Decoding anterior-posterior axis emergence among mouse,monkey, and human embryos

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Disabled-2 is essential for endodermal cell positioning and structure formation during mouse embryogenesis

The signal transduction adapter protein Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The brain-specific Disabled-1 (Dab1) functions in positional organization of brain cells during development. Dab2 is widely distributed and is highly expressed in many epithelial cell types. The dab2 gene was interrupted by in-frame insertion of beta-galactosidase (LacZ) in embryonic stem cells and transgenic mice were produced. Dab2 expression was first observed in the primitive endoderm at E4.5, immediately following implantation. The homozygous Dab2-deficient mutant is embryonic lethal (earlier than E6.5) due to defective cell positioning and structure formation of the visceral endoderm. In E5.5 dab2 (-/-) conceptus, visceral endoderm-like cells are present in the deformed primitive egg cylinder; however, the visceral endoderm cells are not organized, the cells of the epiblast have not expanded, and the proamniotic cavity fails to form. Disorganization of the visceral endodermal layer is evident, as cells with positive visceral endoderm markers are scattered throughout the dab2 (-/-) conceptus. Only degenerated remains were observed at E6.5 for dab2 (-/-) embryos, and by E7.5, the defective embryos were completely reabsorbed. In blastocyst in vitro culture, initially cells with characteristics of endoderm, trophectoderm, and inner cell mass were observed in the outgrowth of the hatched dab2 (-/-) blastocysts. However, the dab2 (-/-) endodermal cells are much more dispersed and disorganized than those from wild-type blastocysts, the inner cell mass fails to expand, and the outgrowth degenerates by day 7. Thus, Dab2 is required for visceral endodermal cell organization during early mouse development. The absence of an organized visceral endoderm in Dab2-deficient conceptus leads to the growth failure of the inner cell mass. We suggest that Dab2 functions in a signal pathway to regulate endodermal cell organization using endocytosis of ligands from the blastocoel cavity as a positioning cue.     

Sfrp1, Sfrp2, and Sfrp5 regulate the Wnt/beta-catenin and the planar cell polarity pathways during early trunk formation in mouse

Sfrp is a secreted Wnt antagonist that directly interacts with Wnt ligand. We show here that inactivation of Sfrp1, Sfrp2, and Sfrp5 leads to fused somites formation in early-somite mouse embryos, simultaneously resulting in defective convergent extension (CE), which causes severe shortening of the anteroposterior axis. These observations indicate the redundant roles of Sfrp1, Sfrp2, and Sfrp5 in early trunk formation. The roles of the Sfrps were genetically distinguished in terms of the regulation of Wnt pathways. Genetic analysis combining Sfrps mutants and Loop-tail mice revealed the involvement of Sfrps in CE through the regulation of the planar cell polarity pathway. Furthermore, Dkk1-deficient embryos carrying Sfrp1 homozygous and Sfrp2 heterozygous mutations display irregular somites and indistinct intersomitic boundaries, which indicates that Sfrps-mediated inhibition of the Wnt/beta-catenin pathway is necessary for somitogenesis. Our results suggest that Sfrps regulation of the canonical and noncanonical pathways is essential for proper trunk formation.     

Sfrp1a and Sfrp5 function as positive regulators of Wnt and BMP signaling during early retinal development

Axial patterning of the developing eye is critically important for proper axonal pathfinding as well as for key morphogenetic events, such as closure of the optic fissure. The dorsal retina is initially specified by the actions of Bone Morphogenetic Protein (BMP) signaling, with such identity subsequently maintained by the Wnt-β catenin pathway. Using zebrafish as a model system, we demonstrate that Secreted frizzled-related protein 1a (Sfrp1a) and Sfrp5 work cooperatively to pattern the retina along the dorso-ventral axis. Sfrp1a/5 depleted embryos display a reduction in dorsal marker gene expression that is consistent with defects in BMP- and Wnt-dependent dorsal retina identity. In accord with this finding, we observe a marked reduction in transgenic reporters of BMP and Wnt signaling within the dorsal retina of Sfrp1a/5 depleted embryos. In contrast to studies in which canonical Wnt signaling is blocked, we note an increase in BMP ligand expression in Sfrp1a/5 depleted embryos, a phenotype similar to that seen in embryos with inhibited BMP signaling. Overexpression of a low dose of sfrp5 mRNA causes an increase in dorsal retina marker gene expression. We propose a model in which Sfrp proteins function as facilitators of both BMP and Wnt signaling within the dorsal retina.     

