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1.  The medaka dhc2 mutant reveals conserved and distinct mechanisms of Hedgehog signaling in teleosts 
Background
Primary cilia are essential for Hedgehog (Hh) signal transduction in vertebrates. Although the core components of the Hh pathway are highly conserved, the dependency on cilia in Hh signaling is considered to be lower in fish than in mice, suggesting the presence of species-specific mechanisms for Hh signal transduction.
Results
To precisely understand the role of cilia in Hh signaling in fish and explore the evolution of Hh signaling, we have generated a maternal-zygotic medaka (Oryzias latipes) mutant that lacks cytoplasmic dynein heavy chain 2 (dhc2; MZdhc2), a component required for retrograde intraflagellar transport. We found that MZdhc2 exhibited the shortened cilia and partial defects in Hh signaling, although the Hh defects were milder than zebrafish mutants which completely lack cilia. This result suggests that Hh activity in fish depends on the length of cilium. However, the activity of Hh signaling in MZdhc2 appeared to be higher than that in mouse Dnchc2 mutants, suggesting a lower requirement for cilia in Hh signaling in fish. We confirmed that Ptch1 receptor is exclusively localized on the cilium in fish as in mammals. Subsequent analyses revealed that Fused, an essential mediator for Hh signaling in Drosophila and fish but not in mammals, augments the activity of Hh signaling in fish as a transcriptional target of Hh signaling.
Conclusions
Ciliary requirement for Hh signaling in fish is lower than that in mammals, possibly due to fused-mediated positive feedback in Hh signaling. The finding of this fish-specific augmentation provides a novel insight into the evolution of Hh signaling.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0057-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0057-x
PMCID: PMC4320493  PMID: 25645819
Hedgehog signaling; Cilia; Medaka fish; Fused; dhc2; Cytoplasmic dynein heavy chain 2; Neural tube
2.  Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation 
Background
Tissue fixation is crucial for preserving the morphology of biological structures and cytological details to prevent postmortem degradation and autolysis. Improper fixation conditions could lead to artifacts and thus incorrect conclusions in immunofluorescence or histology experiments. To resolve reported structural anomalies with respect to Drosophila photoreceptor cell organization we developed and utilized a combination of live imaging and fixed samples to investigate the exact biogenesis and to identify the underlying source for the reported discrepancies in structure.
Results
We found that piperazine-N,N’-bis(ethanesulfonic acid) (PIPES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), two zwitterionic buffers commonly used in tissue fixation, can cause severe lumen and cell morphological defects in Drosophila pupal and adult retina; the inter-rhabdomeral lumen becomes dilated and the photoreceptor cells are significantly reduced in size. Correspondingly, the localization pattern of Eyes shut (EYS), a luminal protein, is severely altered. In contrast, tissues fixed in the phosphate buffered saline (PBS) buffer results in lumen and cell morphologies that are consistent with live imaging.
Conclusions
We suggest that PIPES and HEPES buffers should be utilized with caution for fixation when examining the interplay between cells and their extracellular environment, especially in Drosophila pupal and adult retina research.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0056-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0056-y
PMCID: PMC4320506  PMID: 25645690
PIPES; HEPES; Artifact; Inter-rhabdomeral space; Rhabdomere; Eyes shut; Drosophila; Photoreceptor; Biological tube
3.  Reversible regulation of stem cell niche size associated with dietary control of Notch signalling 
Background
Stem cells can respond to environmental and physiological inputs to adaptively remodel tissues. Little is known about whether stem cell niches are similarly responsive. The Drosophila ovary germline stem cell (GSC) niche is a well-studied model, which is comprised of cap cells that provide anchorage and maintenance signals for GSCs to maintain oogenesis. Previous studies have shown a strong link between diet and the regulation of oogenesis, making this a useful model system in which to investigate dietary regulation of the niche and its associated stem cells.
Results
We show that the Drosophila ovary GSC cap cell niche is a dynamic structure, which can contract and expand in fluctuating dietary conditions. Cap cells are lost when adult flies are shifted to nutrient poor diet and are restored after returning flies to nutrient-rich medium. Notch signalling in cap and escort cells is similarly reduced and restored by dietary shifts to nutrient poor and rich media. In old flies decreased Notch signalling is associated with decreased robustness of the niche to dietary changes. We demonstrated using a Notch temperature sensitive allele that removal and restoration of Notch signalling also leads to a reduction and re-expansion of the niche. Changes in niche size were not associated with apoptosis or cell division. We identified two distinct roles for Notch in the adult germarium. Notch can act in cap cells to prevent their loss while activation of Notch in the flanking escort cells results in expansion of the niche.
Conclusions
We provide evidence that dietary changes alone are sufficient to alter Notch signalling and reversibly change niche size in the adult in wild type flies. We show Notch acts in different cells to maintain and re-expand the niche and propose a model in which cell fate transitions between cap cells and flanking somatic cells accounts for niche dynamics. These findings reveal an unexpected reversible plasticity of the GSC niche whose responses provide an integrated read out of the physiological status of the fly that is modulated by diet and age.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0059-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0059-8
PMCID: PMC4320563  PMID: 25637382
4.  Transcriptome analysis of the hormone-sensing cells in mammary epithelial reveals dynamic changes in early pregnancy 
Background
Alveoli, the milk-producing units of the mammary gland, are generated during pregnancy by collaboration of different epithelial cell types. We present the first analysis of transcriptional changes within the hormone sensing population during pregnancy. Hormone-receptor positive (HR+) cells play a key role in the initiation of alveologenesis as they sense systemic hormonal changes and translate these into local instructions for neighboring HR- cells. We recently showed that IGF2 is produced specifically by HR+ cells in early pregnancy, but is undetectable in the virgin state. Here, we define the transcriptome of HR+ cells in early pregnancy with the aim to elucidate additional changes that are unique for this dynamic developmental time window.
