FGF8 induces chemokinesis and regulates condensation of mouse nephron progenitor cells.

Kidneys develop via iterative branching of the ureteric epithelial tree and subsequent nephrogenesis at the branch points. Nephrons form in the cap mesenchyme as the metanephric mesenchyme (MM) condenses around the epithelial ureteric buds (UBs). Previous work has demonstrated that FGF8 is important for the survival of nephron progenitor cells (NPCs), and early deletion of Fgf8 leads to the cessation of nephron formation, which results in post-natal lethality. We now reveal a previously unreported function of FGF8. By combining transgenic mouse models, quantitative imaging assays and data-driven computational modelling, we show that FGF8 has a strong chemokinetic effect and that this chemokinetic effect is important for the condensation of NPCs to the UB. The computational model shows that the motility must be lower close to the UB to achieve NPC attachment. We conclude that the FGF8 signalling pathway is crucial for the coordination of NPC condensation at the UB. Chemokinetic effects have also been described for other FGFs and may be generally important for the formation of mesenchymal condensates.

Generation of novel in vitro flexible kidney organoid model to investigate the role of extracellular vesicles in induction of nephrogenesis.

BACKGROUND: During kidney organogenesis, metanephric mesenchyme (MM) and ureteric bud (UB) interact reciprocally to form nephrons. Signaling stimuli involved in these interactions include Wnts, growth factors and nano/micro particles. How UB and MM are interacting is not completely understood. Our study investigated the signaling and communication via extracellular vesicles (EVs) during nephrogenesis. Embryonic day (E) 11.5 mouse kidney UB and MM produce very low number of primary cells that have limited ability for proliferation in culture. Such limitations obstruct studying the role of EVs in induction of nephrogenesis. These issues necessitate to generate a nephrogenesis model allowing to study the comprehensive role of EVs during nephrogenesis. RESULTS: Our study generated a UB derived cell line-based in vitro flexible model of nephrogenesis allowing expandable cell culturing, in addition to performing characterization, tracking and blocking of EVs. UB cell line aggregation with E11.5 MM cells induced the formation of segmented nephrons. Most efficient nephrogenesis was obtained by the co-culturing of 30,000 cells of UB cell line with 50,000 MM cells. Results revealed that both the UB and the MM secrete EVs during nephrogenesis. UB cell line derived EVs were characterized by their size, morphology and expression of markers (CD63, TSG101, CD9 and CD81). Furthermore, proteomics data of UB cell line-derived EVs revealed large number of proteins involved in nephrogenesis-related signaling pathways. Palmitoylated GFP-tagged EVs from UB cell line were found in the nephron formation zone in the developing kidney organoid. UB cell line derived EVs did not induce nephrogenesis in MM cells but significantly contributed to the survival and nephrogenesis-competency of MM cells. The secretion of EVs was continuously inhibited during the ongoing nephrogenesis by the knockdown of RalA and RalB gene expression using short hairpin RNAs. This inhibition partially impaired the ability of UB cell line to induce nephrogenesis. Moreover, impaired nephrogenesis was partially rescued by the addition of EVs. CONCLUSION: Our study established a novel in vitro flexible model of nephrogenesis that solved the limitations of primary embryonic kidney cells and mouse embryonic stem cell kidney organoids for the EV research. EVs were found to be an integral part of nephrogenesis process. Video Abstract.

Deciphering the minimal quantity of mouse primary cells to undergo nephrogenesis ex vivo.

BACKGROUND: Tissue organoids derived from primary cells have high potential for studying organ development and diseases in numerous organs. They recreate the morphological structure and mimic the functions of given organ while being compact in size, easy to produce, and suitable for use in various experimental setups. RESULTS: In this study we established the number of cells that form mouse kidney rudiments at E11.5, and generated renal organoids of various sizes from the mouse primary cells of the metanephric mesenchyme (MM). We investigated the ability of renal organoids to undergo nephrogenesis upon Wnt/ ß-catenin pathway-mediated tubule induction with a GSK-3 inhibitor (BIO) or by initiation through the ureteric bud (UB). We found that 5000 cells of MM cells are necessary to successfully form renal organoids with well-structured nephrons as judged by fluorescent microscopy, transmission electron microscopy (TEM), and quantitative Polymerase Chain Reaction (qPCR). These mouse organoids also recapitulated renal secretion function in the proximal tubules. CONCLUSIONS: We show that a significant decrease of cells used to generate renal mouse organoids in a dissociation/re-aggregation assay, does not interfere with development, and goes toward 3Rs. This enables generation of more experimental samples with one mouse litter, limiting the number of animals used for studies.