Requirement for beta-catenin in anterior-posterior axis formation in mice

The anterior-posterior axis of the mouse embryo is defined before formation of the primitive streak, and axis specification and subsequent anterior development involves signaling from both embryonic ectoderm and visceral endoderm. Tauhe Wnt signaling pathway is essential for various developmental processes, but a role in anterior-posterior axis formation in the mouse has not been previously established. Beta-catenin is a central player in the Wnt pathway and in cadherin-mediated cell adhesion. We generated beta-catenin-deficient mouse embryos and observed a defect in anterior-posterior axis formation at embryonic day 5.5, as visualized by the absence of Hex and Hesx1 and the mislocation of cerberus-like and Lim1 expression. Subsequently, no mesoderm and head structures are generated. Intercellular adhesion is maintained since plakoglobin substitutes for beta-catenin. Our data demonstrate that beta-catenin function is essential in anterior-posterior axis formation in the mouse, and experiments with chimeric embryos show that this function is required in the embryonic ectoderm.     

Wnt3 signaling in the epiblast is required for proper orientation of the anteroposterior axis

The establishment of anteroposterior (AP) polarity in the early mouse epiblast is crucial for the initiation of gastrulation and the subsequent formation of the embryonic (head to tail) axis. The localization of anterior and posterior determining genes to the appropriate region of the embryo is a dynamic process that underlies this early polarity. Several studies indicate that morphological and molecular markers which define the early AP axis are first aligned along the short axis of the elliptical egg cylinder. Subsequently, just prior to the time of primitive streak formation, a conformational change in the embryo realigns these markers with the long axis. We demonstrate that embryos lacking the signaling factor Wnt3 exhibit defects in this axial realignment. In addition, chimeric analyses and conditional removal of Wnt3 activity reveal that Wnt3 expression in the epiblast is required for induction of the primitive streak and mesoderm whereas activity in the posterior visceral endoderm is dispensable.     

Sall4 isoforms act during proximal-distal and anterior-posterior axis formation in the mouse embryo

Reciprocal signals from embryonic and extra-embryonic tissues pattern the embryo in proximal-distal (PD) and anterior-posterior (AP) fashion. Here we have analyzed three gene trap mutations of Sall4, of which one (Sall4-1a) led to a hypomorphic and recessive phenotype, demonstrating that Sall4-1a has yet undescribed extra-embryonic and embryonic functions in regulating PD and AP axis formation. In Sall4-1a mutants the self-maintaining autoregulatory interaction between Bmp4, Nodal and Wnt, which determines the PD axis was disrupted because of defects in the extra-embryonic visceral endoderm. More severely, two distinct Sall4 gene-trap mutants (Sall4-1a,b), resembling null mutants, failed to initiate Bmp4 expression in the extra-embryonic ectoderm and Nodal in the epiblast and were therefore unable to initiate PD axis formation. Tetraploid rescue underlined the extra-embryonic nature of the Sall4-1a phenotype and revealed a further embryonic function in Wnt/beta-catenin signaling to elongate the AP axis during gastrulation. This observation was supported through genetic interaction with beta-catenin mutants, since compound heterozygous mutants recapitulated the defects of Wnt3a mutants in posterior development.     

