High-throughput micropatterning platform reveals Nodal-dependent bisection of peri-gastrulation-associated versus preneurulation-associated fate patterning.

In vitro models of postimplantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon bone morphogenetic protein 4 (BMP4) treatment of geometrically confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line-associated heterogeneity was found to be attributable to endogenous Nodal expression, with up-regulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal down-regulation correlated with a preneurulation-associated gene profile expression. We harness this knowledge to establish a platform of preneurulation-like fate patterning in geometrically confined hPSC colonies in which fates arise because of a BMPs signalling gradient conveying positional information. Our work identifies a Nodal signalling-dependent switch in peri-gastrulation versus preneurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of organized fate specification in differentiating epiblast and ectodermal tissues.

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.

Teratogenesis in the chick embryo following post-gastrulation exposure to Y-27632 -effect of Y-27632 on embryonic development.

The pyridine derivative Y-27632 inhibits Rho-associated coiled-coil-containing protein kinase (ROCK) signaling, which is involved in numerous developmental processes during embryogenesis, primarily by controlling actin-cytoskeleton assembly and cell contractility. Somite formation requires rearrangement of the cytoskeleton and assists in major morphological mechanisms, including ventral body wall formation. Administration of Y-27632 impairs cytoskeletal arrangements in post-gastrulation chick embryos leading to ventral body wall defects (VBWD) at later stages of development. The aim of this study was to investigate the effect of Y-27632 on somite development in post-gastrulation chick embryos during early embryogenesis. After 60 h incubation, embryos in shell-less culture were treated with Y-27632 or vehicle for controls. Following administration, abnormality rates were assessed. In treatment groups, embryos showed a kinked longitudinal body axis. Western blot confirmed impaired ROCK downstream signaling by decreased expression of phosphorylated cofilin-2. Histology, Lysotracker studies and RT-PCR demonstrated increased cell death in somites, the neural tube and the ectoderm. RT-PCR and Western blot of factors known to be involved during somitogenesis revealed reduced expression in the treatment group compared to controls. We hypothesize that administration of Y-27632 disrupts somite development causing axial kinking and embryo malformation, which may lead to VBWD.

fus/TLS orchestrates splicing of developmental regulators during gastrulation.

Here we investigated the function of the atypical RNA-binding protein fus/TLS (fused in sarcoma/translocated in sarcoma) during early frog development. We found that fus is necessary for proper mRNA splicing of a set of developmental regulatory genes during early frog development and gastrulation. Upon fus knockdown, embryos fail to gastrulate and show mesodermal differentiation defects that we connect to intron retention in fgf8 (fibroblast growth factor 8) and fgfr2 (fgf receptor 2) transcripts. During gastrulation, the animal and marginal regions dissociate, and we show that this is caused, at least in part, by intron retention in cdh1 transcripts. We confirm the specificity of splicing defects at a genomic level using analysis of RNA sequencing (RNA-seq) and show that 3%-5% of all transcripts display intron retention throughout the pre-mRNA. By analyzing gene ontology slim annotations, we show that the affected genes are enriched for developmental regulators and therefore represent a biologically coherent set of targets for fus regulation in embryogenesis. This shows that fus is central to embryogenesis and may provide information on its function in neurodegenerative disease.

Progesterone modulates microtubule dynamics and epiboly progression during zebrafish gastrulation.

Control of microtubule dynamics is crucial for cell migration. We analyzed regulation of microtubule network dynamics in the zebrafish yolk cell during epiboly, the earliest coordinated gastrulation movement. We labeled microtubules with EMTB-3GFP and EB3-mCherry to visualize and measure microtubule dynamics by TIRF microscopy live imaging. Yolk cell microtubules dynamics is temporally modulated during epiboly progression. We used maternal zygotic Pou5f3 mutant (MZspg) embryos, which develop strong distortions of microtubule network organization and epiboly retardation, to investigate genetic control of microtubule dynamics. In MZspg embryos, microtubule plus-end growth tracks move slower and are less straight compared to wild-type. MZspg embryos have altered steroidogenic enzyme expression, resulting in increased pregnenolone and reduced progesterone levels. We show that progesterone positively affects microtubule plus-end growth and track straightness. Progesterone may thus act as a non-cell-autonomous regulator of microtubule dynamics across the large yolk cell, and may adjust differing demands on microtubule dynamics and stability during initiation and progression phases of epiboly.

