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.     

Anterior visceral endoderm SMAD4 signaling specifies anterior embryonic patterning and head induction in mice

SMAD4 serves as a common mediator for signaling of TGF-β superfamily. Previous studies illustrated that SMAD4-null mice die at embryonic day 6.5 (E6.5) due to failure of mesoderm induction and extraembryonic defects; however, functions of SMAD4 in each germ layer remain elusive. To investigate this, we disrupted SMAD4 in the visceral endoderm and epiblast, respectively, using a Cre-loxP mediated approach. We showed that mutant embryos lack of SMAD4 in the visceral endoderm (Smad4(Co/Co);TTR-Cre) died at E7.5-E9.5 without head-fold and anterior embryonic structures. We demonstrated that TGF-β regulates expression of several genes, such as Hex1, Cer1, and Lim1, in the anterior visceral endoderm (AVE), and the failure of anterior embryonic development in Smad4(Co/Co);TTR-Cre embryos is accompanied by diminished expression of these genes. Consistent with this finding, SMAD4-deficient embryoid bodies showed impaired responsiveness to TGF-β-induced gene expression and morphological changes. On the other hand, embryos carrying Cre-loxP mediated disruption of SMAD4 in the epiblasts exhibited relatively normal mesoderm and head-fold induction although they all displayed profound patterning defects in the later stages of gastrulation. Cumulatively, our data indicate that SMAD4 signaling in the epiblasts is dispensable for mesoderm induction although it remains critical for head patterning, which is significantly different from SMAD4 signaling in the AVE, where it specifies anterior embryonic patterning and head induction.     

Regionalised signalling within the extraembryonic ectoderm regulates anterior visceral endoderm positioning in the mouse embryo

The development of the anterior-posterior (AP) axis in the mammalian embryo is controlled by interactions between embryonic and extraembryonic tissues. It is well established that one of these extraembryonic tissues, the anterior visceral endoderm (AVE), can repress posterior cell fate and that signalling from the other, the extraembryonic ectoderm (ExE), is required for posterior patterning. Here, we show that signals from the prospective posterior ExE repress AVE gene expression and affect the distribution of the AVE cells. Surgical ablation of the prospective posterior, but not the anterior, extraembryonic region at 5.5 days of development (E5.5) perturbs the characteristic distal-to-anterior distribution of AVE cells and leads to a dramatic expansion of the AVE domain. Time-lapse imaging studies show that this increase is due to the ectopic expression of an AVE marker, which results in a symmetrical positioning of the AVE. Surgical ablation of this same ExE region after the distal-to-anterior migration has already commenced, at E5.75, does not affect the localisation of the AVE, indicating that this effect takes place within a short time window. Conversely, transplanting the prospective posterior, but not the anterior, extraembryonic region onto isolated E5.5 embryonic explants drastically reduces the AVE domain. Further, transplantation experiments demonstrate that the signalling regulating AVE gene expression originates from the posterior ExE, rather than its surrounding VE. Together, our results show that signals emanating from the future posterior ExE within a temporal window both restrict the AVE domain and promote its specific positioning. This indicates for the first time that the ExE is already regionalised a day before the onset of gastrulation in order to correctly set the orientation of the AP axis of the mouse embryo. We propose a reciprocal function of the posterior ExE and the AVE in establishing a balance between the antagonistic activities of these two tissues, essential for AP patterning.     

The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation

Initiation of the development of the anterior-posterior axis in the mouse embryo has been thought to take place only when the anterior visceral endoderm (AVE) emerges and starts its asymmetric migration. However, expression of Lefty1, a marker of the AVE, was recently found to initiate before embryo implantation. This finding has raised two important questions: are the cells that show such early, preimplantation expression of this AVE marker the real precursors of the AVE and, if so, how does this contribute to the establishment of the AVE? Here, we address both of these questions. First, we show that the expression of another AVE marker, Cer1, also commences before implantation and its expression becomes consolidated in the subset of ICM cells that comprise the primitive endoderm. Second, to determine whether the cells showing this early Cer1 expression are true precursors of the AVE, we set up conditions to trace these cells in time-lapse studies from early periimplantation stages until the AVE emerges and becomes asymmetrically displaced. We found that Cer1-expressing cells are asymmetrically located after implantation and, as the embryo grows, they become dispersed into two or three clusters. The expression of Cer1 in the proximal domain is progressively diminished, whilst it is reinforced in the distal-lateral domain. Our time-lapse studies demonstrate that this distal-lateral domain is incorporated into the AVE together with cells in which Cer1 expression begins only after implantation. Thus, the AVE is formed from both part of an ancestral population of Cerl-expressing cells and cells that acquire Cer1 expression later. Finally, we demonstrate that when the AVE shifts asymmetrically to establish the anterior pole, this occurs towards the region where the earlier postimplantation expression of Cer1 was strongest. Together, these results suggest that the orientation of the anterior-posterior axis is already anticipated before AVE migration.     