Results
We harvested mammary glands from virgin, 3-day and 7-day pregnant mice and isolated a few hundred hormone-sensing cells per animal by FACS for microarray analysis. There was a high concordance between animals with a clear induction of cell cycle progression genes at day 3 of pregnancy and molecules involved in paracrine signalling at day 7.
Conclusions
These findings underscore the proliferative capacity of HR+ cells upon specific stimuli and elucidate developmentally-restricted changes in cellular communication. Since the majority of breast cancers are HR+, with a variable proportion of HR+ cells per tumor, we anticipate that this data set will aid further studies into the regulation of HR+ cell proliferation and the role of heterotypic signalling within tumors.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0058-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0058-9
PMCID: PMC4314744  PMID: 25623114
Mammary gland; Morphogenesis; Single cell analysis; Proliferation; Estrogen receptor; Microarray
5.  Distinct expression and localization of the type II diacylglycerol kinase isozymes δ, η and κ in the mouse reproductive organs 
Background
We have revealed that the type II diacylglycerol kinases (DGKs) δ, η and κ were expressed in the testis and ovary. However, these enzymes’ functions in the reproductive organs remain unknown.
Results
In this study, we first identified the expression sites of type II DGKs in the mouse reproductive organs in detail. Reverse transcription-polymerase chain reaction and Western blotting confirmed that DGKδ2 (splicing variant 2) but not DGKδ1 (splicing variant 1) and DGKκ were expressed in the testis, ovary and uterus. DGKη1 (splicing variant 1) but not DGKη2 (splicing variant 2) was strongly detected in the ovary and uterus. Interestingly, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the testis. Moreover, we investigated the distribution of type II DGKs in the testis, ovary and uterus through in situ hybridization. DGKδ2 was distributed in the primary spermatocytes of the testis and ovarian follicles. DGKη1 was distributed in the oviductal epithelium of the ovary and the luminal epithelium of the uterus. Intriguingly, DGKη3 was strongly expressed in the secondary spermatocytes and round spermatids of the testis. DGKκ was distributed in the primary and secondary spermatocyte of the testis.
Conclusion
These results indicate that the expression patterns of the type II DGK isoforms δ2, η1, η3 and κ differ from each other, suggesting that these DGK isoforms play specific roles in distinct compartments and developmental stages of the reproductive organs, especially in the processes of spermatogenesis and oocyte maturation.
doi:10.1186/s12861-015-0055-z
PMCID: PMC4308931  PMID: 25613821
Diacylglycerol kinase; Alternative splicing; Spermatocyte; Ovarian follicle; Uterine luminal epithelium
6.  Reviewer acknowledgement 2014 
Contributing reviewers
The editors of BMC Developmental Biology would like to thank all our reviewers who have contributed to the journal in Volume 14 (2014).
doi:10.1186/s12861-015-0052-2
PMCID: PMC4300680  PMID: 25604987
7.  Epigenetic regulation of histone modifications and Gata6 gene expression induced by maternal diet in mouse embryoid bodies in a model of developmental programming 
Background
Dietary interventions during pregnancy alter offspring fitness. We have shown mouse maternal low protein diet fed exclusively for the preimplantation period (Emb-LPD) before return to normal protein diet (NPD) for the rest of gestation, is sufficient to cause adult offspring cardiovascular and metabolic disease. Moreover, Emb-LPD blastocysts sense altered nutrition within the uterus and activate compensatory cellular responses including stimulated endocytosis within extra-embryonic trophectoderm and primitive endoderm (PE) lineages to protect fetal growth rate. However, these responses associate with later disease. Here, we investigate epigenetic mechanisms underlying nutritional programming of PE that may contribute to its altered phenotype, stabilised during subsequent development. We use embryonic stem (ES) cell lines established previously from Emb-LPD and NPD blastocysts that were differentiated into embryoid bodies (EBs) with outer PE-like layer.
Results
Emb-LPD EBs grow to a larger size than NPD EBs and express reduced Gata6 transcription factor (regulator of PE differentiation) at mRNA and protein levels, similar to Emb-LPD PE derivative visceral yolk sac tissue in vivo in later gestation. We analysed histone modifications at the Gata6 promoter in Emb-LPD EBs using chromatin immunoprecipitation assay. We found significant reduction in histone H3 and H4 acetylation and RNA polymerase II binding compared with NPD EBs, all markers of reduced transcription. Other histone modifications, H3K4Me2, H3K9Me3 and H3K27Me3, were unaltered. A similar but generally non-significant histone modification pattern was found on the Gata4 promoter. Consistent with these changes, histone deacetylase Hdac-1, but not Hdac-3, gene expression was upregulated in Emb-LPD EBs.