Development of the aboral domain in Nematostella requires ß-catenin and the opposing activities of Six3/6 and Frizzled5/8.

The development of the oral pole in cnidarians and the posterior pole in bilaterians is regulated by canonical Wnt signaling, whereas a set of transcription factors, including Six3/6 and FoxQ2, controls aboral development in cnidarians and anterior identity in bilaterians. However, it is poorly understood how these two patterning systems are initially set up in order to generate correct patterning along the primary body axis. Investigating the early steps of aboral pole formation in the sea anemone Nematostella vectensis, we found that, at blastula stage, oral genes are expressed before aboral genes and that Nvß-catenin regulates both oral and aboral development. In the oral hemisphere, Nvß-catenin specifies all subdomains except the oral-most, NvSnailA-expressing domain, which is expanded upon Nvß-catenin knockdown. In addition, Nvß-catenin establishes the aboral patterning system by promoting the expression of NvSix3/6 at the aboral pole and suppressing the Wnt receptor NvFrizzled5/8 at the oral pole. NvFrizzled5/8 expression thereby gets restricted to the aboral domain. At gastrula stage, NvSix3/6 and NvFrizzled5/8 are both expressed in the aboral domain, but they have opposing activities, with NvSix3/6 maintaining and NvFrizzled5/8 restricting the size of the aboral domain. At planula stage, NvFrizzled5/8 is required for patterning within the aboral domain and for regulating the size of the apical organ by modulation of a previously characterized FGF feedback loop. Our findings suggest conserved roles for Six3/6 and Frizzled5/8 in aboral/anterior development and reveal key functions for Nvß-catenin in the patterning of the entire oral-aboral axis of Nematostella.

Retinoic acid is required and Fgf, Wnt, and Bmp signaling inhibit posterior lateral line placode induction in zebrafish.

The lateral line system is a mechanosensory systems present in aquatic animals. The anterior and posterior lateral lines develop from anterior and posterior lateral line placodes (aLLp and pLLp), respectively. Although signaling molecules required for the induction of other cranial placodes have been well studied, the molecular mechanisms underlying formation of the lateral line placodes are unknown. In this study we tested the requirement of multiple signaling pathways, such as Wnt, Bmp Fgf, and Retinoic Acid for aLLp and pLLp induction. We determined that aLLp specification requires Fgf signaling, whilst pLLp specification requires retinoic acid which inhibits Fgf signaling. pLLp induction is also independent of Wnt and Bmp activities, even though these pathways limit the boundaries of the pLLp. This is the first report that the aLLp and pLLp depend on different inductive mechanisms and that pLLp induction requires the inhibition of Fgf, Wnt and Bmp signaling.

A parental requirement for dual-specificity phosphatase 6 in zebrafish.

