The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.     

Early embryonic death in mice lacking the beta-catenin-binding protein Duplin

The Wnt signaling pathway plays a pivotal role in vertebrate early development and morphogenesis. Duplin (axis duplication inhibitor) interacts with beta-catenin and prevents its binding to Tcf, thereby inhibiting downstream Wnt signaling. Here we show that Duplin is expressed predominantly from early- to mid-stage mouse embryogenesis, and we describe the generation of mice deficient in Duplin. Duplin(-/-) embryos manifest growth retardation from embryonic day 5.5 (E5.5) and developmental arrest accompanied by massive apoptosis at E7.5. The mutant embryos develop into an egg cylinder but do not form a primitive streak or mesoderm. Expression of beta-catenin target genes, including those for T (brachyury), Axin2, and cyclin D1, was not increased in Duplin(-/-) embryos, suggesting that the developmental defect is not simply attributable to upregulation of Wnt signaling caused by the lack of this inhibitor. These results suggest that Duplin plays an indispensable role, likely by a mechanism independent of inhibition of Wnt signaling, in mouse embryonic growth and differentiation at an early developmental stage.     

BMP receptor IA is required in mammalian neural crest cells for development of the cardiac outflow tract and ventricular myocardium

The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. We have ablated Bmpr1a specifically in the neural crest, beginning at the five-somite stage. We find that most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium.     

Moderate expression of Wnt signaling genes is essential for porcine parthenogenetic embryo development

Parthenogenetic embryos are invariably lost in mid-gestation, possibly due to the lack of the paternal genome and the consequent induction of aberrant gene expression. Wnt signaling is essential for embryonic development; however, the studies of this pathway in porcine parthenogenetic embryos have been limited. Here, the role of Wnt signaling in porcine parthenogenetic embryos was studied. In vivo embryos were used as controls. Single cell quantitative real-time PCR showed that Wnt signaling was down-regulated in porcine parthenogenetic embryos. Furthermore, immunofluorescence staining and real-time PCR demonstrated that porcine parthenogenetic embryo development was largely unaffected by the inhibition of Wnt signaling with IWP-2, but blastocyst hatching and trophectoderm development was blocked. In addition, parthenogenetic blastocyst hatching was improved by the activation of Wnt signaling by BIO. However, the developmental competency of porcine embryos, including blastocyst hatching, was impaired and apoptosis was induced upon the excessive activation of Wnt signaling. These findings constitute novel evidence that Wnt signaling is important for porcine pre-implantation development and that its down-regulation may lead to the low hatching rate of porcine parthenogenetic blastocysts.     

Developmental retardation of XO mouse embryos at mid-gestation.

It is not known why XO mouse embryos, which develop more slowly than XX embryos until early mid-gestation, reach the same stage in their growth and development as their XX littermates at the mid-gestation stage. It is hypothesized that there is an effect of 'litter size' that causes an acceleration of the development of XO embryos at mid-gestation. The present study was performed to determine whether the development of XO embryos is retarded compared with that of their XX litermates at early mid-gestation (day 8 of gestation), before reduction of litter size. The percentage of pre-somite stage XO embryos was greater than the percentage of pre-somite stage XX embryos, and the mean number of somites was greater in XX embryos than it was in XO embryos. These findings indicate that the development of XO embryos was retarded when compared with that of their XX litermates at early mid-gestation. This result is discussed with respect to the compensatory development of XO embryos at mid-gestation and the reduction of litter size shortly after early mid-gestation.     

