Function of Rieger syndrome gene in left-right asymmetry and craniofacial development.

Rieger syndrome, an autosomal dominant disorder, includes ocular, craniofacial and umbilical abnormalities. The pitx2 homeobox gene, which is mutated in Rieger syndrome, has been proposed to be the effector molecule interpreting left-right axial information from the early embryonic trunk to each organ. Here we have used gene targeting in mice to generate a loss-of-function allele that would be predicted to result in organ randomization or isomerization. Although pitx2-/- embryos had abnormal cardiac morphogenesis, mutant hearts looped in the normal direction. Pitx2-/- embryos had correctly oriented, but arrested, embryonic rotation and right pulmonary isomerism. They also had defective development of the mandibular and maxillary facial prominences, regression of the stomodeum and arrested tooth development. Fgf8 expression was absent, and Bmp4 expression was expanded in the branchial-arch ectoderm. These data reveal a critical role for pitx2 in left-right asymmetry but indicate that pitx2 may function at an intermediate step in cardiac morphogenesis and embryonic rotation.     

Type ID unconventional myosin controls left-right asymmetry in Drosophila

Breaking left-right symmetry in Bilateria embryos is a major event in body plan organization that leads to polarized adult morphology, directional organ looping, and heart and brain function. However, the molecular nature of the determinant(s) responsible for the invariant orientation of the left-right axis (situs choice) remains largely unknown. Mutations producing a complete reversal of left-right asymmetry (situs inversus) are instrumental for identifying mechanisms controlling handedness, yet only one such mutation has been found in mice (inversin) and snails. Here we identify the conserved type ID unconventional myosin 31DF gene (Myo31DF) as a unique situs inversus locus in Drosophila. Myo31DF mutations reverse the dextral looping of genitalia, a prominent left-right marker in adult flies. Genetic mosaic analysis pinpoints the A8 segment of the genital disc as a left-right organizer and reveals an anterior-posterior compartmentalization of Myo31DF function that directs dextral development and represses a sinistral default state. As expected of a determinant, Myo31DF has a trigger-like function and is expressed symmetrically in the organizer, and its symmetrical overexpression does not impair left-right asymmetry. Thus Myo31DF is a dextral gene with actin-based motor activity controlling situs choice. Like mouse inversin, Myo31DF interacts and colocalizes with beta-catenin, suggesting that situs inversus genes can direct left-right development through the adherens junction.     

Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis

The pattern of blood flow in the developing heart has long been proposed to play a significant role in cardiac morphogenesis. In response to flow-induced forces, cultured cardiac endothelial cells rearrange their cytoskeletal structure and change their gene expression profiles. To link such in vitro data to the intact heart, we performed quantitative in vivo analyses of intracardiac flow forces in zebrafish embryos. Using in vivo imaging, here we show the presence of high-shear, vortical flow at two key stages in the developing heart, and predict flow-induced forces much greater than might have been expected for micro-scale structures at low Reynolds numbers. To test the relevance of these shear forces in vivo, flow was occluded at either the cardiac inflow or outflow tracts, resulting in hearts with an abnormal third chamber, diminished looping and impaired valve formation. The similarity of these defects to those observed in some congenital heart diseases argues for the importance of intracardiac haemodynamics as a key epigenetic factor in embryonic cardiogenesis.     

Regulation of vertebrate left-right asymmetries by extracellular matrix.

The vertebrate body is organized along three geometric axes: anterior-posterior, dorsal-ventral and left-right. Left-right axis formation, displayed in heart and gut development, is the least understood, even though it has been studied for many years. In Xenopus laevis gastrulae, a fibronectin-rich extracellular matrix is deposited on the basal surface of ectoderm cells over which cardiac and visceral primordia move during development. Here I report experiments in which localized perturbation of a small patch of extracellular matrix by microsurgery was correlated with localized randomization of left-right asymmetries. Global perturbation of the extracellular matrix by microinjection of Arg-Gly-Asp peptides or heparinase into the blastocoel resulted in global randomization of left-right asymmetries. From these observations, I suggest that left-right axial information is contained in the extracellular matrix early in development and is independently transmitted to cardiac and visceral primordia.     