BACKGROUND: Signaling cascades, such as the extracellular signal-regulated kinase (ERK) pathway, play vital roles in early vertebrate development. Signals through these pathways are initiated by a growth factor or hormone, are transduced through a kinase cascade, and result in the expression of specific downstream genes that promote cellular proliferation, growth, or differentiation. Tight regulation of these signals is provided by positive or negative modulators at varying levels in the pathway, and is required for proper development and function. Two members of the dual-specificity phosphatase (Dusp) family, dusp6 and dusp2, are believed to be negative regulators of the ERK pathway and are expressed in both embryonic and adult zebrafish, but their specific roles in embryogenesis remain to be fully understood. RESULTS: Using CRISPR/Cas9 genome editing technology, we generated zebrafish lines harboring germ line deletions in dusp6 and dusp2. We do not detect any overt defects in dusp2 mutants, but we find that approximately 50% of offspring from homozygous dusp6 mutants do not proceed through embryonic development. These embryos are fertilized, but are unable to proceed past the first zygotic mitosis and stall at the 1-cell stage for several hours before dying by 10 h post fertilization. We demonstrate that dusp6 is expressed in gonads of both male and female zebrafish, suggesting that loss of dusp6 causes defects in germ cell production. Notably, the 50% of homozygous dusp6 mutants that complete the first cell division appear to progress through embryogenesis normally and give rise to fertile adults. CONCLUSIONS: The fact that offspring of homozygous dusp6 mutants stall prior to activation of the zygotic genome, suggests that loss of dusp6 affects gametogenesis and/or parentally-directed early development. Further, since only approximately 50% of homozygous dusp6 mutants are affected, we postulate that ERK signaling is tightly regulated and that dusp6 is required to keep ERK signaling within a range that is permissive for proper embryogenesis. Lastly, since dusp6 is expressed throughout zebrafish embryogenesis, but dusp6 mutants do not exhibit defects after the first cell division, it is possible that other regulators of the ERK pathway compensate for loss of dusp6 at later stages.

High salt-induced excess reactive oxygen species production resulted in heart tube malformation during gastrulation.

An association has been proved between high salt consumption and cardiovascular mortality. In vertebrates, the heart is the first functional organ to be formed. However, it is not clear whether high-salt exposure has an adverse impact on cardiogenesis. Here we report high-salt exposure inhibited basement membrane breakdown by affecting RhoA, thus disturbing the expression of Slug/E-cadherin/N-cadherin/Laminin and interfering with mesoderm formation during the epithelial-mesenchymal transition(EMT). Furthermore, the DiI+ cell migration trajectory in vivo and scratch wound assays in vitro indicated that high-salt exposure restricted cell migration of cardiac progenitors, which was caused by the weaker cytoskeleton structure and unaltered corresponding adhesion junctions at HH7. Besides, down-regulation of GATA4/5/6, Nkx2.5, TBX5, and Mef2c and up-regulation of Wnt3a/ß-catenin caused aberrant cardiomyocyte differentiation at HH7 and HH10. High-salt exposure also inhibited cell proliferation and promoted apoptosis. Most importantly, our study revealed that excessive reactive oxygen species(ROS)generated by high salt disturbed the expression of cardiac-related genes, detrimentally affecting the above process including EMT, cell migration, differentiation, cell proliferation and apoptosis, which is the major cause of malformation of heart tubes.

Rho kinase activity controls directional cell movements during primitive streak formation in the rabbit embryo.

During animal gastrulation, the specification of the embryonic axes is accompanied by epithelio-mesenchymal transition (EMT), the first major change in cell shape after fertilization. EMT takes place in disparate topographical arrangements, such as the circular blastopore of amphibians, the straight primitive streak of birds and mammals or in intermediate gastrulation forms of other amniotes such as reptiles. Planar cell movements are prime candidates to arrange specific modes of gastrulation but there is no consensus view on their role in different vertebrate classes. Here, we test the impact of interfering with Rho kinase-mediated cell movements on gastrulation topography in blastocysts of the rabbit, which has a flat embryonic disc typical for most mammals. Time-lapse video microscopy, electron microscopy, gene expression and morphometric analyses of the effect of inhibiting ROCK activity showed - besides normal specification of the organizer region - a dose-dependent disruption of primitive streak formation; this disruption resulted in circular, arc-shaped or intermediate forms, reminiscent of those found in amphibians, fishes and reptiles. Our results reveal a crucial role of ROCK-controlled directional cell movements during rabbit primitive streak formation and highlight the possibility that temporal and spatial modulation of cell movements were instrumental for the evolution of gastrulation forms.

Knockdown of Mns1 Increases Susceptibility to Craniofacial Defects Following Gastrulation-Stage Alcohol Exposure in Mice.

