLR. Both the CLR and elongating spermatid flagella have incomplete C-tubules that form longitudinal sheets associated with the B-tubule wall, unlike axonemes of other organisms in which C-tubules stop growing at the transition between the basal body and the axonemal doublets. Moreover, both the CLR and spermatid flagella lack a structured transition zone, a characteristic feature of ciliated cells. Uncoordinated (unc) mutants that lack C-remnants have short centrioles, suggesting that the C-sheets play a role in the elongation of the centriole after it docks to the cell membrane. The structural similarities between CLR and sperm axoneme suggest that the CLR can be considered as the basal region of the future axoneme and could represent the start point for its elongation.

Drosophila Mgr, a Prefoldin subunit cooperating with von Hippel Lindau to regulate tubulin stability.

Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. We now show that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting ß-tubulin, suggesting Mgr function is required for tubulin stability. Instability of ß-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic ß-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading us to propose a requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not.

Mutations in Cog7 affect Golgi structure, meiotic cytokinesis and sperm development during Drosophila spermatogenesis.

The conserved oligomeric Golgi (COG) complex plays essential roles in Golgi function, vesicle trafficking and glycosylation. Deletions in the human COG7 gene are associated with a rare multisystemic congenital disorder of glycosylation that causes mortality within the first year of life. In this paper, we characterise the Drosophila orthologue of COG7 (Cog7). Loss-of-function Cog7 mutants are viable but male sterile. The Cog7 gene product is enriched in the Golgi stacks and in Golgi-derived structures throughout spermatogenesis. Mutations in the Cog7 gene disrupt Golgi architecture and reduce the number of Golgi stacks in primary spermatocytes. During spermiogenesis, loss of the Cog7 protein impairs the assembly of the Golgi-derived acroblast in spermatids and affects axoneme architecture. Similar to the Cog5 homologue, four way stop (Fws), Cog7 enables furrow ingression during cytokinesis. We show that the recruitment of the small GTPase Rab11 and the phosphatidylinositol transfer protein Giotto (Gio) to the cleavage site requires a functioning wild-type Cog7 gene. In addition, Gio coimmunoprecipitates with Cog7 and with Rab11 in the testes. Our results altogether implicate Cog7 as an upstream component in a gio-Rab11 pathway controlling membrane addition during cytokinesis.

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.

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.

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.

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.

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.