Cortical Cyclin A controls spindle orientation during asymmetric cell divisions in Drosophila.

The coordination between cell proliferation and cell polarity is crucial to orient the asymmetric cell divisions to generate cell diversity in epithelia. In many instances, the Frizzled/Dishevelled planar cell polarity pathway is involved in mitotic spindle orientation, but how this is spatially and temporally coordinated with cell cycle progression has remained elusive. Using Drosophila sensory organ precursor cells as a model system, we show that Cyclin A, the main Cyclin driving the transition to M-phase of the cell cycle, is recruited to the apical-posterior cortex in prophase by the Frizzled/Dishevelled complex. This cortically localized Cyclin A then regulates the orientation of the division by recruiting Mud, a homologue of NuMA, the well-known spindle-associated protein. The observed non-canonical subcellular localization of Cyclin A reveals this mitotic factor as a direct link between cell proliferation, cell polarity and spindle orientation.

A neural progenitor mitotic wave is required for asynchronous axon outgrowth and morphology.

Spatiotemporal mechanisms generating neural diversity are fundamental for understanding neural processes. Here, we investigated how neural diversity arises from neurons coming from identical progenitors. In the dorsal thorax of Drosophila, rows of mechanosensory organs originate from the division of sensory organ progenitor (SOPs). We show that in each row of the notum, an anteromedial located central SOP divides first, then neighbouring SOPs divide, and so on. This centrifugal wave of mitoses depends on cell-cell inhibitory interactions mediated by SOP cytoplasmic protrusions and Scabrous, a secreted protein interacting with the Delta/Notch complex. Furthermore, when this mitotic wave was reduced, axonal growth was more synchronous, axonal terminals had a complex branching pattern and fly behaviour was impaired. We show that the temporal order of progenitor divisions influences the birth order of sensory neurons, axon branching and impact on grooming behaviour. These data support the idea that developmental timing controls axon wiring neural diversity.

Shaping of Drosophila Neural Cell Lineages Through Coordination of Cell Proliferation and Cell Fate by the BTB-ZF Transcription Factor Tramtrack-69.

Cell diversity in multicellular organisms relies on coordination between cell proliferation and the acquisition of cell identity. The equilibrium between these two processes is essential to assure the correct number of determined cells at a given time at a given place. Using genetic approaches and correlative microscopy, we show that Tramtrack-69 (Ttk69, a Broad-complex, Tramtrack and Bric-à-brac - Zinc Finger (BTB-ZF) transcription factor ortholog of the human promyelocytic leukemia zinc finger factor) plays an essential role in controlling this balance. In the Drosophila bristle cell lineage, which produces the external sensory organs composed by a neuron and accessory cells, we show that ttk69 loss-of-function leads to supplementary neural-type cells at the expense of accessory cells. Our data indicate that Ttk69 (1) promotes cell cycle exit of newborn terminal cells by downregulating CycE, the principal cyclin involved in S-phase entry, and (2) regulates cell-fate acquisition and terminal differentiation, by downregulating the expression of hamlet and upregulating that of Suppressor of Hairless, two transcription factors involved in neural-fate acquisition and accessory cell differentiation, respectively. Thus, Ttk69 plays a central role in shaping neural cell lineages by integrating molecular mechanisms that regulate progenitor cell cycle exit and cell-fate commitment.

Escargot and Scratch regulate neural commitment by antagonizing Notch activity in Drosophila sensory organs.

During Notch (N)-mediated binary cell fate decisions, cells adopt two different fates according to the levels of N pathway activation: an Noff-dependent or an Non-dependent fate. How cells maintain these N activity levels over time remains largely unknown. We address this question in the cell lineage that gives rise to the Drosophila mechanosensory organs. In this lineage a primary precursor cell undergoes a stereotyped sequence of oriented asymmetric cell divisions and transits through two neural precursor states before acquiring a neuron identity. Using a combination of genetic and cell biology strategies, we show that Escargot and Scratch, two transcription factors belonging to the Snail superfamily, maintain Noff neural commitment by directly blocking the transcription of N target genes. We propose that Snail factors act by displacing proneural transcription activators from DNA binding sites. As such, Snail factors maintain the Noff state in neural precursor cells by buffering any ectopic variation in the level of N activity. Since Escargot and Scratch orthologs are present in other precursor cells, our findings are fundamental for understanding precursor cell fate acquisition in other systems.

