In vitro teratogenicity testing using a 3D, embryo-like gastruloid system.

Pharmaceuticals intended for use in patients of childbearing potential need to be tested for teratogenicity before marketing. Several pharmaceutical companies use animal-free in vitro models which allow a more rapid selection of lead compounds and contribute to 3Rs principles ('replace, reduce and refine') by streamlining the selection of promising compounds submitted to further regulatory studies in animals. Currently available in vitro models typically rely on adherent monolayer cultures or disorganized 3D structures, both of which lack the spatiotemporal and morphological context of the developing embryo. A newly developed 3D 'gastruloid' model has the potential to achieve a more reliable prediction of teratogenicity by providing a robust recapitulation of gastrulation-like events alongside morphological coordination at relatively high-throughput. In this first proof-of-concept study, we used both mouse and human gastruloids to examine a panel of seven reference compounds, with associated in vivo data and known teratogenic risk, to quantitatively assess in vitro teratogenicity. We observed several gross morphological effects, including significantly reduced elongation or decreased size of the gastruloids, upon exposure to several of the reference compounds. We also observed aberrant gene expression using fluorescent reporters, including SOX2, BRA, and SOX17, suggestive of multi-lineage differentiation defects and disrupted axial patterning. Finally, we saw that gastruloids recapitulated some of the known in vivo species-specific susceptibilities between their mouse and human counterparts. We therefore suggest that gastruloids represent a powerful tool for teratogenicity assessment by enabling relevant physiological recapitulation of early embryonic development, demonstrating their use as a novel in vitro teratogenic model system.

Gastruloids generated without exogenous Wnt activation develop anterior neural tissues.

When stimulated with a pulse from an exogenous WNT pathway activator, small aggregates of mouse embryonic stem cells (ESCs) can undergo embryo-like axial morphogenesis and patterning along the three major body axes. However, these structures, called gastruloids, currently lack the anterior embryonic regions, such as those belonging to the brain. Here, we describe an approach to generate gastruloids that have a more complete antero-posterior development. We used hydrogel microwell arrays to promote the robust derivation of mouse ESCs into post-implantation epiblast-like (EPI) aggregates in a reproducible and scalable manner. These EPI aggregates break symmetry and axially elongate without external chemical stimulation. Inhibition of WNT signaling in early stages of development leads to the formation of gastruloids with anterior neural tissues. Thus, we provide a new tool to study the development of the mouse after implantation in vitro, especially the formation of anterior neural regions.

Dissecting the dynamics of signaling events in the BMP, WNT, and NODAL cascade during self-organized fate patterning in human gastruloids.

During gastrulation, the pluripotent epiblast self-organizes into the 3 germ layers-endoderm, mesoderm and ectoderm, which eventually form the entire embryo. Decades of research in the mouse embryo have revealed that a signaling cascade involving the Bone Morphogenic Protein (BMP), WNT, and NODAL pathways is necessary for gastrulation. In vivo, WNT and NODAL ligands are expressed near the site of gastrulation in the posterior of the embryo, and knockout of these ligands leads to a failure to gastrulate. These data have led to the prevailing view that a signaling gradient in WNT and NODAL underlies patterning during gastrulation; however, the activities of these pathways in space and time have never been directly observed. In this study, we quantify BMP, WNT, and NODAL signaling dynamics in an in vitro model of human gastrulation. Our data suggest that BMP signaling initiates waves of WNT and NODAL signaling activity that move toward the colony center at a constant rate. Using a simple mathematical model, we show that this wave-like behavior is inconsistent with a reaction-diffusion-based Turing system, indicating that there is no stable signaling gradient of WNT/NODAL. Instead, the final signaling state is homogeneous, and spatial differences arise only from boundary effects. We further show that the durations of WNT and NODAL signaling control mesoderm differentiation, while the duration of BMP signaling controls differentiation of CDX2-positive extra-embryonic cells. The identity of these extra-embryonic cells has been controversial, and we use RNA sequencing (RNA-seq) to obtain their transcriptomes and show that they closely resemble human trophoblast cells in vivo. The domain of BMP signaling is identical to the domain of differentiation of these trophoblast-like cells; however, neither WNT nor NODAL forms a spatial pattern that maps directly to the mesodermal region, suggesting that mesoderm differentiation is controlled dynamically by the combinatorial effect of multiple signals. We synthesize our data into a mathematical model that accurately recapitulates signaling dynamics and predicts cell fate patterning upon chemical and physical perturbations. Taken together, our study shows that the dynamics of signaling events in the BMP, WNT, and NODAL cascade in the absence of a stable signaling gradient control fate patterning of human gastruloids.