Retinoic acid coordinates somitogenesis and left-right patterning in vertebrate embryos

A striking feature of the body plan of a majority of animals is bilateral symmetry. Almost nothing is known about the mechanisms controlling the symmetrical arrangement of the left and right body sides during development. Here we report that blocking the production of retinoic acid (RA) in chicken embryos leads to a desynchronization of somite formation between the two embryonic sides, demonstrated by a shortened left segmented region. This defect is linked to a loss of coordination of the segmentation clock oscillations. The lateralization of this defect led us to investigate the relation between somitogenesis and the left-right asymmetry machinery in RA-deficient embryos. Reversal of the situs in chick or mouse embryos lacking RA results in a reversal of the somitogenesis laterality defect. Our data indicate that RA is important in buffering the lateralizing influence of the left-right machinery, thus permitting synchronization of the development of the two embryonic sides.     

Determination of left-right patterning of the mouse embryo by artificial nodal flow

Substantial insight has recently been achieved into the mechanisms responsible for the generation of left-right (L-R) asymmetry in the vertebrate body plan. However, the mechanism that underlies the initial breaking of symmetry has remained unclear. In the mouse, a leftward fluid flow on the ventral side of the node caused by the vortical motion of cilia (referred to as nodal flow) is implicated in symmetry breaking, but direct evidence for the role of this flow has been lacking. Here we describe the development of a system in which mouse embryos are cultured under an artificial fluid flow and with which we have examined how flow affects L-R patterning. An artificial rightward flow that was sufficiently rapid to reverse the intrinsic leftward nodal flow resulted in reversal of situs in wild-type embryos. The artificial flow was also able to direct the situs of mutant mouse embryos with immotile cilia. These results provide the first direct evidence for the role of mechanical fluid flow in L-R patterning.     

Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis

It has been suggested that the failure of parthenogenetic mouse embryos to develop to term is primarily due to their aberrant cytoplasm and homozygosity leading to the expression of recessive lethal genes. The reported birth of homozygous gynogenetic (male pronucleus removed from egg after fertilization) mice and of animals following transplantation of nuclei from parthenogenetic embryos to enucleated fertilized eggs, is indicative of abnormal cytoplasm and not an abnormal genotype of the activated eggs. However, we and others have been unable to obtain such homozygous mice. We investigated this problem further by using reconstituted heterozygous eggs, with haploid parthenogenetic eggs as recipients for a male or female pronucleus. We report here that the eggs which receive a male pronucleus develop to term but those with two female pronuclei develop only poorly after implantation. Therefore, the cytoplasm of activated eggs is fully competent to support development to term but not if the genome is entirely of maternal origin. We propose that specific imprinting of the genome occurs during gametogenesis so that the presence of both a male and a female pronucleus is essential in an egg for full-term development. The paternal imprinting of the genome appears necessary for the normal development of the extraembryonic membranes and the trophoblast.     

Role for sperm in spatial patterning of the early mouse embryo

Despite an apparent lack of determinants that specify cell fate, spatial patterning of the mouse embryo is evident early in development. The axis of the post-implantation egg cylinder can be traced back to organization of the pre-implantation blastocyst. This in turn reflects the organization of the cleavage-stage embryo and the animal-vegetal axis of the zygote. These findings suggest that the cleavage pattern of normal development may be involved in specifying the future embryonic axis; however, how and when this pattern becomes established is unclear. In many animal eggs, the sperm entry position provides a cue for embryonic patterning, but until now no such role has been found in mammals. Here we show that the sperm entry position predicts the plane of initial cleavage of the mouse egg and can define embryonic and abembryonic halves of the future blastocyst. In addition, the cell inheriting the sperm entry position acquires a division advantage and tends to cleave ahead of its sister. As cell identity reflects the timing of the early cleavages, these events together shape the blastocyst whose organization will become translated into axial patterning after implantation. We present a model for axial development that accommodates these findings with the regulative nature of mouse embryos.     