Allophagy: a macroautophagic process degrading spermatozoid-inherited organelles.

In most animals, during oocyte fertilization the spermatozoon provides DNA and centrioles together with some cytoplasm and organelles, but paternal mitochondria are generally eliminated in the embryo. Using the model animal C. elegans we have shown that paternal organelle degradation is dependent on the formation of autophagosomes a few minutes after fertilization. This macroautophagic process is preceded by an active ubiquitination of some spermatozoon-inherited organelles. Analysis of fertilized mouse embryos suggests that this autophagy event is evolutionarily conserved.

Postfertilization autophagy of sperm organelles prevents paternal mitochondrial DNA transmission.

In sexual reproduction of most animals, the spermatozoon provides DNA and centrioles, together with some cytoplasm and organelles, to the oocyte that is being fertilized. Paternal mitochondria and their genomes are generally eliminated in the embryo by an unknown degradation mechanism. We show that, upon fertilization, a Caenorhabditis elegans spermatozoon triggers the recruitment of autophagosomes within minutes and subsequent paternal mitochondria degradation. Whereas the nematode-specific sperm membranous organelles are ubiquitinated before autophagosome formation, the mitochondria are not. The degradation of both paternal structures and mitochondrial DNA requires an LC3-dependent autophagy. Analysis of fertilized mouse embryos shows the localization of autophagy markers, which suggests that this autophagy event is evolutionarily conserved to prevent both the transmission of paternal mitochondrial DNA to the offspring and the establishment of heteroplasmy.

Orientation of mitotic spindles during the 8- to 16-cell stage transition in mouse embryos.

BACKGROUND: Asymmetric cell divisions are involved in the divergence of the first two lineages of the pre-implantation mouse embryo. They first take place after cell polarization (during compaction) at the 8-cell stage. It is thought that, in contrast to many species, spindle orientation is random, although there is no direct evidence for this. METHODOLOGY/PRINCIPAL FINDINGS: Tubulin-GFP and live imaging with a spinning disk confocal microscope were used to directly study spindle orientation in whole embryos undergoing the 8- to 16-cell stage transition. This approach allowed us to determine that there is no predetermined cleavage pattern in 8-cell compacted mouse embryos and that mitotic spindle orientation in live embryo is only modulated by the extent of cell rounding up during mitosis. CONCLUSIONS: These results clearly demonstrate that spindle orientation is not controlled at the 8- to 16-cell transition, but influenced by cell bulging during mitosis, thus reinforcing the idea that pre-implantation development is highly regulative and not pre-patterned.

Inactivation of aPKClambda reveals a context dependent allocation of cell lineages in preimplantation mouse embryos.

BACKGROUND: During mammalian preimplantation development, lineage divergence seems to be controlled by the interplay between asymmetric cell division (once cells are polarized) and positional information. In the mouse embryo, two distinct cell populations are first observed at the 16-cell stage and can be distinguished by both their position (outside or inside) and their phenotype (polarized or non-polarized). Many efforts have been made during the last decade to characterize the molecular mechanisms driving lineage divergence. METHODOLOGY/PRINCIPAL FINDINGS: In order to evaluate the importance of cell polarity in the determination of cell fate we have disturbed the activity of the apical complex aPKC/PAR6 using siRNA to down-regulate aPKClambda expression. Here we show that depletion of aPKClambda results in an absence of tight junctions and in severe polarity defects at the 16-cell stage. Importantly, we found that, in absence of aPKClambda, cell fate depends on the cellular context: depletion of aPKClambda in all cells results in a strong reduction of inner cells at the 16-cell stage, while inhibition of aPKClambda in only half of the embryo biases the progeny of aPKClambda defective blastomeres towards the inner cell mass. Finally, our study points to a role of cell shape in controlling cell position and thus lineage allocation. CONCLUSION: Our data show that aPKClambda is dispensable for the establishment of polarity at the 8-cell stage but is essential for the stabilization of cell polarity at the 16-cell stage and for cell positioning. Moreover, this study reveals that in addition to positional information and asymmetric cell divisions, cell shape plays an important role for the control of lineage divergence during mouse preimplantation development. Cell shape is able to influence both the type of division (symmetric or asymmetric) and the position of the blastomeres within the embryo.