Erbb4 regulates the oocyte microenvironment during folliculogenesis.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders leading to infertility in women affecting reproductive, endocrine and metabolic systems. Recent genomewide association studies on PCOS cohorts revealed a single nucleotide polymorphism (SNP) in the ERBB4 receptor tyrosine kinase 4 gene, but its role in ovary development or during folliculogenesis remains poorly understood. Since no genetic animal models mimicking all PCOS reproductive features are available, we conditionally deleted Erbb4 in murine granulosa cells (GCs) under the control of Amh promoter. While we have demonstrated that Erbb4 deletion displayed aberrant ovarian function by affecting the reproductive function (asynchronous oestrous cycle leading to few ovulations and subfertility) and metabolic function (obesity), their ovaries also present severe structural and functional abnormalities (impaired oocyte development). Hormone analysis revealed an up-regulation of serum luteinizing hormone, hyperandrogenism, increased production of ovarian and circulating anti-Müllerian hormone. Our data implicate that Erbb4 deletion in GCs leads to defective intercellular junctions between the GCs and oocytes, causing changes in the expression of genes regulating the local microenvironment of the follicles. In vitro culture assays reducing the level of Erbb4 via shRNAs confirm that Erbb4 is essential for regulating Amh level. In conclusion, our results indicate a functional role for Erbb4 in the ovary, especially during folliculogenesis and its reduced expression plays an important role in reproductive pathophysiology, such as PCOS development.

Characterization of nucleic acids from extracellular vesicle-enriched human sweat.

BACKGROUND: The human sweat is a mixture of secretions from three types of glands: eccrine, apocrine, and sebaceous. Eccrine glands open directly on the skin surface and produce high amounts of water-based fluid in response to heat, emotion, and physical activity, whereas the other glands produce oily fluids and waxy sebum. While most body fluids have been shown to contain nucleic acids, both as ribonucleoprotein complexes and associated with extracellular vesicles (EVs), these have not been investigated in sweat. In this study we aimed to explore and characterize the nucleic acids associated with sweat particles. RESULTS: We used next generation sequencing (NGS) to characterize DNA and RNA in pooled and individual samples of EV-enriched sweat collected from volunteers performing rigorous exercise. In all sequenced samples, we identified DNA originating from all human chromosomes, but only the mitochondrial chromosome was highly represented with 100% coverage. Most of the DNA mapped to unannotated regions of the human genome with some regions highly represented in all samples. Approximately 5 % of the reads were found to map to other genomes: including bacteria (83%), archaea (3%), and virus (13%), identified bacteria species were consistent with those commonly colonizing the human upper body and arm skin. Small RNA-seq from EV-enriched pooled sweat RNA resulted in 74% of the trimmed reads mapped to the human genome, with 29% corresponding to unannotated regions. Over 70% of the RNA reads mapping to an annotated region were tRNA, while misc. RNA (18,5%), protein coding RNA (5%) and miRNA (1,85%) were much less represented. RNA-seq from individually processed EV-enriched sweat collection generally resulted in fewer percentage of reads mapping to the human genome (7-45%), with 50-60% of those reads mapping to unannotated region of the genome and 30-55% being tRNAs, and lower percentage of reads being rRNA, LincRNA, misc. RNA, and protein coding RNA. CONCLUSIONS: Our data demonstrates that sweat, as all other body fluids, contains a wealth of nucleic acids, including DNA and RNA of human and microbial origin, opening a possibility to investigate sweat as a source for biomarkers for specific health parameters.