BACKGROUND: MNS1 (meiosis-specific nuclear structural protein 1) is necessary for motile cilia function, such as sperm flagella or those found in the embryonic primitive node. While little is known regarding the function or expression pattern of MNS1 in the embryo, co-immunoprecipitation experiments in sperm have determined that MNS1 interacts with ciliary proteins, which are also important during development. Establishment of morphogenic gradients is dependent on normal ciliary motion in the primitive node beginning during gastrulation (gestational day [GD] 7 in the mouse, second-third week of pregnancy in humans), a critical window for face, eye, and brain development and particularly susceptible to perturbations of developmental signals. The current study investigates the role of Mns1 in craniofacial defects associated with gastrulation-stage alcohol exposure. METHODS: On GD7, pregnant Mns1+/- dams were administered 2 doses of ethanol (5.8 g/kg total) or vehicle 4 hours apart to target gastrulation. On GD17, fetuses were examined for ocular defects by scoring each eye on a scale from 1 to 7 (1 = normal, 2 to 7 = defects escalating in severity). Craniofacial and brain abnormalities were also assessed. RESULTS: Prenatal alcohol exposure (PAE) significantly increased the rate of defects in wild-type fetuses, as PAE fetuses had an incidence rate of 41.18% compared to a 10% incidence rate in controls. Furthermore, PAE interacted with genotype to significantly increase the defect rate and severity in Mns1+/- (64.29%) and Mns1-/- mice (92.31%). PAE Mns1-/- fetuses with severe eye defects also presented with craniofacial dysmorphologies characteristic of fetal alcohol syndrome and midline tissue loss in the brain, palate, and nasal septum. CONCLUSIONS: These data demonstrate that a partial or complete knockdown of Mns1 interacts with PAE to increase the susceptibility to ocular defects and correlating craniofacial and brain anomalies, likely though interaction of alcohol with motile cilia function. These results further our understanding of genetic risk factors that may underlie susceptibility to teratogenic exposures.

Effects of low-dose melamine exposure during pregnancy on maternal and fetal kidneys in rats.

Despite the previous reports on melamine contamination in high concentrations some years ago, there were not many studies on low-level exposure in daily life, particularly in pregnancy. We investigated the effect of low-dose melamine on the kidneys of the pregnant rats and their developing embryos/fetuses during various gestational stages namely implantation, gastrulation, organogenesis, maturation and whole pregnancy. Our results showed that the repeated low level of melamine (12.5, 25, and 50 mg/kg bw/d) during pregnancy did not cause obstruction of renal tubules although more precipitating crystals were found in the early gestational periods. Simple hyperplasia in the maternal tubules and pelvic epithelium were more prominent after exposed to melamine during the whole gestational period. Neonatal kidneys significantly suffered more from congestion in glomeruli and interstitium, dilated tubules and interstitial edema after melamine administration to the mother in the late and the whole gestational periods. A trend of advance of glomerular development in fetuses was also observed. We conclude that in utero exposure of low-level melamine could post a risk on the kidneys of the pregnant mother as well as the developing fetuses, which may further increase the possibility of other health problems later in life.

MAPK signaling is necessary for neurogenesis in Nematostella vectensis.