"Fingering" the vertebrate limb

A detailed and precise picture is being pieced together about how the pattern of digits develops in vertebrate limbs. What is particularly exciting is that it will soon be possible to trace the process all the way from establishment of a signalling centre in a small bud of undifferentiated cells right through to final limb anatomy. The development of the vertebrate limb is a traditional model in which to explore mechanisms involved in pattern formation, and there is accelerating knowledge about the genes involved. One reason why the limb is holding its place in the post-genomic age is that it is rich in pre-genomic embryology. Here, we will focus on recent findings about the aspect of vertebrate limb development concerned with digit pattern across the anteroposterior axis of the limb. This process is controlled by a signalling region in the early limb bud known as the polarizing region. Interactions between polarizing region cells and other cells in the limb bud ensure that a thumb develops at one edge of the hand (anterior) and a little finger at the other (posterior).     

猫扣带回前部内脏伤害感受神经元的膜电学特性

目的：研究猫扣带回前部内脏大神经刺激相关神经元的膜电生理特性,以便从神经元水平进一步了解大脑皮质内脏伤害感受的特性及机制,为痛觉理论“特异性学说”提供新的实验依据。方法：应用在体玻璃微电极细胞内电位记录技术及细胞内注入极化电流的方法,测量和计算神经元的膜电学参数。结果：将20只猫扣带回前部176个内脏大神经刺激相关神经元,分为内脏伤害(148个)和非伤害(28个)感受神经元。发现它们在膜电阻、时间常数、膜电容及I—V曲线等方面存在差异。注入去极化电流引发的放电幅值及频率也存在差异。结论：扣带回前部内脏伤害与非伤害感受神经元可能在细胞膜结构、细胞大小等形态学方面存有差别。     

猫扣带回前部内脏伤害感受神经元的诱发反应

应用玻璃微电极细胞内电位记录技术,观察了２０史猫扣带回前部４６１个神经元对电刺激对侧内脏大神经的诱发反应及其电生理特性,在被观察的神经元中,１７６个为刺激相关神经元。根据诱发反应的特性,将其分为特异性内脏伤害感受神经元（１１４个,６４．７７％）、非特异性内脏伤害感受神经元（３４个,１９．３２％）及非内脏伤害感受神经元（２８个,１５．９１％）。诱发反应分为兴奋性（５９．４６％）、抑制性（２２．３０％     

Phosphorylated CaMKII post-synaptic binding to NR2B subunits in the anterior cingulate cortex mediates visceral pain in visceral hypersensitive rats

The NR2B subunit of NMDA receptor in the anterior cingulate cortex (ACC) is up-regulated in viscerally hypersensitive (VH) rats induced by colonic anaphylaxis. It plays a critical role in modulation of ACC sensitization and visceral pain responses. Given the key role of calcium/calmodulin-dependent protein kinase II (CaMKII) in synaptic plasticity and behavior learning and memory, we hypothesize that phosphorylation of CaMKII binding to NR2B mediates visceral pain in VH states. We performed in vivo electroporation of CaMKII siRNA produced inhibition of colorectal distension-induced visceromotor response in the VH rats. The NR2B, CaMKII and P-CaMKII-Thr2?? protein levels were increased in 180%, 220% and 304% fold in the post-synaptic density (PSD) fraction in VH rats separately. Western blotting following co-immunoprecipitation showed that P-CaMKII-Thr2?? bound to NR2B in the PSD, which was increased to 267% of control in VH rats. Administration of CaMKII antagonist Antennapedia-CaMKIINtide suppressed visceromotor response in VH rats in parallel with decrease of NR2B levels and reduction of the NR2B-P-CaMKII-Thr2?? protein complex in PSD. In conclusion, CaMKII is a critical signaling molecule in the ACC glutamatergic synaptic transmission and phosphorylation of CaMKII at Thr286, which binds to NR2B subunit at post-synaptic site, modulates visceral pain in viscerally hypersensitive state.     