In vitro synergy of azole antifungals and methotrexate against Candida albicans.

OBJECTIVE: This study aims to evaluate the effective of azoles and MTX for patients with invasive candidiasis. METHODS: We used the disk diffusion assay and the checkerboard assay to evaluate the in vitro interactions between MTX and antifungals. In addition, we used the transmission electron microscopy to observe the ultrastructure of the effect of MTX and fluconazole on Candida albicans. RESULTS: The rates of synergy for the combination of MTX with fluconazole (FLC), itraconazole (ITC), and voriconazole (VRZ) were 91.3%, 65.2%, and 87% in checkerboard testing. No antagonism was found between methotrexate and azole antifungals in any of the strains. Furthermore, MTX treated C. albicans showed extensive cell wall vacuolations and the inhibition of blastospores growth, as observed using transmission electron microscopy. There was an apparent destruction of the cell membrane and cell wall resulting in the destruction of cytoplasm, a phenomenon observed when MTX was combined with azoles. CONCLUSION: This study provides evidence that the combination of azoles and MTX is effective for patients with invasive candidiasis, which on the other hand, will reduce the side effects of the drugs.

Nodal and Hedgehog synergize in gill slit formation during development of the cephalochordate Branchiostoma floridae.

The larval pharynx of the cephalochordate Branchiostoma (amphioxus) is asymmetrical. The mouth is on the left, and endostyle and gill slits are on the right. At the neurula, Nodal and Hedgehog (Hh) expression becomes restricted to the left. To dissect their respective roles in gill slit formation, we inhibited each pathway separately for 20 min at intervals during the neurula stage, before gill slits penetrate, and monitored the effects on morphology and expression of pharyngeal markers. The results pinpoint the short interval spanning the gastrula/neurula transition as the critical period for specification and positioning of future gill slits. Thus, reduced Nodal signaling shifts the gill slits ventrally, skews the pharyngeal domains of Hh, Pax1/9, Pax2/5/8, Six1/2 and IrxC towards the left, and reduces Hh and Tbx1/10 expression in endoderm and mesoderm, respectively. Nodal auto-regulates. Decreased Hh signaling does not affect gill slit positions or Hh or Nodal expression, but it does reduce the domain of Gli, the Hh target, in the pharyngeal endoderm. Thus, during the neurula stage, Nodal and Hh cooperate in gill slit development - Hh mediates gill slit formation and Nodal establishes their left-right position.

Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation.

During the gastrulation stage in animal embryogenesis, the cells leading the axial mesoderm migrate toward the anterior side of the embryo, vigorously extending cell protrusions such as lamellipodia. It is thought that the leading cells sense gradients of chemoattractants emanating from the ectodermal cells and translate them to initiate and maintain the cell movements necessary for gastrulation. However, it is unclear how the extracellular information is converted to the intracellular chemical reactions that lead to motion. Here we demonstrated that intracellular Ca2+ levels in the protrusion-forming leading cells are markedly higher than those of the following cells and the axial mesoderm cells. We also showed that inhibiting the intracellular Ca2+ significantly retarded the gastrulation cell movements, while increasing the intracellular Ca2+ with an ionophore enhanced the migration. We further found that the ionophore treatment increased the active form of the small GTPase Rac1 in these cells. Our results suggest that transient intracellular Ca2+ signals play an essential role in the active cell migration during gastrulation.