The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry.

In the chick embryo, left-right asymmetric patterns of gene expression in the lateral plate mesoderm are initiated by signals located in and around Hensen's node. Here we show that Caronte (Car), a secreted protein encoded by a member of the Cerberus/Dan gene family, mediates the Sonic hedgehog (Shh)-dependent induction of left-specific genes in the lateral plate mesoderm. Car is induced by Shh and repressed by fibroblast growth factor-8 (FGF-8). Car activates the expression of Nodal by antagonizing a repressive activity of bone morphogenic proteins (BMPs). Our results define a complex network of antagonistic molecular interactions between Activin, FGF-8, Lefty-1, Nodal, BMPs and Car that cooperate to control left-right asymmetry in the chick embryo.     

Tbx6-dependent Sox2 regulation determines neural or mesodermal fate in axial stem cells

The classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. However, recent cell-lineage-tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. Here we demonstrate that Tbx6-dependent regulation of Sox2 determines the fate of axial stem cells. In wild-type embryos, enhancer N1 of the neural primordial gene Sox2 is activated in the caudal lateral epiblast, and the cells staying in the superficial layer sustain N1 activity and activate Sox2 expression in the neural plate. In contrast, the cells destined to become mesoderm activate Tbx6 and turn off enhancer N1 before migrating into the paraxial mesoderm compartment. In Tbx6 mutant embryos, however, enhancer N1 activity persists in the paraxial mesoderm compartment, eliciting ectopic Sox2 activation and transforming the paraxial mesoderm into neural tubes. An enhancer-N1-specific deletion mutation introduced into Tbx6 mutant embryos prevented this Sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that Tbx6 regulates Sox2 via enhancer N1. Tbx6-dependent repression of Wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. Paraxial mesoderm-specific misexpression of a Sox2 transgene in wild-type embryos resulted in ectopic neural tube development. Thus, Tbx6 represses Sox2 by inactivating enhancer N1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells.     

Calcium-releasing activity induced by nuclei of mouse fertilized early embryos

At fertilization,repectitive transient rises of intracellular calcium concentration occur in all mammals studied so far .It has been shown that calcium rises could be induced when mouse fertilized 1-,2-cell nuclei were trans-planted into unfertilized eggs and that the reconstituted embryo could be activated .Howerver,whecther the capability of inducing calcium rises occurs in all stages of mammalian embryos remains unknown ,In this study ,by using the nuclear transplantation technique and measurement of intracellular calcium rises in living cells,we showed that only the nuclei from mouse fertilized 1-cell and 2-cell embryos ,neither the nuclei from 4-,8-cell and ethanol activated parthe-nogenetic embryos nor 2 or 3 nuclei of electrofused 4-cell stage syncytium ,have calcium -releasing activity when they were transferred into unfertilized mature oocytes,Our results indicate that the calcium-releasing activity in nuclei of 1-,2-cell embryos is produced during fertilization and exists at the special stage of fertilized early embryos.These sug-gested that the capacity of inducting calcium release activity in fertilized early embryos is important for normal embryonic development.     

Generation of mutants with developmental defects in zebrafish by ENU mutagenesis

As a good model for studying early development of vertebrates, zebrafish (Danio rerio) is attracting more and more attention. Following ENU mutagenesis, 320 F2 families were established. Mutants, which showed defects in epiboly, axis, somite, head, and cardiac and blood systems, were identified by observing morphological changes in F3 embryos. So far, 35 mutant lines have been established, the majority of which showed anomalies in axis and somite formation. These mutant lines provide useful genetic resources for cloning of the mutant genes and for studying mechanisms of early development of vertebrate embryos.     