Murine endogenous retrovirus MuERV-L is the progenitor of the "orphan" epsilon viruslike particles of the early mouse embryo.

Viruslike particles which displayed a peculiar wheellike appearance that distinguished them from A-, B- or C-type particles had previously been described in the early mouse embryo. The maximum expression of these so-called epsilon particles was observed in two-cell-stage embryos, followed by their rapid decline at later stages of development and no particles detected at the zygote one-cell stage. Here, we show that these particles are in fact produced by a newly discovered murine endogenous retrovirus (ERV) belonging to the widespread family of mammalian ERV-L elements and named MuERV-L. Using antibodies that we raised against the Gag protein of these elements, Western blot analysis and in toto immunofluorescence studies of the embryos at various stages disclosed the same developmental expression profile as that observed for epsilon particles. Using expression vectors for cloned, full-length, entirely coding MuERV-L copies and cell transfection, direct identification of the epsilon particles was finally achieved by high-resolution electron microscopy.

Morphogenesis of the mammalian blastocyst.

The first 4 days of mouse pre-implantation development are characterized by a period of segmentation, including morphogenetic events that are required for the divergence of embryonic and extra-embryonic lineages. These extra-embryonic tissues are essential for the implantation into the maternal uterus and for the development of the foetus. In this review, we first discuss data showing unambiguously that no essential axis of development is set up before the late blastocyst stage, and explain why the pre-patterning described during the early phases (segmentation) of development in other vertebrates cannot apply to mammalian pre-implantation period. Then, we describe important cellular and molecular events that are required for the morphogenesis of the blastocyst.

Where do we stand now? Mouse early embryo patterning meeting in Freiburg, Germany (2005).

Mechanism underlying mammalian preimplantation development has long been a subject of controversy and the central question has been if any "determinants" play a key role in a manner comparable to the non-mammalian "model" system. During the last decade, this issue has been revived (Pearson, 2002; Rossant and Tam, 2004) by claims that the axes of the mouse blastocyst are anticipated at the egg ("prepatterning model"; Gardner, 1997; Gardner, 2001; Piotrowska et al., 2001; Piotrowska and Zernicka-Goetz, 2001; Zernicka-Goetz, 2005), suggesting that a mechanism comparable to that operating in non-mammals may be at work. However, recent studies by other laboratories do not support these claims ("regulative model"; Alarcon and Marikawa, 2003; Chroscicka et al., 2004; Hiiragi and Solter, 2004; Alarcon and Marikawa, 2005; Louvet-Vallee et al., 2005; Motosugi et al., 2005) and the issue is currently under hot debate (Vogel, 2005). Deepening our knowledge of this issue will not only provide an essential basis for understanding mammalian development, but also directly apply to ongoing clinical practices such as intracytoplasmic sperm injection (ICSI) and preimplantation genetic diagnosis (PGD). These practices were originally supported by a classical premise that mammalian preimplantation embryos are highly regulative (Tarkowski, 1959; Tarkowski, 1961; Tarkowski and Wroblewska, 1967; Rossant, 1976), in keeping with the "regulative model". However, if the "prepatterning model" is correct, the latter will require critical reassessment.

Embryology: does prepatterning occur in the mouse egg?