Dynamic morphogenetic events characterize the mouse visceral endoderm

Several lines of evidence suggest that the extraembryonic endoderm of vertebrate embryos plays an important role in the development of rostral neural structures. In mice, neural inductive signals are thought to reside in an area of visceral endoderm that expresses the Hex gene. Here, we have conducted a morphological and lineage analysis of visceral endoderm cells spanning pre- and postprimitive streak stages. Our results show that Hex-expressing cells have a tall, columnar epithelial morphology, which distinguishes them from other visceral endoderm cells. This region of visceral endoderm thickening (VET) is found overlying first the distal and then one side of the epiblast at stages between 5.5 and 5.75 days post coitum (d.p.c.). In addition, we show that the epiblast has an anteroposterior-compressed appearance that is aligned with the position of the VET. Intracellular labeling of VET/Hex-expressing cells reveals an anterior and anterolateral shift from their distal epiblast position. VET/Hex-expressing cells are first localized to the anterior side of the epiblast by 5.75 d.p.c. and form a crescent on the anterior half of the embryo at the onset of gastrulation. Subsequently, VET descendants are distributed along the embryonic/extraembryonic boundary by headfold stages at 7.5 d.p.c. The morphological characteristics and position of VET/Hex-expressing cells distinguishes the future anteroposterior axis of the embryo and provide landmarks to stage mouse embryos at preprimitive streak stages. Moreover, the morphological characteristics of pregastrulation mouse embryos together with the stereotyped shift in the position of visceral endoderm cells reveal similarities among amniote embryos that suggest an evolutionary conservation of the mechanisms that pattern the rostral neurectoderm at pregastrula stages.     

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.     

Requirement for tumor suppressor Apc in the morphogenesis of anterior and ventral mouse embryo

Tumor suppressor Apc (adenomatous polyposis coli) is implicated in the Wnt signaling pathway that is involved in the early embryonic development and tumorigenesis in vertebrates. While the heterozygous null mutant mice develop intestinal polyps, the homozygous embryos die before gastrulation. To investigate the role of Apc in later embryonic development, we constructed a novel hypomorphic Apc allele whose expression was attenuated by approximately 80%. In the hypomorphic Apc homozygous ES cells, reduction in Apc expression caused beta-catenin accumulation and Wnt signaling activation. The homozygous mutant mouse embryos survived 3 days longer than the null mutant embryos. Interestingly, they showed anterior truncation, partial axis duplication, and defective ventral morphogenesis. To determine the tissues where Apc functions for anterior and ventral morphogenesis, we constructed chimeric embryos whose epiblast was derived predominantly from the Apc hypomorphic homozygous cells but the visceral endoderm was from the wild type. Although these chimeric embryos still showed some anterior defects, their ventral morphogenesis was rescued. In addition, marker studies indicated that the axial mesendoderm was also defective in the homozygous embryos. Our results provide genetic evidence that expression of Apc at the normal level is essential for both anterior and ventral development, in the epiblast derivatives and visceral endoderm.     

The anterior tail chamber and survival of Gorgoderina vitelliloba

Mitchell J. B., Mason A. R. and Whalley A. J. S. 1980. The anterior tail chamber and survival of Gorgoderina vitelliloba. International Journal for Parasitology10: 181–182. Survival in pond water of intact cystocercous cercariae of Gorgoderina vitelliloba was significantly better than that of isolated cercarial bodies, although the latter were able to survive in Ringer's Solution. Survival of cercarial bodies in water was improved somewhat when sterility was maintained. It is suggested that the wall of the anterior tail chamber protects the body from the osmotic stress imposed by life in fresh water.     

Differential migration and proliferation of geometrical ensembles of cell clusters

Differential cell migration and growth drives the organization of specific tissue forms and plays a critical role in embryonic development, tissue morphogenesis, and tumor invasion. Localized gradients of soluble factors and extracellular matrix have been shown to modulate cell migration and proliferation. Here we show that in addition to these factors, initial tissue geometry can feedback to generate differential proliferation, cell polarity, and migration patterns. We apply layer by layer polyelectrolyte assembly to confine multicellular organization and subsequently release cells to demonstrate the spatial patterns of cell migration and growth. The cell shapes, spreading areas, and cell–cell contacts are influenced strongly by the confining geometry. Cells within geometric ensembles are morphologically polarized. Symmetry breaking was observed for cells on the circular pattern and cells migrate toward the corners and in the direction parallel to the longest dimension of the geometric shapes. This migration pattern is disrupted when actomyosin based tension was inhibited. Cells near the edge or corner of geometric shapes proliferate while cells within do not. Regions of higher rate of cell migration corresponded to regions of concentrated growth. These findings demonstrate that multicellular organization can result in spatial patterns of migration and proliferation.     

Drosophila female sterile mutation spoonbill interferes with multiple pathways in oogenesis

spoonbill is a Drosophila female-sterile mutation, which displays a range of eggshell and egg chamber patterning defects. Previous analysis has shown that the mutation interfered with the function of two major signaling pathways, GRK/EGFR and DPP. In this report, the nature of spoonbill was further investigated to examine whether it was associated with additional pathways in oogenesis. Clonal analysis, presented here, demonstrated that most of the aberrant phenotypes associated with spoonbill were dependent on a mutant germline. Nevertheless, SPOONBILL may function also in the soma to ensure proper polarization and migration of the border-cell-cluster. Further, genetic interaction studies implicated spoonbill in additional unrelated pathways such as the one(s) involved in actin polymerization/depolymerization. Based on the previous data and the results presented here, it is anticipated that spoonbill may encode a multifunctional protein that perhaps coordinately regulated the activity of multiple signaling pathways during oogenesis.     

