Christoph Viebahn

Cell Movements at Hensen’s Node Establish Left/Right Asymmetric Gene Expression in the Chick

Migration and Asymmetry Although vertebrates show asymmetry in internal body organization, the earliest steps toward establishing different anatomies on the left and right sides are not conserved. How this is achieved in birds has been especially confusing. Gros et al. (p. 941, published online 9 April) show that in chicks some of the earliest left-right asymmetric domains of gene expression, including those of Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8), are produced passively. Genes are activated in bilateral cell populations, followed by rearrangements that shuffle Shh-expressing cells. Asymmetric gene expression is passively set up in the early chick embryo by cell rearrangements. In vertebrates, the readily apparent left/right (L/R) anatomical asymmetries of the internal organs can be traced to molecular events initiated at or near the time of gastrulation. However, the earliest steps of this process do not seem to be universally conserved. In particular, how this axis is first defined in chicks has remained problematic. Here we show that asymmetric cell rearrangements take place within chick embryos, creating a leftward movement of cells around the node. It is the relative displacement of cells expressing sonic hedgehog (Shh) and fibroblast growth factor 8 (Fgf8) that is responsible for establishing their asymmetric expression patterns. The creation of asymmetric expression domains as a passive effect of cell movements represents an alternative strategy for breaking L/R symmetry in gene activity.

Rabbit as a reproductive model for human health

The renaissance of the laboratory rabbit as a reproductive model for human health is closely related to the growing evidence of periconceptional metabolic programming and its determining effects on offspring and adult health. Advantages of rabbit reproduction are the exact timing of fertilization and pregnancy stages, high cell numbers and yield in blastocysts, relatively late implantation at a time when gastrulation is already proceeding, detailed morphologic and molecular knowledge on gastrulation stages, and a hemochorial placenta structured similarly to the human placenta. To understand, for example, the mechanisms of periconceptional programming and its effects on metabolic health in adulthood, these advantages help to elucidate even subtle changes in metabolism and development during the pre- and peri-implantation period and during gastrulation in individual embryos. Gastrulation represents a central turning point in ontogenesis in which a limited number of cells program the development of the three germ layers and, hence, the embryo proper. Newly developed transgenic and molecular tools offer promising chances for further scientific progress to be attained with this reproductive model species.

Blastocyst elongation, trophoblastic differentiation, and embryonic pattern formation

The molecular basis of ungulate and non-rodent conceptus elongation and gastrulation remains poorly understood; however, use of state-of-the-art genomic technologies is beginning to elucidate the mechanisms regulating these complicated processes. For instance, transcriptome analysis of elongating porcine concepti indicates that protein synthesis and trafficking, cell growth and proliferation, and cellular morphology are major regulated processes. Furthermore, potential autocrine roles of estrogen and interleukin-1-beta in regulating porcine conceptus growth and remodeling and metabolism have become evident. The importance of estrogen in pig is emphasized by the altered expression of essential steroidogenic and trophoblast factors in lagging ovoid concepti. In ruminants, the characteristic mononucleate trophoblast cells differentiate into a second lineage important for implantation, the binucleate trophoblast, and transcriptome profiling of bovine concepti has revealed a gene cluster associated with rapid trophoblast proliferation and differentiation. Gene cluster analysis has also provided evidence of correlated spatiotemporal expression and emphasized the significance of the bovine trophoblast cell lineage and the regulatory mechanism of trophoblast function. As a part of the gastrulation process in the mammalian conceptus, specification of the germ layers and hence definitive body axes occur in advance of primitive streak formation. Processing of the transforming growth factor-beta-signaling molecules nodal and BMP4 by specific proteases is emerging as a decisive step in the initial patterning of the pre-gastrulation embryo. The topography of expression of these and other secreted molecules with reference to embryonic and extraembryonic tissues determines their local interaction potential. Their ensuing signaling leads to the specification of axial epiblast and hypoblast compartments through cellular migration and differentiation and, in particular, the specification of the early germ layer tissues in the epiblast via gene expression characteristic of endoderm and mesoderm precursor cells.