Secreted Extracellular Vesicle Molecular Cargo as a Novel Liquid Biopsy Diagnostics of Central Nervous System Diseases.

Secreted extracellular vesicles (EVs) are heterogeneous cell-derived membranous granules which carry a large diversity of molecules and participate in intercellular communication by transferring these molecules to target cells by endocytosis. In the last decade, EVs' role in several pathological conditions, from etiology to disease progression or therapy evasion, has been consolidated, including in central nervous system (CNS)-related disorders. For this review, we performed a systematic search of original works published, reporting the presence of molecular components expressed in the CNS via EVs, which have been purified from plasma, serum or cerebrospinal fluid. Our aim is to provide a list of molecular EV components that have been identified from both nonpathological conditions and the most common CNS-related disorders. We discuss the methods used to isolate and enrich EVs from specific CNS-cells and the relevance of its components in each disease context.

HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis.

Kidney development depends crucially on proper ureteric bud branching giving rise to the entire collecting duct system. The transcription factor HNF1B is required for the early steps of ureteric bud branching, yet the molecular and cellular events regulated by HNF1B are poorly understood. We report that specific removal of Hnf1b from the ureteric bud leads to defective cell-cell contacts and apicobasal polarity during the early branching events. High-resolution ex vivo imaging combined with a membranous fluorescent reporter strategy show decreased mutant cell rearrangements during mitosis-associated cell dispersal and severe epithelial disorganization. Molecular analysis reveals downregulation of Gdnf-Ret pathway components and suggests that HNF1B acts both upstream and downstream of Ret signaling by directly regulating Gfra1 and Etv5 Subsequently, Hnf1b deletion leads to massively mispatterned ureteric tree network, defective collecting duct differentiation and disrupted tissue architecture, which leads to cystogenesis. Consistently, mRNA-seq analysis shows that the most impacted genes encode intrinsic cell-membrane components with transporter activity. Our study uncovers a fundamental and recurring role of HNF1B in epithelial organization during early ureteric bud branching and in further patterning and differentiation of the collecting duct system in mouse.

Novel fixed z-direction (FiZD) kidney primordia and an organoid culture system for time-lapse confocal imaging.

Tissue, organ and organoid cultures provide suitable models for developmental studies, but our understanding of how the organs are assembled at the single-cell level still remains unclear. We describe here a novel fixed z-direction (FiZD) culture setup that permits high-resolution confocal imaging of organoids and embryonic tissues. In a FiZD culture a permeable membrane compresses the tissues onto a glass coverslip and the spacers adjust the thickness, enabling the tissue to grow for up to 12 days. Thus, the kidney rudiment and the organoids can adjust to the limited z-directional space and yet advance the process of kidney morphogenesis, enabling long-term time-lapse and high-resolution confocal imaging. As the data quality achieved was sufficient for computer-assisted cell segmentation and analysis, the method can be used for studying morphogenesis ex vivo at the level of the single constituent cells of a complex mammalian organogenesis model system.

Extracellular Vesicles as Innovative Tool for Diagnosis, Regeneration and Protection against Neurological Damage.

Extracellular vesicles (EVs) have recently attracted a great deal of interest as they may represent a new biosignaling paradigm. According to the mode of biogenesis, size and composition, two broad categories of EVs have been described, exosomes and microvesicles. EVs have been shown to carry cargoes of signaling proteins, RNA species, DNA and lipids. Once released, their content is selectively taken up by near or distant target cells, influencing their behavior. Exosomes are involved in cell-cell communication in a wide range of embryonic developmental processes and in fetal-maternal communication. In the present review, an outline of the role of EVs in neural development, regeneration and diseases is presented. EVs can act as regulators of normal homeostasis, but they can also promote either neuroinflammation/degeneration or tissue repair in pathological conditions, depending on their content. Since EV molecular cargo constitutes a representation of the origin cell status, EVs can be exploited in the diagnosis of several diseases. Due to their capability to cross the blood-brain barrier (BBB), EVs not only have been suggested for the diagnosis of central nervous system disorders by means of minimally invasive procedures, i.e., "liquid biopsies", but they are also considered attractive tools for targeted drug delivery across the BBB. From the therapeutic perspective, mesenchymal stem cells (MSCs) represent one of the most promising sources of EVs. In particular, the neuroprotective properties of MSCs derived from the dental pulp are here discussed.

Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk.

Ovarian hormones increase breast cancer risk by poorly understood mechanisms. We assess the role of progesterone on global stem cell function by serially transplanting mouse mammary epithelia. Progesterone receptor (PR) deletion severely reduces the regeneration capacity of the mammary epithelium. The PR target, receptor activator of Nf-κB ligand (RANKL), is not required for this function, and the deletion of Wnt4 reduces the mammary regeneration capacity even more than PR ablation. A fluorescent reporter reveals so far undetected perinatal Wnt4 expression that is independent of hormone signaling. Pubertal and adult Wnt4 expression is specific to PR+ luminal cells and requires intact PR signaling. Conditional deletion of Wnt4 reveals that this early, previously unappreciated, Wnt4 expression is functionally important. We provide genetic evidence that canonical Wnt signaling in the myoepithelium required PR and Wnt4, whereas the canonical Wnt signaling activities observed in the embryonic mammary bud and in the stroma around terminal end buds are independent of Wnt4. Thus, progesterone and Wnt4 control stem cell function through a luminal-myoepithelial crosstalk with Wnt4 acting independent of PR perinatally.

The CapZ interacting protein Rcsd1 is required for cardiogenesis downstream of Wnt11a in Xenopus laevis.

Wnt proteins are critical for embryonic cardiogenesis and cardiomyogenesis by regulating different intracellular signalling pathways. Whereas canonical Wnt/ß-catenin signalling is required for mesoderm induction and proliferation of cardiac progenitor cells, ß-catenin independent, non-canonical Wnt signalling regulates cardiac specification and terminal differentiation. Although the diverse cardiac malformations associated with the loss of non-canonical Wnt11 in mice such as outflow tract (OFT) defects, reduced ventricular trabeculation, myofibrillar disorganization and reduced cardiac marker gene expression are well described, the underlying molecular mechanisms are still not completely understood. Here we aimed to further characterize Wnt11 mediated signal transduction during vertebrate cardiogenesis. Using Xenopus as a model system, we show by loss of function and corresponding rescue experiments that the non-canonical Wnt signalling mediator Rcsd1 is required downstream of Wnt11 for ventricular trabeculation, terminal differentiation of cardiomyocytes and cardiac morphogenesis. We here place Rcsd1 downstream of Wnt11 during cardiac development thereby providing a novel mechanism for how non-canonical Wnt signalling regulates vertebrate cardiogenesis.

Wnt4 coordinates directional cell migration and extension of the Müllerian duct essential for ontogenesis of the female reproductive tract.

The Müllerian duct (MD) is the anlage of the oviduct, uterus and upper part of the vagina, the main parts of the female reproductive tract. Several wingless-type mouse mammary tumor virus (MMTV) integration site family member (Wnt) genes, including Wnt4, Wnt5a and Wnt7a, are involved in the development of MD and its derivatives, with Wnt4 particularly critical, since the MD fails to develop in its absence. We use, here, Wnt4(EGFPCre)-based fate mapping to demonstrate that the MD tip cells and the subsequent MD cells are derived from Wnt4+ lineage cells. Moreover, Wnt4 is required for the initiation of MD-forming cell migration. Application of anti-Wnt4 function-blocking antibodies after the initiation of MD elongation indicated that Wnt4 is necessary for the elongation as well, and consistent with this, cell culture wound-healing assays with NIH3T3 cells overexpressing Wnt4 promoted cell migration by comparison with controls. In contrast to the Wnt4 null embryos, some Wnt4(monomeric cherry/monomeric cherry) (Wnt4(mCh/mCh)) hypomorphic mice survived to adulthood and formed MD in ∼45% of cases. Nevertheless, the MD of the Wnt4(mCh/mCh) females had altered cell polarization and basement membrane deposition relative to the controls. Examination of the reproductive tract of the Wnt4(mCh/mCh) females indicated a poorly coiled oviduct, absence of the endometrial glands and an undifferentiated myometrium, and these mice were prone to develop a hydro-uterus. In conclusion, the results suggest that the Wnt4 gene encodes signals that are important for various aspects of female reproductive tract development.