Conclusions
First, these data demonstrate ES cells and EBs retain and propagate nutritional programming adaptations in vitro, suitable for molecular analysis of mechanisms, reducing animal use. Second, they reveal maternal diet induces persistent changes in histone modifications to regulate Gata6 expression and PE growth and differentiation that may affect lifetime health.
doi:10.1186/s12861-015-0053-1
PMCID: PMC4305257  PMID: 25609498
Maternal low protein diet; Embryoid body; Mouse blastocyst; Histone epigenetics; Metabolic disease; Gata6; Primitive endoderm; Chromatin immunoprecipitation
8.  Development of the follicular basement membrane during human gametogenesis and early folliculogenesis 
Background
In society, there is a clear need to improve the success rate of techniques to restore fertility. Therefore a deeper knowledge of the dynamics of the complex molecular environment that regulates human gametogenesis and (early) folliculogenesis in vivo is necessary. Here, we have studied these processes focusing on the formation of the follicular basement membrane (BM) in vivo.
Results
The distribution of the main components of the extracellular matrix (ECM) collagen IV, laminin and fibronectin by week 10 of gestation (W10) in the ovarian cortex revealed the existence of ovarian cords and of a distinct mesenchymal compartment, resembling the organization in the male gonads. By W17, the first primordial follicles were assembled individually in that (cortical) mesenchymal compartment and were already encapsulated by a BM of collagen IV and laminin, but not fibronectin. In adults, in the primary and secondary follicles, collagen IV, laminin and to a lesser extent fibronectin were prominent in the follicular BM.
Conclusions
The ECM-molecular niche compartimentalizes the female gonads from the time of germ cell colonization until adulthood. This knowledge may contribute to improve methods to recreate the environment needed for successful folliculogenesis in vitro and that would benefit a large number of infertility patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0054-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-015-0054-0
PMCID: PMC4307144  PMID: 25605128
Human; Germ cells; Adult ovary; Follicles; Extracellular matrix; Gonads
9.  Novel monoclonal antibodies to study tissue regeneration in planarians 
Background
Planarians are an attractive model organism for studying stem cell-based regeneration due to their ability to replace all of their tissues from a population of adult stem cells. The molecular toolkit for planarian studies currently includes the ability to study gene function using RNA interference (RNAi) and observe gene expression via in situ hybridizations. However, there are few antibodies available to visualize protein expression, which would greatly enhance analysis of RNAi experiments as well as allow further characterization of planarian cell populations using immunocytochemistry and other immunological techniques. Thus, additional, easy-to-use, and widely available monoclonal antibodies would be advantageous to study regeneration in planarians.
Results
We have created seven monoclonal antibodies by inoculating mice with formaldehyde-fixed cells isolated from dissociated 3-day regeneration blastemas. These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema. We have tested these antibodies using eight variations of a formaldehyde-based fixation protocol and determined reliable protocols for immunolabeling whole planarians with each antibody. We found that labeling efficiency for each antibody varies greatly depending on the addition or removal of tissue processing steps that are used for in situ hybridization or immunolabeling techniques. Our experiments show that a subset of the antibodies can be used alongside markers commonly used in planarian research, including anti-SYNAPSIN and anti-SMEDWI, or following whole-mount in situ hybridization experiments.
Conclusions
The monoclonal antibodies described in this paper will be a valuable resource for planarian research. These antibodies have the potential to be used to better understand planarian biology and to characterize phenotypes following RNAi experiments. In addition, we present alterations to fixation protocols and demonstrate how these changes can increase the labeling efficiencies of antibodies used to stain whole planarians.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-014-0050-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-014-0050-9
PMCID: PMC4307677  PMID: 25604901
Planaria; Regeneration; Schmidtea mediterranea; Monoclonal antibodies; Immunostaining; Immunohistochemistry
10.  Bioelectric patterning during oogenesis: stage-specific distribution of membrane potentials, intracellular pH and ion-transport mechanisms in Drosophila ovarian follicles 
Background
Bioelectric phenomena have been found to exert influence on various developmental and regenerative processes. Little is known about their possible functions and the cellular mechanisms by which they might act during Drosophila oogenesis. In developing follicles, characteristic extracellular current patterns and membrane-potential changes in oocyte and nurse cells have been observed that partly depend on the exchange of protons, potassium ions and sodium ions. These bioelectric properties have been supposed to be related to various processes during oogenesis, e. g. pH-regulation, osmoregulation, cell communication, cell migration, cell proliferation, cell death, vitellogenesis and follicle growth. Analysing in detail the spatial distribution and activity of the relevant ion-transport mechanisms is expected to elucidate the roles that bioelectric phenomena play during oogenesis.
Results
To obtain an overview of bioelectric patterning along the longitudinal and transversal axes of the developing follicle, the spatial distributions of membrane potentials (Vmem), intracellular pH (pHi) and various membrane-channel proteins were studied systematically using fluorescent indicators, fluorescent inhibitors and antisera. During mid-vitellogenic stages 9 to 10B, characteristic, stage-specific Vmem-patterns in the follicle-cell epithelium as well as anteroposterior pHi-gradients in follicle cells and nurse cells were observed. Corresponding distribution patterns of proton pumps (V-ATPases), voltage-dependent L-type Ca2+-channels, amiloride-sensitive Na+-channels and Na+,H+-exchangers (NHE) and gap-junction proteins (innexin 3) were detected. In particular, six morphologically distinguishable follicle-cell types are characterized on the bioelectric level by differences concerning Vmem and pHi as well as specific compositions of ion channels and carriers. Striking similarities between Vmem-patterns and activity patterns of voltage-dependent Ca2+-channels were found, suggesting a mechanism for transducing bioelectric signals into cellular responses. Moreover, gradients of electrical potential and pH were observed within single cells.