BACKGROUND: The nerve net of Nematostella is generated using a conserved cascade of neurogenic transcription factors. For example, NvashA, a homolog of the achaete-scute family of basic helix-loop-helix transcription factors, is necessary and sufficient to specify a subset of embryonic neurons. However, positive regulators required for the expression of neurogenic transcription factors remain poorly understood. RESULTS: We show that treatment with the MEK/MAPK inhibitor U0126 severely reduces the expression of known neurogenic genes, Nvath-like, NvsoxB(2), and NvashA, and known markers of differentiated neurons, suggesting that MAPK signaling is necessary for neural development. Interestingly, ectopic NvashA fails to rescue the expression of neural markers in U0126-treated animals. Double fluorescence in situ hybridization and transgenic analysis confirmed that NvashA targets represent both unique and overlapping populations of neurons. Finally, we used a genome-wide microarray to identify additional patterning genes downstream of MAPK that might contribute to neurogenesis. We identified 18 likely neural transcription factors, and surprisingly identified ~40 signaling genes and transcription factors that are expressed in either the aboral domain or animal pole that gives rise to the endomesoderm at late blastula stages. CONCLUSIONS: Together, our data suggest that MAPK is a key early regulator of neurogenesis, and that it is likely required at multiple steps. Initially, MAPK promotes neurogenesis by positively regulating expression of NvsoxB(2), Nvath-like, and NvashA. However, we also found that MAPK is necessary for the activity of the neurogenic transcription factor NvashA. Our forward molecular approach provided insight about the mechanisms of embryonic neurogenesis. For instance, NvashA suppression of Nvath-like suggests that inhibition of progenitor identity is an active process in newly born neurons, and we show that downstream targets of NvashA reflect multiple neural subtypes rather than a uniform neural fate. Lastly, analysis of the MAPK targets in the early embryo suggests that MAPK signaling is critical not only to neurogenesis, but also endomesoderm formation and aboral patterning.

Adverse reproductive effects of maternal low-dose melamine exposure during pregnancy in rats.

Melamine is a heterocyclic, aromatic amine and nitrogen-enriched environmental toxicant, found in not only adulterated foodstuffs but also industrial household tableware and paints. Previous studies demonstrated adverse effects of high-dose melamine on human infants and pregnant animals, but effects of low-dose melamine on pregnancy have not been reported. In this study, reproductive effects of low-dose melamine were investigated in pregnant rats. Melamine in the range of 12.5-50 mg/kg was administered to pregnant rats at different gestational stages. Maternal weight gain was not significantly affected, and other maternal morbidity was not observed. Low-dose melamine exposure during pregnancy increased fetal size but reduced somite number in gastrulation (GD8.5-GD10.5) and organogenesis (GD10.5-GD16.5) periods, and increased incidence of stillbirth in whole gestational period (GD0.5 to delivery). Embryotoxicity of melamine was further confirmed by whole embryo culture in vitro that melamine retarded embryonic growth, impaired development of brain and heart, and induced open neural tube and atrioventricular defects with increased apoptosis. In conclusion, adverse reproductive effects of low-dose melamine during pregnancy were identified in the developing rat embryos and the perinatal effects of melamine were gestational and developmental stage dependent. Detailed hazard and risk assessment of melamine in reproduction system are warrant. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 131-138, 2017.

Identification of anti-cancer chemical compounds using Xenopus embryos.

Cancer tissues have biological characteristics similar to those observed in embryos during development. Many types of cancer cells acquire pro-invasive ability through epithelial-mesenchymal transition (EMT). Similar processes (gastrulation and migration of cranial neural crest cells [CNCC]) are observed in the early stages of embryonic development in Xenopus during which cells that originate from epithelial sheets through EMT migrate to their final destinations. The present study examined Xenopus embryonic tissues to identify anti-cancer compounds that prevent cancer invasion. From the initial test of known anti-cancer drugs, AMD3100 (an inhibitor of CXCR4) and paclitaxel (a cytoskeletal drug targeting microtubules) effectively prevented migration during gastrulation or CNCC development. Blind-screening of 100 synthesized chemical compounds was performed, and nine candidates that inhibited migration of these embryonic tissues without embryonic lethality were selected. Of these, C-157 (an analog of podophyllotoxin) and D-572 (which is an indole alkaroid) prevented cancer cell invasion through disruption of interphase microtubules. In addition, these compounds affected progression of mitotic phase and induced apoptosis of SAS oral cancer cells. SAS tumors were reduced in size after intratumoral injection of C-157, and peritoneal dissemination of melanoma cells and intracranial invasion of glioma cells were inhibited by C-157 and D-572. When the other analogues of these chemicals were compared, those with subtle effect on embryos were not tumor suppressive. These results suggest that a novel chemical-screening approach based on Xenopus embryos is an effective method for isolating anti-cancer drugs and, in particular, targeting cancer cell invasion and proliferation.