Expression of Notch signaling pathway genes in mouse embryos lacking beta4galactosyltransferase-1

A requirement for beta4galactosyltransferase-1 (beta4GalT-1) activity in the modulation of Notch signaling by the glycosyltransferase Fringe was previously identified in a mammalian co-culture assay. Notch signaling is necessary for the formation of somites in mammals. We therefore investigated the expression of eleven Notch pathway and somitogenic genes in E9.5 mouse embryos lacking beta4GalT-1. Four of these genes were altered in expression pattern or expression level. The Notch target genes Hes5 and Mesp2 were affected to some degree in all mutant embryos. The Notch ligand genes Dll1 and Dll3 were reduced or altered in expression in a significant proportion of mutants. While there were no differences in the number or morphology of somites in E9.5 B4galt1 null embryos, the number of lumbar vertebrae in mutant embryos differed from control littermates (P < or = 0.01). The subtlety of the in vivo phenotype may be due to redundancy since several B4galt genes related to B4galt1 are expressed during embryogenesis.     

Sfrp5 is not essential for axis formation in the mouse

Secreted frizzled related protein (Sfrp) genes encode extracellular factors that can modulate Wnt signaling. During early post-implantation mouse development Sfrp5 is expressed in the anterior visceral endoderm (AVE) and the ventral foregut endoderm. The AVE is important in anterior-posterior axis formation and the ventral foregut endoderm contributes to multiple gut tissues. Here to determine the essential role of Sfrp5 in early mouse development we generated Sfrp5-deficient mice by gene targeting. We report that Sfrp5-deficient mice are viable and fertile. To determine whether the absence of an axis phenotype might be due to genetic redundancy with Dkk1 in the AVE we generated Sfrp5;Dkk1 double mutant mice. AVE development and primitive streak formation appeared normal in Sfrp5(-/-);Dkk1(-/-) embryos. These results indicate that Sfrp5 is not essential for axis formation or foregut morphogenesis in the mouse and also imply that Sfrp5 and Dkk1 together are not essential for AVE development.     

Analysis of cytogenetic and developmental effects on pre-implantation, mid-gestation and near-term mouse embryos after treatment with trichlorfon during zygote stage

Trichlorfon has been widely used in agriculture as a broad spectrum insecticide. We examined cytogenetic and developmental effects on early mouse zygotes exposed to trichlorfon in vivo. Pregnant female mice were intraperitoneally administered a single dose of trichlorfon (100 or 200mg/kg) at 6h post presumed conception and either sacrificed on day of gestation (dg) 3, 9 or 17 to assess the developmental toxicity and mutagenic effects on embryos.Mean cell number (dg 3) and somite number (dg 9) of embryos in the two trichlorfon-treated groups were significantly fewer than in the control group and the mean micronucleus (MN) number (dg 3) and the frequency of mosaic aneuploidies including monosomic or trisomic cell lines (dg 9) was significantly increased in both trichlorfon-treated groups compared with the control group. However, there was no difference in fetal body weight (dg 17) between the control and trichlorfon-treated groups and no increased incidence of external malformations was observed in the trichlorfon-treated groups. These findings suggest that acute exposure of trichlorfon around fertilization induces a high frequency of MN, mosaic aneuploidies and developmental retardation in pre-implantation and mid-gestation embryos, and thereafter these embryos with MN or chromosome damage appear to develop past mid-gestation and catch up with normal embryos by near-term.     

Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning.

Despite a wealth of experimental data implicating fibroblast growth factor (FGF) signaling in various developmental processes, genetic inactivation of individual genes encoding specific FGFs or their receptors (FGFRs) has generally failed to demonstrate their role in vertebrate organogenesis due to early embryonic lethality or functional redundancy. Here we show that broad mid-gestational expression of a novel secreted kinase-deficient receptor, specific for a defined subset of the FGF superfamily, caused agenesis or severe dysgenesis of kidney, lung, specific cutaneous structures, exocrine and endocrine glands, and craniofacial and limb abnormalities reminiscent of human skeletal disorders associated with FGFR mutations. Analysis of diagnostic molecular markers revealed that this soluble dominant-negative mutant disrupted early inductive signaling in affected tissues, indicating that FGF signaling is required for growth and patterning in a broad array of organs and in limbs. In contrast, transgenic mice expressing a membrane-tethered kinase-deficient FGFR were viable. Our results demonstrate that secreted FGFR mutants are uniquely effective as dominant-negative agents in vivo, and suggest that related soluble receptor isoforms expressed in wild-type mouse embryos may help regulate FGF activity during normal development.     