Conclusions
Our data suggest that spatial patterning of Vmem, pHi and specific membrane-channel proteins results in bioelectric signals that are supposed to play important roles during oogenesis, e. g. by influencing spatial coordinates, regulating migration processes or modifying the cytoskeletal organization. Characteristic stage-specific changes of bioelectric activity in specialized cell types are correlated with various developmental processes.
doi:10.1186/s12861-015-0051-3
PMCID: PMC4302609  PMID: 25591552
Drosophila melanogaster; Oogenesis; Bioelectricity; Cell communication; Pattern formation; Membrane potential; Ion channel; Ion pump; Gap junction; Live-cell imaging
11.  Denervation impairs regeneration of amputated zebrafish fins 
Background
Zebrafish are able to regenerate many of its tissues and organs after damage. In amphibians this process is regulated by nerve fibres present at the site of injury, which have been proposed to release factors into the amputated limbs/fins, promoting and sustaining the proliferation of blastemal cells. Although some candidate factors have been proposed to mediate the nerve dependency of regeneration, the molecular mechanisms involved in this process remain unclear.
Results
We have used zebrafish as a model system to address the role of nerve fibres in fin regeneration. We have developed a protocol for pectoral fin denervation followed by amputation and analysed the regenerative process under this experimental conditions. Upon denervation fins were able to close the wound and form a wound epidermis, but could not establish a functional apical epithelial cap, with a posterior failure of blastema formation and outgrowth, and the accumulation of several defects. The expression patterns of genes known to be key players during fin regeneration were altered upon denervation, suggesting that nerves can contribute to the regulation of the Fgf, Wnt and Shh pathways during zebrafish fin regeneration.
Conclusions
Our results demonstrate that proper innervation of the zebrafish pectoral fin is essential for a successful regenerative process, and establish this organism as a useful model to understand the molecular and cellular mechanisms of nerve dependence, during vertebrate regeneration.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-014-0049-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-014-0049-2
PMCID: PMC4333893  PMID: 25551555
12.  Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine 
BMC Developmental Biology  2014;14(1):781.
Background
Mapping of tissue structure at the cellular, circuit, and organ-wide scale is important for understanding physiological and biological functions. A bio-electrochemical technique known as CLARITY used for three-dimensional anatomical and phenotypical mapping within transparent intact tissues has been recently developed. This method provided a major advance in understanding the structure-function relationships in circuits of the nervous system and organs by using whole-body clearing. Thus, in the present study, we aimed to improve the original CLARITY procedure and developed specific CLARITY protocols for various intact organs.
Results
We determined the optimal conditions for reducing bubble formation, discoloration, and depositing of black particles on the surface of tissue, which allowed production of clearer organ images. We also determined the appropriate replacement cycles of clearing solution for each type of organ, and convincingly demonstrated that 250–280 mA is the ideal range of electrical current for tissue clearing. We then acquired each type of cleared organs including brain, pancreas, liver, lung, kidney, and intestine. Additionally, we determined the images of axon fibers of hippocampal region, the Purkinje layer of cerebellum, and vessels and cellular nuclei of pancreas.
Conclusions
CLARITY is an innovative biochemical technology for the structural and molecular analysis of various types of tissue. We developed improved CLARITY methods for clearing of the brain, pancreas, lung, intestine, liver, and kidney, and identified the appropriate experimental conditions for clearing of each specific tissue type. These optimized methods will be useful for the application of CLARITY to various types of organs.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-014-0048-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-014-0048-3
PMCID: PMC4281481  PMID: 25528649
CLARITY; Brain; Nervous system; Electrophoretic tissue clearing; 3D Reconstruction; Purkinje layer
13.  Generation of cell type-specific monoclonal antibodies for the planarian and optimization of sample processing for immunolabeling 
BMC Developmental Biology  2014;14(1):45.
Background
Efforts to elucidate the cellular and molecular mechanisms of regeneration have required the application of methods to detect specific cell types and tissues in a growing cohort of experimental animal models. For example, in the planarian Schmidtea mediterranea, substantial improvements to nucleic acid hybridization and electron microscopy protocols have facilitated the visualization of regenerative events at the cellular level. By contrast, immunological resources have been slower to emerge. Specifically, the repertoire of antibodies recognizing planarian antigens remains limited, and a more systematic approach is needed to evaluate the effects of processing steps required during sample preparation for immunolabeling.
Results
To address these issues and to facilitate studies of planarian digestive system regeneration, we conducted a monoclonal antibody (mAb) screen using phagocytic intestinal cells purified from the digestive tracts of living planarians as immunogens. This approach yielded ten antibodies that recognized intestinal epitopes, as well as markers for the central nervous system, musculature, secretory cells, and epidermis. In order to improve signal intensity and reduce non-specific background for a subset of mAbs, we evaluated the effects of fixation and other steps during sample processing. We found that fixative choice, treatments to remove mucus and bleach pigment, as well as methods for tissue permeabilization and antigen retrieval profoundly influenced labeling by individual antibodies. These experiments led to the development of a step-by-step workflow for determining optimal specimen preparation for labeling whole planarians as well as unbleached histological sections.