A recurring question in developmental biology has been whether localized determinants play any role in mammalian preimplantation development. This is a controversial issue that brings back the idea of prepatterning and is explored further by Plusa et al., who claim it is the first cleavage of the mouse zygote that predicts the blastocyst axis, rather than the animal pole or sperm entry point, as previously suggested. However, other evidence indicates that the blasotcyst axis is not predetermined and there is no prepatterning in the mouse egg. Here we investigate the origin of these different views and conclude that they arise from differences in the data themselves and in their interpretation.

Vezatin, a protein associated to adherens junctions, is required for mouse blastocyst morphogenesis.

Cell-cell interactions play a major role during preimplantation development of the mouse embryo. The formation of adherens junctions is a major feature of compaction, the first morphogenetic event that takes place at the 8-cell stage. Then, during the following two cell cycles, tight junctions form, and the outer layer of cells differentiate into a functional epithelium, leading to the formation of the blastocoel cavity. Until now, E-cadherin was the only transmembrane molecule localized in adherens junctions and required for early development. Vezatin is a transmembrane protein of adherens junctions, interacting with the E-cadherin-catenins complex. Here, we show that vezatin is expressed very early during mouse preimplantation development. It co-localizes with E-cadherin throughout development, being found all around the cell cortex before compaction and basolaterally in adherens junctions thereafter. In addition, vezatin is also detected in nuclei during most of the cell cycle. Finally, using a morpholino-oligonucleotide approach to inhibit vezatin function during preimplantation development, we observed that inhibition of vezatin synthesis leads to a cell cycle arrest with limited cell-cell interactions. This phenotype can be rescued when mRNAs coding for vezatin missing the 5'UTR are co-injected with the anti-vezatin morpholino-oligonucleotide. Cells derived from blastomeres injected with morpholino-oligonucleotide had a reduced amount of vezatin concomitantly with a decrease in the quantity of E-cadherin and beta-catenin localized in the areas of intercellular contact. Shift in E-cadherin cortical distribution was correlated with a strong decrease in E-cadherin mRNA and protein contents. Altogether, these observations demonstrate that vezatin is required for morphogenesis of the preimplantation mouse embryo.

Asymmetric distribution of PAR proteins in the mouse embryo begins at the 8-cell stage during compaction.

In many organisms, like Caenorhabditis elegans and Drosophila melanogaster, establishment of spatial patterns and definition of cell fate are driven by the segregation of determinants in response to spatial cues, as early as oogenesis or fertilization. In these organisms, a family of conserved proteins, the PAR proteins, is involved in the asymmetric distribution of cytoplasmic determinants and in the control of asymmetric divisions. In the mouse embryo, it is only at the 8-cell stage during compaction that asymmetries, leading to cellular diversification and blastocyst morphogenesis, are first observed. However, it has been suggested that developmentally relevant asymmetries could be established already in the oocyte and during fertilization. This led us to study the PAR proteins during the early stages of mouse development. We observed that the homologues of the different members of the PAR/aPKC complex and PAR1 are expressed in the preimplantation mouse embryo. During the first embryonic cleavages, before compaction, PARD6b and EMK1 are observed on the spindle. The localization of these two proteins becomes asymmetric during compaction, when blastomeres flatten upon each other and polarize. PARD6b is targeted to the apical pole, whereas EMK1 is distributed along the baso-lateral domain. The targeting of EMK1 is dependent upon cell-cell interactions while the apical localization of PARD6b is independent of cell contacts. At the 16-cell stage, aPKCzeta colocalizes with PARD6b and a colocalization of the three proteins (PARD6b/PARD3/aPKCzeta can occur in blastocysts, only at tight junctions. This choreography suggests that proteins of the PAR family are involved in the setting up of blastomere polarity and blastocyst morphogenesis in the early mammalian embryo although the interactions between the different players differ from previously studied systems. Finally, they reinforce the idea that the first developmentally relevant asymmetries are set up during compaction.