Kidney development and perspectives for organ engineering.

Organ transplantation is currently the best strategy for treating end stage renal disease (ESRD) but the numbers of donor kidneys available are not sufficient to meet the needs of the ever-increasing ESRD population. Therefore, developments in the field of tissue engineering are necessary to provide alternative treatments. Decellularization and three-dimensional (3D) bioprinting strategies may serve as attractive novel options. Since successful tissue engineering requires an in -depth understanding of organ development and regulatory pathways, we discuss signaling in renal development and the composition of the renal extracellular matrix before presenting progress in the decellularization and 3D bioprinting fields.

Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis.

BACKGROUND: Wnt11 is a member of the Wnt family of secreted signals controlling the early steps in ureteric bud (UB) branching. Due to the reported lethality of Wnt11 knockout embryos in utero, its role in later mammalian kidney organogenesis remains open. The presence of Wnt11 in the emerging tubular system suggests that it may have certain roles later in the development of the epithelial ductal system. RESULTS: The Wnt11 knockout allele was backcrossed with the C57Bl6 strain for several generations to address possible differences in penetrance of the kidney phenotypes. Strikingly, around one third of the null mice with this inbred background survived to the postnatal stages. Many of them also reached adulthood, but urine and plasma analyses pointed out to compromised kidney function. Consistent with these data the tubules of the C57Bl6 Wnt11 (-/-) mice appeared to be enlarged, and the optical projection tomography indicated changes in tubular convolution. Moreover, the C57Bl6 Wnt11 (-/-) mice developed secondary glomerular cysts not observed in the controls. The failure of Wnt11 signaling reduced the expression of several genes implicated in kidney development, such as Wnt9b, Six2, Foxd1 and Hox10. Also Dvl2, an important PCP pathway component, was downregulated by more than 90 % due to Wnt11 deficiency in both the E16.5 and NB kidneys. Since all these genes take part in the control of UB, nephron and stromal progenitor cell differentiation, their disrupted expression may contribute to the observed anomalies in the kidney tubular system caused by Wnt11 deficiency. CONCLUSIONS: The Wnt11 signal has roles at the later stages of kidney development, namely in coordinating the development of the tubular system. The C57Bl6 Wnt11 (-/-) mouse generated here provides a model for studying the mechanisms behind tubular anomalies and glomerular cyst formation.

CD146(+) cells are essential for kidney vasculature development.

The kidney vasculature is critical for renal function, but its developmental assembly mechanisms remain poorly understood and models for studying its assembly dynamics are limited. Here, we tested whether the embryonic kidney contains endothelial cells (ECs) that are heterogeneous with respect to VEGFR2/Flk1/KDR, CD31/PECAM, and CD146/MCAM markers. Tie1Cre;R26R(YFP)-based fate mapping with a time-lapse in embryonic kidney organ culture successfully depicted the dynamics of kidney vasculature development and the correlation of the process with the CD31(+) EC network. Depletion of Tie1(+) or CD31(+) ECs from embryonic kidneys, with either Tie1Cre-induced diphtheria toxin susceptibility or cell surface marker-based sorting in a novel dissociation and reaggregation technology, illustrated substantial EC network regeneration. Depletion of the CD146(+) cells abolished this EC regeneration. Fate mapping of green fluorescent protein (GFP)-marked CD146(+)/CD31(-) cells indicated that they became CD31(+) cells, which took part in EC structures with CD31(+) wild-type ECs. EC network development depends on VEGF signaling, and VEGF and erythropoietin are expressed in the embryonic kidney even in the absence of any external hypoxic stimulus. Thus, the ex vivo embryonic kidney culture models adopted here provided novel ways for targeting renal EC development and demonstrated that CD146(+) cells are critical for kidney vasculature development.