Conclusions
We generated a collection of monoclonal antibodies recognizing the planarian intestine and other tissues; these antibodies will facilitate studies of planarian tissue morphogenesis. We also developed a protocol for optimizing specimen processing that will accelerate future efforts to generate planarian-specific antibodies, and to extend functional genetic studies of regeneration to post-transcriptional aspects of gene expression, such as protein localization or modification. Our efforts demonstrate the importance of systematically testing multiple approaches to species-specific idiosyncracies, such as mucus removal and pigment bleaching, and may serve as a template for the development of immunological resources in other emerging model organisms.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-014-0045-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-014-0045-6
PMCID: PMC4299570  PMID: 25528559
Planarian; Regeneration; Intestine; Monoclonal antibody screen; Immunohistochemistry; Immunofluorescence
14.  The role of Sox9 in mouse mammary gland development and maintenance of mammary stem and luminal progenitor cells 
BMC Developmental Biology  2014;14(1):47.
Background
Identification and characterization of molecular controls that regulate mammary stem and progenitor cell homeostasis are critical to our understanding of normal mammary gland development and its pathology.
Results
We demonstrate that conditional knockout of Sox9 in the mouse mammary gland results in impaired postnatal development. In short-term lineage tracing in the postnatal mouse mammary gland using Sox9-CreER driven reporters, Sox9 marked primarily the luminal progenitors and bipotent stem/progenitor cells within the basal mammary epithelial compartment. In contrast, long-term lineage tracing studies demonstrate that Sox9+ precursors gave rise to both luminal and myoepithelial cell lineages. Finally, fate mapping of Sox9 deleted cells demonstrates that Sox9 is essential for luminal, but not myoepithelial, lineage commitment and proliferation.
Conclusions
These studies identify Sox9 as a key regulator of mammary gland development and stem/progenitor maintenance.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-014-0047-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-014-0047-4
PMCID: PMC4297388  PMID: 25527186
SOX9; Stem cells; Luminal progenitor cells; Mammary gland development; Cre-lox; Knockout
15.  Isoform-specific functions of Mud/NuMA mediate binucleation of Drosophila male accessory gland cells 
BMC Developmental Biology  2014;14(1):46.
Background
In standard cell division, the cells undergo karyokinesis and then cytokinesis. Some cells, however, such as cardiomyocytes and hepatocytes, can produce binucleate cells by going through mitosis without cytokinesis. This cytokinesis skipping is thought to be due to the inhibition of cytokinesis machinery such as the central spindle or the contractile ring, but the mechanisms regulating it are unclear. We investigated them by characterizing the binucleation event during development of the Drosophila male accessory gland, in which all cells are binucleate.
Results
The accessory gland cells arrested the cell cycle at 50 hours after puparium formation (APF) and in the middle of the pupal stage stopped proliferating for 5 hours. They then restarted the cell cycle and at 55 hours APF entered the M-phase synchronously. At this stage, accessory gland cells binucleated by mitosis without cytokinesis. Binucleating cells displayed the standard karyokinesis progression but also showed unusual features such as a non-round shape, spindle orientation along the apico-basal axis, and poor assembly of the central spindle. Mud, a Drosophila homolog of NuMA, regulated the processes responsible for these three features, the classical isoform MudPBD and the two newly characterized isoforms MudL and MudS regulated them differently: MudL repressed cell rounding, MudPBD and MudS oriented the spindle along the apico-basal axis, and MudS and MudL repressed central spindle assembly. Importantly, overexpression of MudS induced binucleation even in standard proliferating cells such as those in imaginal discs.
Conclusions
We characterized the binucleation in the Drosophila male accessory gland and examined mechanisms that regulated unusual morphologies of binucleating cells. We demonstrated that Mud, a microtubule binding protein regulating spindle orientation, was involved in this binucleation. We suggest that atypical functions exerted by three structurally different isoforms of Mud regulate cell rounding, spindle orientation and central spindle assembly in binucleation. We also propose that MudS is a key regulator triggering cytokinesis skipping in binucleation processes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-014-0046-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-014-0046-5
PMCID: PMC4300151  PMID: 25527079
Drosophila; Male accessory gland; Binucleation; Cytokinesis; Central spindle; Cell rounding; Spindle orientation; Mud
16.  Perturbing the developing skull: using laser ablation to investigate the robustness of the infraorbital bones in zebrafish (Danio rerio) 
BMC Developmental Biology  2014;14(1):44.
Background
The development of the craniofacial skeleton from embryonic mesenchyme is a complex process that is not yet completely understood, particularly for intramembranous bones. This study investigates the development of the neural crest derived infraorbital (IO) bones of the zebrafish (Danio rerio) skull. Located under the orbit, the IO bones ossify in a set sequence and are closely associated with the lateral line system. We conducted skeletogenic condensation and neuromast laser ablation experiments followed by shape analyses in order to investigate the relationship between a developing IO bone and the formation of the IO series as well as to investigate the highly debated inductive potential of neuromasts for IO ossification.