Mitotic spindles and cleavage planes are oriented randomly in the two-cell mouse embryo.

Most experimental embryological studies performed on the early mouse embryo have led to the conclusion that there are no mosaically distributed developmental determinants in the zygote and early embryo (for example see [1-6]). It has been suggested recently that "the cleavage pattern of the early mouse embryo is not random and that the three-dimensional body plan is pre-patterned in the egg" (in [7] for review see [8-10]). Two major spatial cues influencing the pattern of cleavage divisions have been proposed: the site of the second meiotic division [11, 12] and the sperm entry point [13-14], although the latter is controversial [15-17]. An implication of this hypothesis is that the orientations of the first few cleavage divisions are stereotyped. Such a define cleavage pattern, leading to the segregation of developmental determinants, is observed in many species [18]. Recently, it was shown that the first cleavage plane is not predetermined but defined by the topology of the two apposing pronuclei [19]. Because the position of the female pronucleus is dependent upon the site of polar body extrusion and the position of the male pronuclei is dependent upon the sperm entry point [19-20], this observation leaves open the possibility that the sperm may provide some kind of directionality [7]. But, even if asymmetries were set up only after fertilization, a stereotyped cleavage pattern should take place during the following cleavage divisions. Thus, we studied the cleavage pattern of two-cell embryos by videomicroscopy to distinguish between the two hypotheses. After the mitotic spindle formed, its orientation did not change until cleavage. During late metaphase and anaphase, the spindle poles appear to be anchored to the cortex through astral microtubules and PARD6a. Only at the time of cleavage, during late anaphase, do the forming daughter cells change their relative positions. These studies show that cleavage planes are oriented randomly in two-cell embryos. This argues against a prepatterning of the mouse embryo before compaction.

Expression and role of cubilin in the internalization of nutrients during the peri-implantation development of the rodent embryo.

Histiotrophic nutrition is essential during the peri-implantation development in rodents, but little is known about receptors involved in protein and lipid endocytosis derived from the endometrium and the uterine glands. Previous studies suggested that cubilin, a multiligand receptor for vitamin, iron, and protein uptake in the adult, might be important in this process, but the onset of its expression and function is not known. In this study, we analyzed the expression of cubilin in the pre- and early post-implantation rodent embryo and tested its potential function in protein and cholesterol uptake. Using morphological and Western blot analysis, we showed that cubilin first appeared at the eight-cell stage. It was expressed by the maternal-fetal interfaces, trophectoderm and visceral endoderm, but also by the future neuroepithelial cells and the developing neural tube. At all these sites, cubilin was localized at the apical pole of the cells exposed to the maternal environment or to the amniotic and neural tube cavities, and had a very similar distribution to megalin, a member of the LDLR gene family and a coreceptor for cubilin in adult tissues. To analyze cubilin function, we followed endocytosis of apolipoprotein A-I and HDL cholesterol, nutrients normally present in the uterine glands and essential for embryonic growth. We showed that internalization of both ligands was cubilin dependent during the early rodent gestation. In conclusion, the early cubilin expression and its function in protein and cholesterol uptake suggest an important role for cubilin in the development of the peri-implantation embryo.

Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo.