Epithelio-Mesenchymal Transformation during Formation of the Mesoderm in the Mammalian Embryo

The earliest example of epithelio-mesenchymal transformation during embryonic development is the generation of the third germ layer, the mesoderm, from the epiblast (or primitive ectoderm), which marks the beginning of gastrulation. Although it has been regarded as most likely that the principles of this transformation in invertebrates and lower vertebrates also apply to amniotes, morphological and molecular details of mesoderm formation in birds and, in particular, in mammals, which may support this assumption, have only recently been clarified. This chapter thus brings together the light- and electron-microscopical morphology of epithelio-mesenchymal transformation during initial mesoderm formation in the mammalian embryo. Cellular differentiation during this process with regard to the cytoskeleton, cell adhesion molecules and the extracellular matrix are also covered as are cell kinetic studies and the candidate growth factors and genes most likely to be involved in the regulation of mesoderm formation in mammals. Finally, a model is presented which summarizes these morphological and molecular changes and which links the promoting and inhibiting influences of regulatory factors to some of the changes observed during epithelio-mesenchymal transformation.

The mouse homeobox gene Noto regulates node morphogenesis, notochordal ciliogenesis, and left–right patterning

The mouse homeobox gene Noto represents the homologue of zebrafish floating head (flh) and is expressed in the organizer node and in the nascent notochord. Previous analyses suggested that Noto is required exclusively for the formation of the caudal part of the notochord. Here, we show that Noto is also essential for node morphogenesis, controlling ciliogenesis in the posterior notochord, and the establishment of laterality, whereas organizer functions in anterior–posterior patterning are apparently not compromised. In mutant embryos, left–right asymmetry of internal organs and expression of laterality markers was randomized. Mutant posterior notochord regions were variable in size and shape, cilia were shortened with highly irregular axonemal microtubuli, and basal bodies were, in part, located abnormally deep in the cytoplasm. The transcription factor Foxj1, which regulates the dynein gene Dnahc11 and is required for the correct anchoring of basal bodies in lung epithelial cells, was down-regulated in mutant nodes. Likewise, the transcription factor Rfx3, which regulates cilia growth, was not expressed in Noto mutants, and various other genes important for cilia function or assembly such as Dnahc5 and Nphp3 were down-regulated. Our results establish Noto as an essential regulator of node morphogenesis and ciliogenesis in the posterior notochord, and suggest Noto acts upstream of Foxj1 and Rfx3.

Keratin and vimentin expression in early organogenesis of the rabbit embryo

SummaryThe expression of vimentin and keratins is analysed in the early postimplantation embryo of the rabbit at 11 days post conceptionem (d.p.c.) using a panel of monoclonal antibodies specific for single intermediate filament polypeptides (keratins 7, 8, 18, 19 and vimentin) and a “pan-epithelial” monoclonal keratin antibody. Electrophoretic separation of cytoskeletal preparations obtained from embryonic tissues, in combination with immunoblotting of the resulting polypeptide bands, demonstrates the presence of the rabbit equivalents of human keratins 8, 18, and vimentin in 11-day-old rabbit embryonic tissues. Immunohistochemical staining shows that several embryonic epithelia such as notochord, surface ectoderm, primitive intestinal tube, and mesonephric duct, express keratins, while others (neural tube, dermomyotome) express vimentin, and a third group (coelomic epithelia) can express both. Similarly, of the mesenchymal tissues sclerotomal mesenchyme expresses vimentin, while somatopleuric mesenchyme (abdominal wall) expresses keratins, and splanchnopleuric mesenchyme (dorsal mesentery) expresses both keratins and vimentin. While these results are in accordance with most results of keratin and vimentin expression in embryos of other species, they stand against the common concept of keratin and vimentin specificity in adult vertebrate tissues. Furthermore, keratin and vimentin are not expressed in accordance with germ layer origin of tissues in the mammalian embryo; rather the expression of these proteins seems to be related to cellular function during embryonic development.