Requirement for the Xrcc1 DNA base excision repair gene during early mouse development

Surveillance and repair of DNA damage are essential for maintaining the integrity of the genetic information that is needed for normal development. Several multienzyme pathways, including the excision repair of damaged or missing bases, carry out DNA repair in mammals. We determined the developmental role of the X-ray cross-complementing (Xrcc)-1 gene, which is central to base excision repair, by generating a targeted mutation in mice. Heterozygous matings produced Xrcc1-/- embryos at early developmental stages, but not Xrcc1-/- late-stage fetuses or pups. Histology showed that mutant (Xrcc1-/-) embryos arrested at embryonic day (E) 6.5 and by E7.5 were morphologically abnormal. The most severe abnormalities observed in mutant embryos were in embryonic tissues, which showed increased cell death in the epiblast and an altered morphology in the visceral embryonic endoderm. Extraembryonic tissues appeared relatively normal at E6.5-7.5. Even without exposure to DNA-damaging agents, mutant embryos showed increased levels of unrepaired DNA strand breaks in the egg cylinder compared with normal embryos. Xrcc1-/- cell lines derived from mutant embryos were hypersensitive to mutagen-induced DNA damage. Xrcc1 mutant embryos that were also made homozygous for a null mutation in Trp53 underwent developmental arrest after only slightly further development, thus revealing a Trp53-independent mechanism of embryo lethality. These results show that an intact base excision repair pathway is essential for normal early postimplantation mouse development and implicate an endogenous source of DNA damage in the lethal phenotype of embryos lacking this repair capacity.     

Notch2 is required for formation of the placental circulatory system, but not for cell-type specification in the developing mouse placenta

We have previously reported that a mutation in the ankyrin repeats of mouse Notch2 results in embryonic lethality by embryonic day 11.5 (E11.5), showing developmental retardation at E10.5. This indicated that Notch2 plays an essential role in postimplantation development in mice. Here, we demonstrate that whole embryo culture can circumvent developmental retardation of Notch2 mutant embryos for up to 1 day, suggesting that the lethality was primarily caused by extraembryonic defects. Histological examinations revealed delayed entry of maternal blood into the mutant placenta and poor blood sinus formation at later stages. Notch2-expressing cells appeared around maternal blood sinuses. Specification of trophoblast subtypes appeared not to be drastically disturbed and expression of presumptive downstream genes of Notch2 signaling was not altered by the Notch2 mutation. Thus, in the developing mouse placenta, Notch2 is unlikely to be involved in cell fate decisions, but rather participates in formation of maternal blood sinuses. In aggregation chimeras with wild-type tetraploid embryos, the mutant embryos developed normally until E12.5, but died before E13.5. The chimeric placentas showed a restored maternal blood sinus formation when compared with the mutant placentas, but not at the level of wild-type diploid placentas. Therefore, it was concluded that the mutant suffers from defects in maternal blood sinus formation. Thus, Notch2 is not cell autonomously required for the early cell fate determination of subtypes of trophoblast cells, but plays an indispensable role in the formation of maternal blood sinuses in the developing mouse placenta.     

Genetic dissection of nodal function in patterning the mouse embryo

Loss-of-function analysis has shown that the transforming growth factor-like signaling molecule nodal is essential for mouse mesoderm development. However, definitive proof of nodal function in other developmental processes in the mouse embryo has been lacking because the null mutation blocks gastrulation. We describe the generation and analysis of a hypomorphic nodal allele. Mouse embryos heterozygous for the hypomorphic allele and a null allele undergo gastrulation but then display abnormalities that fall into three distinct mutant phenotypic classes, which may result from expression levels falling below critical thresholds in one or more domains of nodal expression. Our analysis of each of these classes provides conclusive evidence for nodal-mediated regulation of several developmental processes in the mouse embryo, beyond its role in mesoderm formation. We find that nodal signaling is required for correct positioning of the anteroposterior axis, normal anterior and midline patterning, and the left-right asymmetric development of the heart, vasculature, lungs and stomach.     