Determination of the effect of Pinellia ternata (Thunb.) Breit. on nervous system development by proteomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Banxia (BX) is the dried tuber of Pinellia ternata (Thunb.) Breit., a commonly prescribed Chinese medicinal herb for the treatment of cough, phlegm, and vomiting in pregnant women. However, raw BX has been demonstrated to exert toxic effects on reproduction and the precise and comprehensive mechanisms remain elusive. AIM OF THE STUDY: We applied an iTRAQ (isobaric tags for relative and absolute quantitation, iTRAQ)-based proteomic method to explore the mechanisms of raw BX-induced fetal toxicity in mice. MATERIALS AND METHODS: The mice were separated into two groups, control mice and BX-treated mice. From gestation days 6-8, the control group was treated with normal saline and the BX group was exposed to BX suspension (2.275g/kg/day). Gastrulae were obtained and analyzed using the quantitative proteomic approach of iTRAQ coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS). A multi-omics data analysis tool, OmicsBean (http://www.omicsbean.cn), was employed to conduct bioinformatic analysis of differentially abundant proteins (DAPs). Quantitative real-time PCR (qRT-PCR) and western blotting methods were applied to detect the protein expression levels and validate the quality of the proteomics. RESULTS: A total of 1245 proteins were identified with < 1% false discovery rate (FDR) and 583 protein abundance changes were confidently assessed. Moreover, 153 proteins identified in BX-treated samples showed significant differences in abundance. Bioinformatics analysis showed that the functions of 37 DAPs were predominantly related to nervous system development. The expression levels of the selected proteins for quantification by qRT-PCR or western blotting were consistent with the results in iTRAQ-labeled proteomics data. CONCLUSION: The results suggested that oral administration of BX in mice may cause fetal abnormality of the nervous system. The findings may be helpful to elucidate the underlying mechanisms of BX-induced embryotoxicity.

Zebrafish Znfl1 proteins control the expression of hoxb1b gene in the posterior neuroectoderm by acting upstream of pou5f3 and sall4 genes.

Transcription factors play crucial roles in patterning posterior neuroectoderm. Previously, zinc finger transcription factor znfl1 was reported to be expressed in the posterior neuroectoderm of zebrafish embryos. However, its roles remain unknown. Here, we report that there are 13 copies of znfl1 in the zebrafish genome, and all the paralogues share highly identical protein sequences and cDNA sequences. When znfl1s are knocked down using a morpholino to inhibit their translation or dCas9-Eve to inhibit their transcription, the zebrafish gastrula displays reduced expression of hoxb1b, the marker gene for the posterior neuroectoderm. Further analyses reveal that diminishing znfl1s produces the decreased expressions of pou5f3, whereas overexpression of pou5f3 effectively rescues the reduced expression of hoxb1b in the posterior neuroectoderm. Additionally, knocking down znfl1s causes the reduced expression of sall4, a direct regulator of pou5f3, in the posterior neuroectoderm, and overexpression of sall4 rescues the expression of pou5f3 in the knockdown embryos. In contrast, knocking down either pou5f3 or sall4 does not affect the expressions of znfl1s Taken together, our results demonstrate that zebrafish znfl1s control the expression of hoxb1b in the posterior neuroectoderm by acting upstream of pou5f3 and sall4.

Imidacloprid Exposure Suppresses Neural Crest Cells Generation during Early Chick Embryo Development.

Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests found on crops and fleas on pets. However, it is still unclear whether imidacloprid exposure could affect early embryo development-despite some studies having been conducted on the gametes. In this study, we demonstrated that imidacloprid exposure could lead to abnormal craniofacial osteogenesis in the developing chick embryo. Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull. We found that the imidacloprid exposure retards the development of gastrulating chick embryos. HNK-1, PAX7, and Ap-2α immunohistological stainings indicated that cranial NCCs generation was inhibited after imidacloprid exposure. Double immunofluorescent staining (Ap-2α and PHIS3 or PAX7 and c-Caspase3) revealed that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in the developing neural tube and by altering expression of EMT-related adhesion molecules (Cad6B, E-Cadherin, and N-cadherin) in the developing neural crests. We also determined that imidacloprid exposure suppressed cranial NCCs migration and their ability to differentiate. In sum, we have provided experimental evidence that imidacloprid exposure during embryogenesis disrupts NCCs development, which in turn causes defective cranial bone development.

Glypican1/2/4/6 and sulfated glycosaminoglycans regulate the patterning of the primary body axis in the cnidarian Nematostella vectensis.