The effects of knee joint kinematics on anterior cruciate ligament injury and articular cartilage damage

This study determined which knee joint motions lead to anterior cruciate ligament (ACL) rupture with the knee at 25° of flexion. The knee was subjected to internal and external rotations, as well as varus and valgus motions. A failure locus representing the relationship between these motions and ACL rupture was established using finite element simulations. This study also considered possible concomitant injuries to the tibial articular cartilage prior to ACL injury. The posterolateral bundle of the ACL demonstrated higher rupture susceptibility than the anteromedial bundle. The average varus angular displacement required for ACL failure was 46.6% lower compared to the average valgus angular displacement. Femoral external rotation decreased the frontal plane angle required for ACL failure by 27.5% compared to internal rotation. Tibial articular cartilage damage initiated prior to ACL failure in all valgus simulations. The results from this investigation agreed well with other experimental and analytical investigations. This study provides a greater understanding of the various knee joint motion combinations leading to ACL injury and articular cartilage damage.     

Growth differentiation factor 11 locally controls anterior–posterior patterning of the axial skeleton

Growth and differentiation factor 11 (GDF11) is a transforming growth factor β family member that has been identified as the central player of anterior–posterior (A–P) axial skeletal patterning. Mice homozygous for Gdf11 deletion exhibit severe anterior homeotic transformations of the vertebrae and craniofacial defects. During early embryogenesis, Gdf11 is expressed predominantly in the primitive streak and tail bud regions, where new mesodermal cells arise. On the basis of this expression pattern of Gdf11 and the phenotype of Gdf11 mutant mice, it has been suggested that GDF11 acts to specify positional identity along the A–P axis either by local changes in levels of signaling as development proceeds or by acting as a morphogen. To further investigate the mechanism of action of GDF11 in the vertebral specification, we used a Cdx2-Cre transgene to generate mosaic mice in which Gdf11 expression is removed in posterior regions including the tail bud, but not in anterior regions. The skeletal analysis revealed that these mosaic mice display patterning defects limited to posterior regions where Gdf11 expression is deficient, whereas displaying normal skeletal phenotype in anterior regions where Gdf11 is normally expressed. Specifically, the mosaic mice exhibited seven true ribs, a pattern observed in wild-type (wt) mice (vs. 10 true ribs in Gdf11^−/− mice), in the anterior axis and nine lumbar vertebrae, a pattern observed in Gdf11 null mice (vs. six lumbar vertebrae in wt mice), in the posterior axis. Our findings suggest that GDF11, rather than globally acting as a morphogen secreted from the tail bud, locally regulates axial vertebral patterning.     

The anterior tooth development of cattle presented for slaughter: an analysis of age,sex and breed

In a cross-sectional study, data from records of cattle slaughtered over a 1-year period at a large abattoir in South West England were analysed using an ordered category response model to investigate the inter-relationships between age, sex and breed on development of the permanent anterior (PA) teeth. Using the model, transition points at which there was a 50% probability of membership of each category of paired PA teeth were identified. Data from ∼60 000 animals were initially analysed for age and sex effect. The age transition was found to be ∼23 months moving from zero to two teeth; 30 months for two to four teeth; 37 months for four to six teeth and 42 months for six to eight teeth. Males were found to develop, on average, ∼22 days earlier than females across all stages. A reduced data set of ∼23 000 animals registered as pure-bred only was used to compare breed and type interactions and to investigate sex effects within the sub-categories. Breeds were grouped into dairy and beef-type and beef breeds split into native and continental. It was found that dairy-types moved through the transition points earlier than beef-types across all stages (interval varying between ∼8 and 12 weeks) and that collectively, native beef breeds moved through the transition points by up to 3 weeks earlier than the continental beef breeds. Interestingly, in contrast to beef animals, dairy females matured before dairy males. However, the magnitude of the difference between dairy females and males diminished at the later stages of development. Differences were found between breeds. Across the first three stages, Ayrshires and Guernseys developed between 3 and 6 weeks later than Friesian/Holsteins and Simmental, Limousin and Blonde Aquitaine 6 and 8 weeks later than Aberdeen Angus. Herefords, Charolais and South Devon developed later but by a smaller interval and Red Devon and Galloway showed the largest individual effect with transition delayed by 8 to 12 weeks.