Disruption of Smad4 in neural crest cells leads to mid-gestation death with pharyngeal arch, craniofacial and cardiac defects

TGFβ/BMP signaling pathways are essential for normal development of neural crest cells (NCCs). Smad4 encodes the only common Smad protein in mammals, which is a critical nuclear mediator of TGFβ/BMP signaling. In this work, we sought to investigate the roles of Smad4 for development of NCCs. To overcome the early embryonic lethality of Smad4 null mice, we specifically disrupted Smad4 in NCCs using a Cre/loxP system. The mutant mice died at mid-gestation with defects in facial primordia, pharyngeal arches, outflow tract and cardiac ventricles. Further examination revealed that mutant embryos displayed severe molecular defects starting from E9.5. Expression of multiple genes, including Msx1, 2, Ap-2α, Pax3, and Sox9, which play critical roles for NCC development, was downregulated by NCC disruption of Smad4. Moreover, increased cell death was observed in pharyngeal arches from E10.5. However, the cell proliferation rate in these areas was not substantially altered. Taken together, these findings provide compelling genetic evidence that Smad4-mediated activities of TGFβ/BMP signals are essential for appropriate NCC development.     

Impaired neural development caused by inducible expression of Axin in transgenic mice

Ablations of the Axin family genes demonstrated that they modulate Wnt signaling in key processes of mammalian development. The ubiquitously expressed Axin1 plays an important role in formation of the embryonic neural axis, while Axin2 is essential for craniofacial skeletogenesis. Although Axin2 is also highly expressed during early neural development, including the neural tube and neural crest, it is not essential for these processes, apparently due to functional redundancy with Axin1. To further investigate the role of Wnt signaling during early neural development, and its potential regulation by Axins, we developed a mouse model for conditional gene activation in the Axin2-expressing domains. We show that gene expression can be successfully targeted to the Axin2-expressing cells in a spatially and temporally specific fashion. High levels of Axin in this domain induce a region-specific effect on the patterning of neural tube. In the mutant embryos, only the development of midbrain is severely impaired even though the transgene is expressed throughout the neural tube. Axin apparently regulates beta-catenin in coordinating cell cycle progression, cell adhesion and survival of neuroepithelial precursors during development of ventricles. Our data support the conclusion that the development of embryonic neural axis is highly sensitive to the level of Wnt signaling.     

Rescuing the N-cadherin knockout by cardiac-specific expression of N- or E-cadherin

Cell-cell adhesion mediated by some members of the cadherin family is essential for embryonic survival. The N-cadherin-null embryo dies during mid-gestation, with multiple developmental defects. We show that N-cadherin-null embryos expressing cadherins using muscle-specific promoters, alpha- or beta-myosin heavy chain, are partially rescued. Somewhat surprisingly, either N-cadherin or E-cadherin was effective in rescuing the embryos. The rescued embryos exhibited an increased number of somites, branchial arches and the presence of forelimb buds; however, in contrast, brain development was severely impaired. In rescued animals, the aberrant yolk sac morphology seen in N-cadherin-null embryos was corrected, demonstrating that this phenotype was secondary to the cardiac defect. Dye injection studies and analysis of chimeric animals that have both wild-type and N-cadherin-null cells support the conclusion that obstruction of the cardiac outflow tract represents a major defect that is likely to be the primary cause of pericardial swelling seen in null embryos. Although rescued embryos were more developed than null embryos, they were smaller than wild-type embryos, even though the integrity of the cardiovascular system appeared normal. The smaller size of rescued embryos may be due, at least in part, to increased apoptosis observed in tissues not rescued by transgene expression, indicating that N-cadherin-mediated cell adhesion provides an essential survival signal for embryonic cells. Our data provide in vivo evidence that cadherin adhesion is essential for cell survival and for normal heart development. Our data also show that E-cadherin can functionally substitute for N-cadherin during cardiogenesis, suggesting a critical role for cadherin-mediated cell-cell adhesion, but not cadherin family member-specific signaling, at the looping stage of heart development.     