Glypicans are members of the heparan sulfate (HS) subfamily of proteoglycans that can function in cell adhesion, cell crosstalk and as modulators of the major developmental signalling pathways in bilaterians. The evolutionary origin of these multiple functions is not well understood. In this study we investigate the role of glypicans in the embryonic and larval development of the sea anemone Nematostella vectensis, a member of the non-bilaterian clade Cnidaria. Nematostella has two glypican (gpc) genes that are expressed in mutually exclusive ectodermal domains, NvGpc1/2/4/6 in a broad aboral domain, and NvGpc3/5 in narrow oral territory. The endosulfatase NvSulf (an extracellular modifier of HS chains) is expressed in a broad oral domain, partially overlapping with both glypicans. Morpholino-mediated knockdown of NvGpc1/2/4/6 leads to an expansion of the expression domains of aboral marker genes and a reduction of oral markers at gastrula stage, strikingly similar to knockdown of the Wnt receptor NvFrizzled5/8. We further show that treatment with sodium chlorate, an inhibitor of glycosaminoglycan (GAG) sulfation, phenocopies knockdown of NvGpc1/2/4/6 at gastrula stage. At planula stage, knockdown of NvGpc1/2/4/6 and sodium chlorate treatment result in alterations in aboral marker gene expression that suggest additional roles in the fine-tuning of patterning within the aboral domain. These results reveal a role for NvGpc1/2/4/6 and sulfated GAGs in the patterning of the primary body axis in Nematostella and suggest an ancient function in regulating Frizzled-mediated Wnt signalling.

The chick embryo as a model for the effects of prenatal exposure to alcohol on craniofacial development.

Prenatal exposure to ethanol results in fetal alcohol spectrum disorder (FASD), a syndrome characterised by a broad range of clinical manifestations including craniofacial dysmorphologies and neurological defects. The characterisation of the mechanisms by which ethanol exerts its teratogenic effects is difficult due to the pleiotropic nature of its actions. Different experimental model systems have been employed to investigate the aetiology of FASD. Here, I will review studies using these different model organisms that have helped to elucidate how ethanol causes the craniofacial abnormalities characteristic of FASD. In these studies, ethanol was found to impair the prechordal plate-an important embryonic signalling centre-during gastrulation and to negatively affect the induction, migration and survival of the neural crest, a cell population that generates the cartilage and most of the bones of the skull. At the cellular level, ethanol appears to inhibit Sonic hedgehog signalling, alter levels of retionoic acid activity, trigger a Ca(2+)-CamKII-dependent pathway that antagonises WNT signalling, affect cytoskeletal dynamics and increase oxidative stress. Embryos of the domestic chick Gallus gallus domesticus have played a central role in developing a working model for the effects of ethanol on craniofacial development because they are easily accessible and because key steps in craniofacial development are particularly well established in the avian embryo. I will finish this review by highlighting some potential future avenues of fetal alcohol research.

Biochemical biomarkers of sublethal effects in Rhinella arenarum late gastrula exposed to the organophosphate chlorpyrifos.

We determined the biochemical and molecular effects of the organophosphate insecticide chlorpyrifos (CPF) in the late gastrula embryonic stage of the South American toad Rhinella arenarum continuously exposed from fertilization (24 h). Our objective was to evaluate these responses as potential biomarkers at low, sublethal levels of the toxicant. We first established the EC50 for embryo arrest in 21.3 mg/L, with a LOEC of 16 mg/L. At 4 mg/L CPF, some embryos were unable to complete the dorsal lip of the blastopore and the yolk plug became blur, probably because of abnormal cell migration. Acetylcholinesterase activity, the specific biomarker for organophosphates, was unaffected by any of the tested concentrations of CPF (2-14 mg/L). In turn, 2 mg/L CPF increased the reduced glutathione levels and inhibited glutathione-S-transferase activity, suggesting an oxidative stress and antioxidant response. Catalase was induced by CPF exposure at higher concentrations (8 and 14 mg/L). We also studied transcription factor c-Fos as a signaling event related to development in early embryogenesis. Analysis of nuclear c-Fos protein showed two bands, both enhanced in embryos exposed to 2 and 8 mg/L CPF. While nuclear Erk protein was practically unaffected, Mek protein levels were induced by the OP. Transcription factor c-Fos may be then linking oxidative stress with developmental alterations observed due to CPF exposure. These molecular and biochemical responses observed in R. arenarum gastrula at sublethal CPF exposures may replace non-responsive AChE as very early biomarkers in toad gastrula.