The preimplantation development of the mouse embryo leads to the divergence of the first two cell lineages, the inner cell mass and the trophectoderm. The formation of a microvillus pole during compaction at the eight-cell stage and its asymmetric inheritance during mitosis are key events in the emergence of these two cell populations. Ezrin, a member of the ERM protein family, seems to be involved in the formation and stabilization of this apical microvillus pole. To further characterize its function in early development, we mutated the key residue T567, which was reported to be essential for regulation of ezrin function through phosphorylation. Here, we show that expression of ezrin mutants in which the COOH-terminal threonine T567 was replaced by an aspartate (to mimic a phosphorylated residue; T567D) or by an alanine (to avoid phosphorylation; T567A) interferes with E-cadherin function and disrupts the first morphogenetic events of development: compaction and cavitation. The active mutant ezrin-T567D induces the formation of numerous and abnormally long microvilli at the surface of blastomeres. Moreover, it localizes all around the cell cortex and inhibits cell-cell adhesion and cell polarization at the eight-cell stage. During the following stages, only half of the embryos are able to compact and finally to cavitate. In those embryos, the amount of ezrin-T567D decreases in the basolateral areas, while the proportion of adherens junctions increases. The reverse inactive mutant ezrin-T567A is mainly cytoplasmic and does not perturb compaction at the eight-cell stage. However, at the 16-cell stage, it relocalizes at the basolateral cortex, leading to a strong decrease in the surface of adherens junctions, and finally, embryos abort development. Our results show that ezrin is directly involved in the formation of microvilli in the early mouse embryo. Moreover, they indicate that maintenance of ezrin in basolateral areas prevents microvilli breakdown and inhibits the formation of normal cell-cell contacts mediated by E-cadherin, thereby impairing blastomeres polarization and morphogenesis of the blastocyst.

Two PAR6 proteins become asymmetrically localized during establishment of polarity in mouse oocytes.

Meiotic maturation in mammals is characterized by two asymmetric divisions, leading to the formation of two polar bodies and the female gamete. Whereas the mouse oocyte is a polarized cell, molecules implicated in the establishment of this polarity are still unknown. PAR proteins have been demonstrated to play an important role in cell polarity in many cell types, where they control spindle positioning and asymmetric distribution of determinants. Here we show that two PAR6-related proteins have distinct polarized distributions in mouse oocytes. mPARD6a is first localized on the spindle and then accumulates at the pole nearest the cortex during spindle migration. In the absence of microtubules, the chromosomes still migrate to the cortex, and mPARD6a was found associated with the chromosomes and was facing the cortex. mPARD6a is the first identified protein to associate with the spindle during spindle migration and to relocalize to the chromosomes in the absence of microtubule behavior, suggesting a role in spindle migration. The other protein, mPARD6b, was found on spindle microtubules until entry into meiosis II and relocalized to the cortex at the animal pole during metaphase II arrest. mPARD6b is the first identified protein to localize to the animal pole of the mouse oocyte and likely contributes to the polarization of the cortex.

Control of vulval competence and centering in the nematode Oscheius sp. 1 CEW1.

To compare vulva development mechanisms in the nematode Oscheius sp. 1 to those known in Caenorhabditis elegans, we performed a genetic screen for vulva mutants in Oscheius sp. 1 CEW1. Here we present one large category of mutations that we call cov, which affect the specification of the Pn.p ventral epidermal cells along the antero-posterior axis. The Pn.p cells are numbered from 1 to 12 from anterior to posterior. In wild-type Oscheius sp. 1 CEW1, the P(4-8).p cells are competent to form the vulva and the progeny of P(5-7).p actually form the vulva, with the descendants of P6.p adopting a central vulval fate. Among the 17 mutations (defining 13 genes) that we characterize here, group 1 mutations completely or partially abolish P(4-8).p competence, and this correlates with early fusion of the Pn.p cells to the epidermal syncytium. In this group, we found a putative null mutation in the lin-39 HOM-C homolog, the associated phenotype of which could be weakly mimicked by injection of a morpholino against Osp1-lin-39 in the mother's germ line. Using cell ablation in a partially penetrant competence mutant, we show that vulval competence is partially controlled by a gonadal signal. Most other mutants found in the screen display phenotypes unknown in C. elegans. Group 2 mutants show a partial penetrance of Pn.p competence loss and an abnormal centering of the vulva on P5.p, suggesting that these two processes are coregulated by the same pathway in Oscheius sp. 1. Group 3 mutants display an enlarged competence group that includes P3.p, thus demonstrating the existence of a specific mechanism inhibiting P3.p competence. Group 4 mutants display an abnormal centering of the vulval pattern on P7.p and suggest that a specific mechanism centers the vulval pattern on a single Pn.p cell.