Anteroposterior and dorsoventral patterning are coordinated by an identical patterning clock.

Establishment of the body plan in vertebrates depends on the temporally coordinated patterning of tissues along the body axes. We have previously shown that dorsoventral (DV) tissues are temporally patterned progressively from anterior to posterior by a BMP signaling pathway. Here we report that DV patterning along the zebrafish anteroposterior (AP) axis is temporally coordinated with AP patterning by an identical patterning clock. We altered AP patterning by inhibiting or activating FGF, Wnt or retinoic acid signaling combined with inhibition of BMP signaling at a series of developmental time points, which revealed that the temporal progression of DV patterning is directly coordinated with AP patterning. We investigated how these signaling pathways are integrated and suggest a model for how DV and AP patterning are temporally coordinated. It has been shown that in Xenopus dorsal tissues FGF and Wnt signaling quell BMP signaling by degrading phosphorylated (P) Smad1/5, the BMP pathway signal transducer, via phosphorylation of the Smad1/5 linker region. We show that in zebrafish FGF/MAPK, but not Wnt/GSK3, phosphorylation of the Smad1/5 linker region localizes to a ventral vegetal gastrula region that could coordinate DV patterning with AP patterning ventrally without degrading P-Smad1/5. Furthermore, we demonstrate that alteration of the MAPK phosphorylation sites in the Smad5 linker causes precocious patterning of DV tissues along the AP axis during gastrulation. Thus, DV and AP patterning are intimately coordinated to allow cells to acquire both positional and temporal information simultaneously.

Acute alcohol exposure during mouse gastrulation alters lipid metabolism in placental and heart development: Folate prevention.

BACKGROUND: Embryonic acute exposure to ethanol (EtOH), lithium, and homocysteine (HCy) induces cardiac defects at the time of exposure; folic acid (FA) supplementation protects normal cardiogenesis (Han et al., , ; Serrano et al., ). Our hypothesis is that EtOH exposure and FA protection relate to lipid and FA metabolism during mouse cardiogenesis and placentation. METHODS: On the morning of conception, pregnant C57BL/6J mice were placed on either of two FA-containing diets: a 3.3 mg health maintenance diet or a high FA diet of 10.5 mg/kg. Mice were injected a binge level of EtOH, HCy, or saline on embryonic day (E) 6.75, targeting gastrulation. On E15.5, cardiac and umbilical blood flow were examined by ultrasound. Embryonic cardiac tissues were processed for gene expression of lipid and FA metabolism; the placenta and heart tissues for neutral lipid droplets, or for medium chain acyl-dehydrogenase (MCAD) protein. RESULTS: EtOH exposure altered lipid-related gene expression on E7.5 in comparison to control or FA-supplemented groups and remained altered on E15.5 similarly to changes with HCy, signifying FA deficiency. In comparison to control tissues, the lipid-related acyl CoA dehydrogenase medium length chain gene and its protein MCAD were altered with EtOH exposure, as were neutral lipid droplet localization in the heart and placenta. CONCLUSION: EtOH altered gene expression associated with lipid and folate metabolism, as well as neutral lipids, in the E15.5 abnormally functioning heart and placenta. In comparison to controls, the high FA diet protected the embryo and placenta from these effects allowing normal development. Birth Defects Research (Part A) 106:749-760, 2016. © 2016 The Authors Birth Defects Research Part A: Clinical and Molecular Teratology Published by Wiley Periodicals, Inc.

Involvement of l(-)-rhamnose in sea urchin gastrulation. Part II: α-l-Rhamnosidase.