Tetraploid embryos rescue the early defects of tw5/tw5 mouse embryos

tclw5 is a t-complex recessive lethal mutation of the tw5-haplotype. Since tw5/tw5 embryos die soon after implantation, the tclw5 gene is thought to play an important role in early embryogenesis. Previous histological studies have demonstrated that tw5 homozygotes do not survive past the gastrulation stage due to extensive death of the embryonic ectoderm, whereas the extraembryonic tissues were less affected. In the present study, we demonstrate that tw5/tw5 embryos may be distinguished from wildtype littermates at embryonic (E) day 5.5. At this stage, the visceral endoderm of tw5/tw5 embryos appeared to be different, possessing smaller and fewer vacuoles compared to normal littermates. This led us to hypothesize that the visceral endoderm may be affected by tclw5. Confirmation was provided by the rescue of tw5/tw5 embryos following aggregation with tetraploid embryos. However, rescued embryos did not survive past E9.0 and displayed an underdeveloped posterior region. This would indicate that the actions of tclw5 extend beyond the midgestation stage.     

Protein kinase CK2 is required for dorsal axis formation in Xenopus embryos

Dorsal axis formation in Xenopus embryos is dependent upon asymmetrical localization of beta-catenin, a transducer of the canonical Wnt signaling pathway. Recent biochemical experiments have implicated protein kinase CK2 as a regulator of members of the Wnt pathway including beta-catenin. Here, we have examined the role of CK2 in dorsal axis formation. CK2 was present in the developing embryo at an appropriate time and place to participate in dorsal axis formation. Overexpression of mRNA encoding CK2 in ventral blastomeres was sufficient to induce a complete ectopic axis, mimicking Wnt signaling. A kinase-inactive mutant of CK2alpha was able to block ectopic axis formation induced by XWnt8 and beta-catenin and was capable of suppressing endogenous axis formation when overexpressed dorsally. Taken together, these studies demonstrate that CK2 is a bona fide member of the Wnt pathway and has a critical role in the establishment of the dorsal embryonic axis.     

The transmembrane receptor Uncoordinated5 (Unc5) is essential for heart lumen formation in Drosophila melanogaster

Transport of liquids or gases in biological tubes is fundamental for many physiological processes. Our knowledge on how tubular organs are formed during organogenesis and tissue remodeling has increased dramatically during the last decade. Studies on different animal systems have helped to unravel some of the molecular mechanisms underlying tubulogenesis. Tube architecture varies dramatically in different organs and different species, ranging from tubes formed by several cells constituting the cross section, tubes formed by single cells wrapping an internal luminal space or tubes that are formed within a cell. Some tubes display branching whereas others remain linear without intersections. The modes of shaping, growing and pre-patterning a tube are also different and it is still not known whether these diverse architectures and modes of differentiation are realized by sharing common signaling pathways or regulatory networks. However, several recent investigations provide evidence for the attractive hypothesis that the Drosophila cardiogenesis and heart tube formation shares many similarities with primary angiogenesis in vertebrates. Additionally, another important step to unravel the complex system of lumen formation has been the outcome of recent studies that junctional proteins, matrix components as well as proteins acting as attractant and repellent cues play a role in the formation of the Drosophila heart lumen. In this study we show the requirement for the repulsively active Unc5 transmembrane receptor to facilitate tubulogenesis in the dorsal vessel of Drosophila. Unc5 is localized in the luminal membrane compartment of cardiomyocytes and animals lacking Unc5 fail to form a heart lumen. Our findings support the idea that Unc5 is crucial for lumen formation and thereby represents a repulsive cue acting during Drosophila heart tube formation.     

Bone morphogenetic protein 4 signaling regulates development of the anterior visceral endoderm in the mouse embryo

The extraembryonic ectoderm (ExE) of the mouse conceptus is known to play a role in embryo patterning by signaling to the underlying epiblast and surrounding visceral endoderm. Bmp4 is one of the key ExE signaling molecules and has been recently implicated to participate in regulating development and migration of the anterior visceral endoderm (AVE). However, it remains unclear when exactly BMP4 signaling starts to regulate AVE positioning. To examine this, we have chosen to affect BMP4 function at two different time points, at embryonic day 5.25 (E5.25), thus before AVE migration, and E5.75, just after AVE migration. To this end, an RNAi technique was used, which consisted of the injection of Bmp4 dsRNA into the proamniotic cavity of the egg cylinder followed by its targeted electroporation into the ExE. This resulted in specific knockdown of Bmp4 . It was found that Bmp4 RNAi at E5.25, but not at E5.75, led to an abnormal pattern of expression of the AVE marker Cerberus-like . Thus, BMP4 signaling appears to affect the expression of Cer1 at a specific time window. This RNAi approach provides a convenient means to study spatial and temporal function of genes shortly after embryo implantation.     