Autophagy is involved in high glucose-induced heart tube malformation.

Both pre-gestational and gestational diabetes have an adverse impact on heart development, but little is known about the influence on the early stage of heart tube formation. Using early gastrulating chick embryos, we investigated the influence of high glucose on the process of heart tube formation, specifically during the primary heart field phase. We demonstrated that high-glucose exposure resulted in 3 types of heart tube malformation: 1) ventricular hypertrophy, 2) ventricular hypertrophy with dextrocardia and 3) ventricular hypertrophy and dextrocardia with the fusion anomaly of a bilateral primary heart tube. Next, we found that these malformation phenotypes of heart tubes might mainly originate from the migratory anomaly of gastrulating precardiac mesoderm cells rather than cell proliferation in the developmental process of bilateral primary heart field primordia. The treatment of rapamycin (RAPA), an autophagy inducer, led to a similar heart tube malformation phenotype as high glucose. Additionally, high-glucose exposure promoted the expression of the key autophagy protein LC3B in early chick tissue. Atg7 is strongly expressed in the fusion site of bilateral primary heart tubes. All of these data imply that autophagy could be involved in the process of high-glucose-induced malformation of the heart tube.

[Influence of the Concentration of Dissolved Oxygen on Embryonic Development of the Common Toad (Bufo bufo)].

Several series of experiments investigating the influence of dissolved oxygen concentrations on the growth rates and mortality in the embryogenesis of the common toad Bufo bufo were carried out. The experiments showed that, when the eggs develop singly, the lack of oxygen does not lead to an increase in mortality by the time of hatching and results only in a change in the dynamics of mortality: mortality occurs at an earlier stage of development than in the conditions of normal access to oxygen. Taking into account the combined effect of the density of eggs and the dissolved oxygen concentration, we increase the accuracy of analysis of the experimental results and improve the interpretation of the results. In the conditions of different initial density of eggs, the impact of the concentration of dissolved oxygen on mortality and rates of development of the common toad embryos is manifested in different ways. At high density, only a small percentage of embryos survives by the time of hatching, and the embryos are significantly behind in their development compared with the individuals that developed in normal oxygen conditions. The lack of oxygen dissolved in the water slows down the development of embryos of the common toad.

Involvement of L(-)-rhamnose in sea urchin gastrulation: a live embryo assay.

The sea urchin embryo is a National Institutes of Health model system that has provided major developments, and is important in human health and disease. To obtain initial insights to identify glycans that mediate cellular interactions, Lytechinus pictus sea urchin embryos were incubated at 24 or 30 h post-fertilization with 0.0009-0.03 M alpha-cyclodextrin, melibiose, L(-)-rhamnose, trehalose, D(+)-xylose or L(-)-xylose in lower-calcium artificial sea water (pH 8.0, 15°C), which speeds the entry of molecules into the interior of the embryos. While α-cyclodextrin killed the embryos, and L(-)-xylose had small effects at one concentration tested, L(-)-rhamnose caused substantially increased numbers of unattached archenterons and exogastrulated embryos at low glycan concentrations after 18-24 h incubation with the sugar. The results were statistically significant compared with the control embryos in the absence of sugar (P < 0.05). The other sugars (melibiose, trehalose, D(+)-xylose) had no statistically significant effects whatsoever at any of the concentrations tested. In total, in the current study, 39,369 embryos were examined. This study is the first demonstration that uses a live embryo assay for a likely role for L(-)-rhamnose in sea urchin gastrula cellular interactions, which have interested investigators for over a century.

Ca²âº influx-linked protein kinase C activity regulates the ß-catenin localization, micromere induction signalling and the oral-aboral axis formation in early sea urchin embryos.

Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal-vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the ß-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal-vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of ß-catenin. To this end, we surveyed the expression pattern of ß-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of ß-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of ß-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.