Results
We demonstrate that when skeletogenic condensations recover from laser ablation, the resulting bone differs in shape compared to controls. Interestingly, neighbouring IO bones in the bone series are unaffected. In addition, we show that the amount of canal wall mineralization is significantly decreased following neuromast laser ablation at juvenile and larval stages.
Conclusions
These results highlight the developmental robustness of the IO bones and provide direct evidence that canal neuromasts play a role in canal wall development in the head. Furthermore, we provide evidence that the IO bones may be two distinct developmental modules. The mechanisms underlying developmental robustness are rarely investigated and are important to increase our understanding of evolutionary developmental biology of the vertebrate skull.
doi:10.1186/s12861-014-0044-7
PMCID: PMC4282728  PMID: 25516292
Intramembranous ossification; Morphometrics; Robustness; Lateral line canal walls; Craniofacial development
17.  Mechanistic target of rapamycin complex 1 signaling regulates cell proliferation, cell survival, and differentiation in regenerating zebrafish fins 
BMC Developmental Biology  2014;14(1):42.
Background
The mechanistic target of rapamycin complex1 (mTORC1) signaling pathway has been implicated in functions of multicellular processes, including cell growth and metabolism. Although recent reports showed that many signaling pathways, including Activin, Bmp, Fgf, sonic hedgehog, Insulin-like growth factor (IGF), Notch, retinoic acid, and Wnt, are implicated in non-mammalian vertebrate regeneration, also known as epimorphic regeneration, mTORC1 function remains unknown.
Results
To investigate the role of mTORC1 signaling pathway in zebrafish caudal fin, we examined the activation and function of mTORC1 signaling using an antibody against phosphorylated S6 kinase and a specific inhibitor, rapamycin. mTORC1 signaling is activated in proliferative cells of intra-ray and wound epidermal cells before blastema formation, as well as in proliferative blastema cells, wound epidermal cells, and osteoblasts during regenerative outgrowth. Before blastema formation, proliferation of intra-ray and wound epidermal cells is suppressed, but cell death is not affected by mTORC1 signaling inhibition with rapamycin. Moreover, rapamycin treatment inhibits blastema and wound epidermal cell proliferation and survival during blastema formation and regenerative outgrowth, as well as osteoblast proliferation and differentiation during regenerative outgrowth. We further determined that mTORC1 signaling is regulated through IGF-1 receptor/phosphatidylinositol-3 kinase and Wnt pathways during fin regeneration.
Conclusion
Taken together, our findings reveal that mTORC1 signaling regulates proliferation, survival, and differentiation of intra-ray cells, wound epidermis, blastema cells, and/or osteoblasts in various fin regeneration stages downstream of IGF and Wnt signaling pathways.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-014-0042-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-014-0042-9
PMCID: PMC4264545  PMID: 25480380
Mechanistic target of rapamycin; Fin; Regeneration; Zebrafish; Osteoblast; Cell proliferation; Cell survival; Differentiation
18.  TGF-β signaling can act from multiple tissues to regulate C. elegans body size 
BMC Developmental Biology  2014;14(1):43.
Background
Regulation of organ and body size is a fundamental biological phenomenon, requiring tight coordination between multiple tissues to ensure accurate proportional growth. In C. elegans, a TGF-β pathway is the major regulator of body size and also plays a role in the development of the male tail, and is thus referred to as the TGF-β/Sma/Mab (for small and male abnormal) pathway. Mutations in components of this pathway result in decreased growth of animals during larval stages, with Sma mutant adults of the core pathway as small as ~60-70% the length of normal animals. The currently accepted model suggests that TGF-β/Sma/Mab pathway signaling in the C. elegans hypodermis is both necessary and sufficient to control body length. However, components of this signaling pathway are expressed in other organs, such as the intestine and pharynx, raising the question of what the function of the pathway is in these organs.
Results
Here we show that TGF-β/Sma/Mab signaling is required for the normal growth of the pharynx. We further extend the current model and show that the TGF-β/Sma/Mab pathway can function in multiple tissues to regulate body and organ length. Specifically, we find that pharyngeal expression of the SMAD protein SMA-3 partially rescues both pharynx length and body length of sma-3 mutants.
Conclusions
Overall, our results support a model in which the TGF-β/Sma/Mab signaling pathway can act in multiple tissues, activating one or more downstream secreted signals that act non cell-autonomously to regulate overall body length in C. elegans.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-014-0043-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-014-0043-8
PMCID: PMC4278669  PMID: 25480452
Caenorhabditis elegans; TGF-β; Sma/Mab; Body size; Pharynx; Hypodermis
19.  A vertebrate-conserved cis-regulatory module for targeted expression in the main hypothalamic regulatory region for the stress response 
BMC Developmental Biology  2014;14(1):41.
Background
The homeodomain transcription factor orthopedia (Otp) is an evolutionarily conserved regulator of neuronal fates. In vertebrates, Otp is necessary for the proper development of different regions of the brain and is required in the diencephalon to specify several hypothalamic cell types, including the cells that control the stress response. To understand how this widely expressed transcription factor accomplishes hypothalamus-specific functions, we performed a comprehensive screening of otp cis-regulatory regions in zebrafish.