Laminin alpha 5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung

Laminin alpha/beta/gamma heterotrimers are the major noncollagenous components of all basement membranes. To date, five alpha, three beta, and three gamma chains have been identified. Laminin alpha 5 is expressed early in lung development and colocalizes with laminin alpha1. While laminin alpha1 expression in the lung is restricted to the embryonic period, laminin alpha 5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin alpha 5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5(-/-) mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin alpha 4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin alpha 5, which was found to be the only laminin alpha chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.     

The cerberus-related gene, Cerr1, is not essential for mouse head formation

The Xenopus cerberus gene encodes a secreted factor expressed in the Spemann organizer that can cause ectopic head formation when its mRNA is injected into Xenopus embryos. In mouse, the cerberus-related gene, Cerr1, is expressed in the anterior mesendoderm that underlies the presumptive anterior neural plate and its expression is downregulated in Lim1 headless embryos. To determine whether Cerr1 is required for head formation we generated a null mutation in Cerr1 by gene targeting in mouse embryonic stem cells. We found that head formation is normal in Cerr1(-/-) embryos and we detected no obvious phenotypic defects in adult Cerr1(-/-) mice. However, in embryonic tissue layer recombination assays, Cerr1(-/-) presomitic/somitic mesoderm, unlike Cerr1-expressing wild-type presomitic/somitic mesoderm, was unable to maintain expression of the anterior neural marker gene Otx2 in ectoderm explants. These findings suggest that establishment of anterior identity in the mouse may involve the action of multiple functionally redundant factors.     

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.     

Heading forwards: anterior visceral endoderm migration in patterning the mouse embryo

The elaboration of anterior–posterior (A–P) pattern is one of the earliest events during development and requires the precisely coordinated action of several players at the level of molecules, cells and tissues. In mammals, it is controlled by a specialized population of migratory extraembryonic epithelial cells, the anterior visceral endoderm (AVE). The AVE is a signalling centre that is responsible for several important patterning events during early development, including specifying the orientation of the A–P axis and the position of the heart with respect to the brain. AVE cells undergo a characteristic stereotypical migration which is crucial to their functions.     

The fibroblast growth factor-2 is not essential for melanoma formation in a transgenic mouse model

Fibroblast growth factor 2 (FGF2) has been assigned a role in melanocyte proliferation and in development of human cutaneous melanoma. We have used a transgenic mouse melanoma model in combination with mice lacking mouse FGF2 to analyse the possible implication of FGF2 in melanomagenesis. Tyr::N-rasQ61K transgenic mice which are deficient for FGF2 and the tumor suppressors p16INK4a and p19ARF are hyperpigmented and develop cutaneous metastasizing melanoma, with no difference to mice wildtype for FGF2. We conclude from our data, that FGF2 is not essential for melanoma progression and metastasis.     

Expression of HSG is essential for mouse blastocyst formation

It has been shown recently that hyperplasia suppressor gene (HSG) is a powerful regulator for cell proliferation and has a critical role in mitochondrial fusion in many cells. However, little is known about its expression, localization, and function during oocyte maturation and early embryogenesis. In this study, with indirect immunofluorescent staining and Western blotting, we found that HSG was expressed in mouse oocytes and preimplantation embryos which primarily exhibited a submembrane distribution pattern in the cytoplasm. Moreover, HSG mainly associated with beta-tubulin during oocyte maturation and early embryonic development. When mouse zygotes were injected with HSG antisense plasmid and cultured in vitro, their capacity to form blastocysts was severely impaired. Our results indicate that HSG plays an essential role in mouse preimplantation development.