Par‐aPKC‐dependent and ‐independent mechanisms cooperatively control cell polarity,Hippo signaling,and cell positioning in 16‐cell stage mouse embryos

In preimplantation mouse embryos, the Hippo signaling pathway plays a central role in regulating the fates of the trophectoderm (TE) and the inner cell mass (ICM). In early blastocysts with more than 32 cells, the Par‐aPKC system controls polarization of the outer cells along the apicobasal axis, and cell polarity suppresses Hippo signaling. Inactivation of Hippo signaling promotes nuclear accumulation of a coactivator protein, Yap, leading to induction of TE‐specific genes. However, whether similar mechanisms operate at earlier stages is not known. Here, we show that slightly different mechanisms operate in 16‐cell stage embryos. Similar to 32‐cell stage embryos, disruption of the Par‐aPKC system activated Hippo signaling and suppressed nuclear Yap and Cdx2 expression in the outer cells. However, unlike 32‐cell stage embryos, 16‐cell stage embryos with a disrupted Par‐aPKC system maintained apical localization of phosphorylated Ezrin/Radixin/Moesin (p‐ERM), and the effects on Yap and Cdx2 were weak. Furthermore, normal 16‐cell stage embryos often contained apolar cells in the outer position. In these cells, the Hippo pathway was strongly activated and Yap was excluded from the nuclei, thus resembling inner cells. Dissociated blastomeres of 8‐cell stage embryos form polar–apolar couplets, which exhibit different levels of nuclear Yap, and the polar cell engulfed the apolar cell. These results suggest that cell polarization at the 16‐cell stage is regulated by both Par‐aPKC‐dependent and ‐independent mechanisms. Asymmetric cell division is involved in cell polarity control, and cell polarity regulates cell positioning and most likely controls Hippo signaling.     

Aberrant profile of gene expression in cloned mouse embryos derived from donor cumulus nuclei

Somatic cell nuclear transfer has successfully been used to clone several mammalian species including the mouse, albeit with extremely low efficiency. This study investigated gene expression in cloned mouse embryos derived from cumulus cell donor nuclei, in comparison with in vivo fertilized mouse embryos, at progressive developmental stages. Enucleation was carried out by the conventional puncture method rather than by the piezo-actuated technique, whereas nuclear transfer was achieved by direct cumulus nuclear injection. Embryonic development was monitored from chemically induced activation on day 0 until the blastocyst stage on day 4. Poor developmental competence of cloned embryos was observed, which was confirmed by lower cell counts in cloned blastocysts, compared with the in vivo fertilized controls. Subsequently, real-time polymerase chain reaction was used to analyze and compare embryonic gene expression at the 2-cell, 4-cell, and blastocyst stages, between the experimental and control groups. The results showed reduced expression of the candidate genes in cloned 2-cell stage embryos, as manifested by poor developmental competence, compared with expression in the in vivo fertilized controls. Cloned 4-cell embryos and blastocysts, which had overcome the developmental block at the 2-cell stage, also showed up-regulated and down-regulated expression of several genes, strongly suggesting incomplete nuclear reprogramming. We have therefore demonstrated that aberrant embryonic gene expression is associated with low developmental competence of cloned mouse embryos. To improve the efficiency of somatic cell nuclear transfer, strategies to rectify aberrant gene expression in cloned embryos should be investigated.This project was funded mainly by the National University of Singapore (grant number: R-174-000-065-112/303).