Results
Here, we report the identification of an evolutionarily conserved vertebrate enhancer module with activity in a restricted area of the forebrain, which includes the region of the hypothalamus that controls the stress response. This region includes neurosecretory cells producing Corticotropin-releasing hormone (Crh), Oxytocin (Oxt) and Arginine vasopressin (Avp), which are key components of the stress axis. Lastly, expression of the bacterial nitroreductase gene under this specific enhancer allowed pharmacological attenuation of the stress response in zebrafish larvae.
Conclusion
Vertebrates share many cellular and molecular components of the stress response and our work identified a striking conservation at the cis-regulatory level of a key hypothalamic developmental gene. In addition, this enhancer provides a useful tool to manipulate and visualize stress-regulatory hypothalamic cells in vivo with the long-term goal of understanding the ontogeny of the stress axis in vertebrates.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-014-0041-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-014-0041-x
PMCID: PMC4248439  PMID: 25427861
Otp; Evolutionarily conserved non-coding regions; cis-regulatory module; Hypothalamic-pituitary-adrenal axis; Neurosecretory preoptic area; Cortisol; Zebrafish; Stress response
20.  Expression of maternally derived KHDC3, NLRP5, OOEP and TLE6 is associated with oocyte developmental competence in the ovine species 
BMC Developmental Biology  2014;14(1):40.
Background
The sub-cortical maternal complex (SCMC), located in the subcortex of mouse oocytes and preimplantation embryos, is composed of at least four proteins encoded by maternal effect genes: OOEP, NLRP5/MATER, TLE6 and KHDC3/FILIA. The SCMC assembles during oocyte growth and was seen to be essential for murine zygote progression beyond the first embryonic cell divisions; although roles in chromatin reprogramming and embryonic genome activation were hypothesized, the full range of functions of the complex in preimplantation development remains largely unknown.
Results
Here we report the expression of the SCMC genes in ovine oocytes and pre-implantation embryos, describing for the first time its expression in a large mammalian species.
We report sheep-specific patterns of expression and a relationship with the oocyte developmental potential in terms of delayed degradation of maternal SCMC transcripts in pre-implantation embryos derived from developmentally incompetent oocytes.
In addition, by determining OOEP full length cDNA by Rapid Amplification of cDNA Ends (RACE) we identified two different transcript variants (OOEP1 and OOEP2), both expressed in oocytes and early embryos, but with different somatic tissue distributions.
In silico translation showed that 140 aminoacid peptide OOEP1 shares an identity with orthologous proteins ranging from 95% with the bovine to 45% with mouse. Conversely, OOEP2 contains a premature termination codon, thus representing an alternative noncoding transcript and supporting the existence of aberrant splicing during ovine oogenesis.
Conclusions
These findings confirm the existence of the SCMC in sheep and its key role for the oocyte developmental potential, deepening our understanding on the molecular differences underlying cytoplasmic vs nuclear maturation of the oocytes.
Describing differences and overlaps in transcriptome composition between model organisms advance our comprehension of the diversity/uniformity between mammalian species during early embryonic development and provide information on genes that play important regulatory roles in fertility in nonmurine models, including the human.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-014-0040-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-014-0040-y
PMCID: PMC4247878  PMID: 25420964
Ovine embryo; Oocyte developmental competence; Subcortical Maternal Complex
22.  DNAJB13, a type II HSP40 family member, localizes to the spermatids and spermatozoa during mouse spermatogenesis 
Background
Hundreds of HSP40s derived from various species have been identified, of which several proteins are involved in spermatogenesis. DNAJB13 is a type II HSP40/DnaJ protein. In a previous study, we cloned mouse Dnajb13, which is up-regulated in cryptorchidism. To date, however, little is known about the localization and functions of DNAJB13 during spermatogenesis. This study intends to identify the expression pattern of DNAJB13 during mammalian spermatogenesis.
Results
RT-PCR and western blot revealed that the Dnajb13 gene and DNAJB13 protein were expressed not only in the testis but also in several other ciliated cell-containing tissues like the trachea, lung and oviduct. Quantitative PCR showed that the expression of Dnajb13 mRNA in testis was detectable as early as postnatal week 1, and sharply increased from postnatal week 3. Western blotting and immunohistochemistry determined that the DNAJB13 protein, which was located in the cytoplasm of spermatids and the sperm flagellum, was detectable from postnatal week 4.
Conclusions
Based on the spatiotemporal expression observed in the cytoplasm of spermatids and sperm flagella, we suggest that DNAJB13 participates in spermiogenesis and the motility of mature spermatozoa.
doi:10.1186/s12861-014-0038-5
PMCID: PMC4236558  PMID: 25233908
Dnajb13; Spermatid; Flagella; Spermiogenesis; Motility
23.  Vascularization of primary and secondary ossification centres in the human growth plate 
Background
The switch from cartilage template to bone during endochondral ossification of the growth plate requires a dynamic and close interaction between cartilage and the developing vasculature. Vascular invasion of the primarily avascular hypertrophic chondrocyte zone brings chondroclasts, osteoblast- and endothelial precursor cells into future centres of ossification.