Autophagy functions on EMT in gastrulation of avian embryo.

Autophagy is important for cell renewing for its contribution to the degradation of bulk cytoplasm, long-lived proteins, and entire organelles and its role in embryonic development is largely unknown. In our study, we investigated the function of autophagy in gastrulation of the chick embryo using both in vivo and in vitro approaches, especially in the EMT process, and we found that autophagy gene Atg7 was expressed on the apical side of the ectoderm and endoderm. Over-expression of Atg7 could enhance the expression of Atg8 and the E-cadherin, the latter of which is a crucial marker of the EMT process. We also found that the disturbance of autophagy could retard the development of chick embryos in HH4 with shorter primitive steak than that in the control group, which is a newly formed structure during EMT process. So we assumed that autophagy could affect EMT process by adhesion molecule expression. Moreover, more molecules, such as slug, chordin, shh et., which were all involved in EMT process, were detected to address the mechanism of this phenomena. We established that the inhibition of autophagy could cause developmental delay by affecting EMT process in gastrulation of chick embryos.

Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage.

Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 µg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 µg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay.

MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate.

Pluripotent mouse embryonic stem cells (mESCs), maintained in the presence of the leukemia inhibitory factor (LIF) cytokine, provide a powerful model with which to study pluripotency and differentiation programs. Extensive microarray studies on cultured cells have led to the identification of three LIF signatures. Here we focus on muscle ras oncogene homolog (MRAS), which is a small GTPase of the Ras family encoded within the Pluri gene cluster. To characterise the effects of Mras on cell pluripotency and differentiation, we used gain- and loss-of-function strategies in mESCs and in the Xenopus laevis embryo, in which Mras gene structure and protein sequence are conserved. We show that persistent knockdown of Mras in mESCs reduces expression of specific master genes and that MRAS plays a crucial role in the downregulation of OCT4 and NANOG protein levels upon differentiation. In Xenopus, we demonstrate the potential of Mras to modulate cell fate at early steps of development and during neurogenesis. Overexpression of Mras allows gastrula cells to retain responsiveness to fibroblast growth factor (FGF) and activin. Collectively, these results highlight novel conserved and pleiotropic effects of MRAS in stem cells and early steps of development.

Towards gene banking amphibian maternal germ lines: short-term incubation, cryoprotectant tolerance and cryopreservation of embryonic cells of the frog, Limnodynastes peronii.

Gene banking is arguably the best method available to prevent the loss of genetic diversity caused by declines in wild populations, when the causes of decline cannot be halted or reversed. For one of the most impacted vertebrate groups, the amphibians, gene banking technologies have advanced considerably, and gametes from the male line can be banked successfully for many species. However, cryopreserving the female germ line remains challenging, with attempts at cryopreserving oocytes unsuccessful due to their large size and yolk content. One possible solution is to target cryopreservation of early embryos that contain the maternal germ line, but consist of smaller cells. Here, we investigate the short term incubation, cryoprotectant tolerance, and cryopreservation of dissociated early embryonic cells from gastrulae and neurulae of the Striped Marsh Frog, Limnodynastes peronii. Embryos were dissociated and cells were incubated for up to 24 hours in various media. Viability of both gastrula and neurula cells remained high (means up to 40-60%) over 24 hours of incubation in all media, although viability was maintained at a higher level in Ca(2+)-free Simplified Amphibian Ringer; low speed centrifugation did not reduce cell viability. Tolerance of dissociated embryonic cells was tested for two cryoprotectants, glycerol and dimethyl sulphoxide; dissociated cells of both gastrulae and neurulae were highly tolerant to both-indeed, cell viability over 24 hours was higher in media containing low-to-medium concentrations than in equivalent cryoprotectant-free media. Viability over 24 hours was lower in concentrations of cryoprotectant higher than 10%. Live cells were recovered following cryopreservation of both gastrula and neurula cells, but only at low rates. Optimal cryodiluents were identified for gastrula and neurula cells. This is the first report of a slow cooling protocol for cryopreservation of amphibian embryonic cells, and sets future research directions for cryopreserving amphibian maternal germ lines.