Large-scale screening of disease model through ENU mutagenesis in mice

Manipulation of mouse genome has merged as one of the most important approaches for studying gene function and establishing the disease model because of the high homology between human genome and mouse genome.In this study, the chemical mutagen ethylnitrosourea (ENU) was employed for inducing germ cell mutations in male C57BL/6J mice. The first generation (G1) of the backcross of these mutated mice, totally 3172, was screened for abnormal phenotypes on gross morphology, behavior, learning and memory, auditory brainstem response (ABR), electrocardiogram (ECG), electroretinogram (ERG), flash-visual evoked potential (F-VEP), bone mineral density, and blood sugar level. 595 mice have been identified with specific dominant abnormalities. Fur color changes, eye defects and hearing loss occurred at the highest frequency. Abnormalities related to metabolism alteration are least frequent. Interestingly, eye defects displayed significant left-right asymmetry and sex preference. Sex preference is also observed in mice with abnormal bone mineral density. Among 104 G1 generation mutant mice examined for inheritability, 14 of them have been confirmed for passing abnormal phenotypes to their progenies. However, we did not observe behavior abnormalities of G1 mice to be inheritable, suggesting multi-gene control for these complicated functions in mice. In conclusion,the generation of these mutants paves the way for understanding molecular and cellular mechanisms of these abnormal phenotypes, and accelerates the cloning of diseaserelated genes.     

Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation

Vertebrate gastrulation involves the specification and coordinated movement of large populations of cells that give rise to the ectodermal, mesodermal and endodermal germ layers. Although many of the genes involved in the specification of cell identity during this process have been identified, little is known of the genes that coordinate cell movement. Here we show that the zebrafish silberblick (slb) locus encodes Wnt11 and that Slb/Wnt11 activity is required for cells to undergo correct convergent extension movements during gastrulation. In the absence of Slb/Wnt11 function, abnormal extension of axial tissue results in cyclopia and other midline defects in the head. The requirement for Slb/Wnt11 is cell non-autonomous, and our results indicate that the correct extension of axial tissue is at least partly dependent on medio-lateral cell intercalation in paraxial tissue. We also show that the slb phenotype is rescued by a truncated form of Dishevelled that does not signal through the canonical Wnt pathway, suggesting that, as in flies, Wnt signalling might mediate morphogenetic events through a divergent signal transduction cascade. Our results provide genetic and experimental evidence that Wnt activity in lateral tissues has a crucial role in driving the convergent extension movements underlying vertebrate gastrulation.     

Effects of different nuclear recipients on developmental potential of mouse somatic nuclear transfer embryos

Somatic cell nuclear transfer has been succeeded in procedures of nuclear transfer. One is single nucleartransfer, the other is serial nuclear transfer. Viable animals have been cloned in different species using both me-thods[1—6]. Different nuclear recipients and donors wereused in serial nuclear transfer, namely, transferring thenuclear of reconstructed embryo into enucleated MⅡoocytes[7], transferring the nuclear of reconstructed em-bryos at one cell stage into enucleated zygote[4] and t…     

微注射人TCP10L基因对斑马鱼形态的影响

利用模式生物斑马鱼,通过微注射的方法,研究了人肝脏特异性转录因子TCP10L对斑马鱼早期胚胎发育的影响.结果显示:人TCP10L基因对斑马鱼胚胎发育有一定的毒性.表现为早期的脊索发育异常,个体严重畸形;当血液循环出现后,表现为血流延缓和围心腔水肿等症状.该项研究表明:人肝脏特异性转录因子TCP10L在斑马鱼胚胎中的过表达会引起显著的形态异常和心血管障碍,并且这种影响与其是否在斑马鱼肝内的特异表达有关,为进一步探明该转录因子在人肝脏内发挥的功能奠定了基础.