Identification of the genes regulated by Wnt-4, a critical signal for commitment of the ovary.

The indifferent mammalian embryonic gonad generates an ovary or testis, but the factors involved are still poorly known. The Wnt-4 signal represents one critical female determinant, since its absence leads to partial female-to-male sex reversal in mouse, but its signalling is as well implicated in the testis development. We used the Wnt-4 deficient mouse as a model to identify candidate gonadogenesis genes, and found that the Notum, Phlda2, Runx-1 and Msx1 genes are typical of the wild-type ovary and the Osr2, Dach2, Pitx2 and Tacr3 genes of the testis. Strikingly, the expression of these latter genes becomes reversed in the Wnt-4 knock-out ovary, suggesting a role in ovarian development. We identified the transcription factor Runx-1 as a Wnt-4 signalling target gene, since it is expressed in the ovary and is reduced upon Wnt-4 knock-out. Consistent with this, introduction of the Wnt-4 signal into early ovary cells ex vivo induces Runx-1 expression, while conversely Wnt-4 expression is down-regulated in the absence of Runx-1. We conclude that the Runx-1 gene can be a Wnt-4 signalling target, and that Runx-1 and Wnt-4 are mutually interdependent in their expression. The changes in gene expression due to the absence of Wnt-4 in gonads reflect the sexually dimorphic role of this signal and its complex gene network in mammalian gonad development.

Wnt5a cooperates with canonical Wnts to generate midbrain dopaminergic neurons in vivo and in stem cells.

Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1(-/-) mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1(-/-);Wnt5a(-/-) mice revealed a greater loss of Nurr1(+) cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson's disease.

Functional genetic targeting of embryonic kidney progenitor cells ex vivo.

The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor-treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting.

ATGme: Open-source web application for rare codon identification and custom DNA sequence optimization.

BACKGROUND: Codon usage plays a crucial role when recombinant proteins are expressed in different organisms. This is especially the case if the codon usage frequency of the organism of origin and the target host organism differ significantly, for example when a human gene is expressed in E. coli. Therefore, to enable or enhance efficient gene expression it is of great importance to identify rare codons in any given DNA sequence and subsequently mutate these to codons which are more frequently used in the expression host. RESULTS: We describe an open-source web-based application, ATGme, which can in a first step identify rare and highly rare codons from most organisms, and secondly gives the user the possibility to optimize the sequence. CONCLUSIONS: This application provides a simple user-friendly interface utilizing three optimization strategies: 1. one-click optimization, 2. bulk optimization (by codon-type), 3. individualized custom (codon-by-codon) optimization. ATGme is an open-source application which is freely available at: http://atgme.org.

Wnt4, a pleiotropic signal for controlling cell polarity, basement membrane integrity, and antimüllerian hormone expression during oocyte maturation in the female follicle.

Wnt4 is a key signal that channels the developmental fate of the indifferent mammalian gonad toward the ovary, but whether Wnt4 has later roles during ovary development remains unknown. To investigate this, we inactivated the Wnt4 gene by crossing Amhr2Cre and doxycycline-inducible Rosa(rtTA)-knock-in Cre mice with mice carrying a floxed Wnt4 allele and used a novel Wnt4(mCherry)-knock-in mouse. In these models, ovarian folliculogenesis was compromised, and female fertility was severely reduced, and Wnt4 deficiency eventually led to premature ovarian failure. These anomalies were associated with cell polarity defects in the follicle. Within the follicle, laminin and type IV collagen assembled ectopic basement membrane-like structures, the cell adherens junction components N-cadherin and ß-catenin lost their polarized expression pattern, and expression of the gap junction protein connexin 43 was reduced by ~30% when compared with that of the controls. Besides these changes, expression of antimüllerian hormone (Amh) was inhibited in the absence of Wnt4 signaling in vivo. Consistent with this, Wnt4 signaling up-regulated Amh gene expression in KK1 cells in vitro. Thus, Wnt4 signaling is necessary during maturation of the ovarian follicles, where it coordinates expression of Amh, cell survival, and polarized organization of the follicular cells.