The sea urchin embryo is recognized as a model system to reveal developmental mechanisms involved in human health and disease. In Part I of this series, six carbohydrates were tested for their effects on gastrulation in embryos of the sea urchin Lytechinus pictus. Only l-rhamnose caused dramatic increases in the numbers of unattached archenterons and exogastrulated archenterons in living, swimming embryos. It was found that at 30 h post-fertilization the l-rhamnose had an unusual inverse dose-dependent effect, with low concentrations (1-3 mM) interfering with development and higher concentrations (30 mM) having little to no effect on normal development. In this study, embryos were examined for inhibition of archenteron development after treatment with α-l-rhamnosidase, an endoglycosidase that removes terminal l-rhamnose sugars from glycans. It was observed that the enzyme had profound effects on gastrulation, an effect that could be suppressed by addition of l-rhamnose as a competitive inhibitor. The involvement of l-rhamnose-containing glycans in sea urchin gastrulation was unexpected, since there are no characterized biosynthetic pathways for rhamnose utilization in animals. It is possible there exists a novel l-rhamnose-containing glycan in sea urchins, or that the enzyme and sugar interfere with the function of rhamnose-binding lectins, which are components of the innate immune system in many vertebrate and invertebrate species.

Loss of Porcupine impairs convergent extension during gastrulation in zebrafish.

Porcupine (Porcn), an O-acyltransferase located in the endoplasmic reticulum (ER), is required for lipidation of Wnt proteins to enable their trafficking from the ER in mammalian cell culture. However, it is unclear whether Porcn is required for trafficking of all members of the Wnt family. In this study, we investigated the function of Porcn in zebrafish embryos. We identified two zebrafish homologs of porcupine, porcn and porcupine-like (porcn-l). Zebrafish porcn, but not porcn-l, restores secretion of Wnt proteins in porcn-deficient mouse L cells. Morpholino-mediated knockdown of porcn in zebrafish embryos impairs convergence and extension (CE) during gastrulation without changing embryonic patterning. Moreover, porcn interacts genetically with wnt5b and wnt11 in regulating CE. By contrast, porcn-deficient embryos do not exhibit phenotypes caused by failure in canonical Wnt signaling, which is activated by several Wnt ligands, including Wnt3a. Furthermore, expression of genes regulated by the canonical Wnt signaling pathway is not perturbed in knockdown embryos relative to that in controls. Although the trafficking and lipidation of ectopically expressed zebrafish Wnt5b and mouse Wnt5a are impaired in porcn-deficient embryos, those of ectopically expressed Wnt3a are less or not affected. In addition, the secretion of Wnt5a is inhibited by less Porcn inhibitor than that of Wnt3a in HEK293T cells. Thus, a decrease of Porcn activity does not equivalently affect trafficking and lipidation of different Wnt proteins in zebrafish embryos and in cultured mammalian cells.

Upon Wnt stimulation, Rac1 activation requires Rac1 and Vav2 binding to p120-catenin.

A role for Rac1 GTPase in canonical Wnt signaling has recently been demonstrated, showing that it is required for ß-catenin translocation to the nucleus. In this study, we investigated the mechanism of Rac1 stimulation by Wnt. Upregulation of Rac1 activity by Wnt3a temporally correlated with enhanced p120-catenin binding to Rac1 and Vav2. Vav2 and Rac1 association with p120-catenin was modulated by phosphorylation of this protein, which was stimulated upon serine/threonine phosphorylation by CK1 and inhibited by tyrosine phosphorylation by Src or Fyn. Acting on these two post-translational modifications, Wnt3a induced the release of p120-catenin from E-cadherin, enabled the interaction of p120-catenin with Vav2 and Rac1, and facilitated Rac1 activation by Vav2. Given that p120-catenin depletion disrupts gastrulation in Xenopus, we analyzed p120-catenin mutants for their ability to rescue this phenotype. In contrast to the wild-type protein or other controls, p120-catenin point mutants that were deficient in the release from E-cadherin or in Vav2 or Rac1 binding failed to rescue p120-catenin depletion. Collectively, these results indicate that binding of p120-catenin to Vav2 and Rac1 is required for the activation of this GTPase upon Wnt signaling.