Vascularization of human growth plates of polydactylic digits was studied by immunohistochemistry, confocal-laser-scanning-microscopy and RT-qPCR using markers specific for endothelial cells CD34 and CD31, smooth muscle cells α-SMA, endothelial progenitor cells CD133, CXCR4, VEGFR-2 and mesenchymal progenitor cells CD90 and CD105. In addition, morphometric analysis was performed to quantify RUNX2+ and DLX5+ hypertrophic chondrocytes, RANK+ chondro- and osteoclasts, and CD133+ progenitors in different zones of the growth plate.
Results
New vessels in ossification centres were formed by sprouting of CD34+ endothelial cells that did not co-express the mature endothelial cell marker CD31. These immature vessels in the growth plate showed no abluminal coverage with α-SMA+ smooth muscle cells, but in their close proximity single CD133+ precursor cells were found that did not express VEGFR-2, a marker for endothelial lineage commitment. In periosteum and in the perichondrial groove of Ranvier that harboured CD90+/CD105+ chondro-progenitors, in contrast, mature vessels were found stabilized by α-SMA+ smooth muscle cells.
Conclusion
Vascularization of ossification centres of the growth plate was mediated by sprouting of capillaries coming from the bone collar or by intussusception rather than by de-novo vessel formation involving endothelial progenitor cells. Vascular invasion of the joint anlage was temporally delayed compared to the surrounding joint tissue.
doi:10.1186/s12861-014-0036-7
PMCID: PMC4236517  PMID: 25164565
Growth plate; vascularisation; Primary ossification centres; Secondary ossification centres; Progenitor cells
24.  Changes in expression of Class 3 Semaphorins and their receptors during development of the rat retina and superior colliculus 
Background
Members of the Semaphorin 3 family (Sema3s) influence the development of the central nervous system, and some are implicated in regulating aspects of visual system development. However, we lack information about the timing of expression of the Sema3s with respect to different developmental epochs in the mammalian visual system. In this time-course study in the rat, we document for the first time changes in the expression of RNAs for the majority of Class 3 Semaphorins (Sema3s) and their receptor components during the development of the rat retina and superior colliculus (SC).
Results
During retinal development, transcript levels changed for all of the Sema3s examined, as well as Nrp2, Plxna2, Plxna3, and Plxna4a. In the SC there were also changes in transcript levels for all Sema3s tested, as well as Nrp1, Nrp2, Plxna1, Plxna2, Plxna3, and Plxna4a. These changes correlate with well-established epochs, and our data suggest that the Sema3s could influence retinal ganglion cell (RGC) apoptosis, patterning and connectivity in the maturing retina and SC, and perhaps guidance of RGC and cortical axons in the SC. Functionally we found that SEMA3A, SEMA3C, SEMA3E, and SEMA3F proteins collapsed purified postnatal day 1 RGC growth cones in vitro. Significantly this is a developmental stage when RGCs are growing into and within the SC and are exposed to Sema3 ligands.
Conclusion
These new data describing the overall temporal regulation of Sema3 expression in the rat retina and SC provide a platform for further work characterising the functional impact of these proteins on the development and maturation of mammalian visual pathways.
doi:10.1186/s12861-014-0034-9
PMCID: PMC4121511  PMID: 25062604
Retinal ganglion cells; Collapse assay; qPCR; Neuropilins; Plexins; Cell adhesion molecules
25.  14-3-3 epsilon prevents G2/M transition of fertilized mouse eggs by binding with CDC25B 
Background
The 14-3-3 (YWHA) proteins are highly conserved in higher eukaryotes, participate in various cellular signaling pathways including cell cycle regulation, development and growth. Our previous studies demonstrated that 14-3-3ε (YWHAE) is responsible for maintaining prophase | arrest in mouse oocyte. However, roles of 14-3-3ε in the mitosis of fertilized mouse eggs have remained unclear. Here, we showed that 14-3-3ε interacts and cooperates with CDC25B phosphorylated at Ser321 regulating G2/M transition of mitotic progress of fertilized mouse eggs.
Results
Disruption of 14-3-3ε expression by RNAi prevented normal G2/M transition by inhibition of MPF activity and leaded to the translocation of CDC25B into the nucleus from the cytoplasm. Overexpression of 14-3-3ε-WT and unphosphorylatable CDC25B mutant (CDC25B-S321A) induced mitotic resumption in dbcAMP-arrested eggs. In addition, we examined endogenous and exogenous distribution of 14-3-3ε and CDC25B. Endogenous 14-3-3ε and CDC25B were co-localized primarily in the cytoplasm at the G1, S, early G2 and M phases whereas CDC25B was found to accumulate in the nucleus at the late G2 phase. Upon coexpression with RFP–14-3-3ε, GFP–CDC25B–WT and GFP–CDC25B–S321A were predominantly cytoplasmic at early G2 phase and then GFP–CDC25B–S321A moved to the nucleus whereas CDC25B-WT signals were observed in the cytoplasm without nucleus accumulation at late G2 phase at presence of dbcAMP.
Conclusions
Our data indicate that 14-3-3ε is required for the mitotic entry in the fertilized mouse eggs. 14-3-3ε is primarily responsible for sequestering the CDC25B in cytoplasm and 14-3-3ε binding to CDC25B-S321 phosphorylated by PKA induces mitotic arrest at one-cell stage by inactivation of MPF in fertilized mouse eggs.
doi:10.1186/s12861-014-0033-x
PMCID: PMC4222595  PMID: 25059436
14-3-3ε; CDC25B; MPF; Fertilized mouse eggs

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