FGF8 Acts as a Right Determinant during Establishment of the Left-Right Axis in the Rabbit

BACKGROUND FGF8 has been implicated in the transfer of left-right (L-R) asymmetry from the embryonic midline (node) to the lateral plate mesoderm (LPM). Surprisingly, opposite roles have been described in chick and mouse. In mouse, FGF8 is required for the left-asymmetric expression of nodal, lefty2, and Pitx2. In chick, FGF8 represses nodal and Pitx2 on the right side. This discrepancy could reflect evolutionary differences between birds and mammals. Alternatively, the right-asymmetric expression of fgf8, which is not found in mouse, at the chick node may be a prerequisite of right-sided function. Finally, chick (blastodisc) and mouse (egg cylinder) differ with respect to the topology of the early gastrula/neurula embryo. RESULTS The rabbit blastodisc was investigated as an additional mammalian L-R model system. While nodal, lefty, and Pitx2 showed asymmetric expression in the left LPM, fgf8 and all other midline marker genes were symmetrically expressed at the node like in mouse. Left-sided application of FGF8 repressed the endogenous transcription of nodal as well as ectopic expression induced by the parallel administration of BMP4. Right-sided inhibition of FGF8 signaling induced bilateral marker gene expression, demonstrating that, in rabbit, FGF8 acts as a right determinant like in chick. CONCLUSIONS These findings suggest that the anatomy of the early embryo (blastodisc versus egg cylinder) rather than taxonomical differences or asymmetry in expression constitutes an important determinant of FGF8 function in L-R axis formation. The rabbit may provide a useful model for early human embryogenesis, as human embryos develop via a blastodisc as well.

Brachyury expression predicts poor prognosis at early stages of colorectal cancer

Although survival rates of colon cancer patients diagnosed at an early stage (T1-2N0M0; Dukes A) vary considerably according to the studies cited, several studies indicate development of distant metastases already occurring in a considerable percentage of these patients leading to the death of the patients. This particular high risk group cannot be identified properly as no marker exists to identify these patients. As the Wnt/Win pathway plays a crucial role in metastasis formation in colorectal carcinoma, we analysed whether the transcription factor brachyury critically involved in this pathway may predict metastasis formation in these patients. The expression of brachyury-homologous (T) was immunohistochemically analysed in 748 patients and the data were correlated with classical and newer prognostic markers in colorectal cancer. Early stages colorectal cancer patients (T1-2N0M0, Dukes A) showed a significantly decreased survival when brachyury was expressed in the tumour tissue while no correlation was observed in later tumour stages. Hence a subset of colorectal cancers exists in which the ability to metastasise is already present at early stages of tumour growth and this high risk group can now be detected by immunohistochemistry.

Epithelial-mesenchymal transition in colonies of rhesus monkey embryonic stem cells : A model for processes involved in gastrulation

Rhesus monkey embryonic stem (rhES) cells were grown on mouse embryonic fibroblast (MEF) feeder layers for up to 10 days to form multilayered colonies. Within this period, stem cell colonies differentiated transiently into complex structures with a disc‐like morphology. These complex colonies were characterized by morphology, immunohistochemistry, and marker mRNA expression to identify processes of epithelialization as well as epithelial–mesenchymal transition (EMT) and pattern formation. Typically, differentiated colonies were comprised of an upper and a lower ES cell layer, the former growing on top of the layer of MEF cells whereas the lower ES cell layer spread out underneath the MEF cells. Interestingly, in the central part of the colonies, a roundish pit developed. Here the feeder layer disappeared, and upper layer cells seemed to ingress and migrate through the pit downward to form the lower layer while undergoing a transition from the epithelial to the mesenchymal phenotype, which was indicated by the loss of the marker proteins E‐cadherin and ZO‐1 in the lower layer. In support of this, we found a concomitant 10‐fold upregulation of the gene Snail2, which is a key regulator of the EMT process. Conversion of epiblast to mesoderm was also indicated by the regulated expression of the mesoderm marker Brachyury. An EMT is a characteristic process of vertebrate gastrulation. Thus, these rhES cell colonies may be an interesting model for studies on some basic processes involved in early primate embryogenesis and may open new ways to study the regulation of EMT in vitro.

Signs of the principle body axes prior to primitive streak formation in the rabbit embryo

An early common element during anterior-posterior axis formation amongst amniotes is the primitive streak, running longitudinally in the two-layered embryonic disc. In mammals the primordium of this transient structure is the first definite morphological sign of the anterior-posterior axis, while in avian embryos the axis is visible and apparently defined earlier. Here we scrutinize suggestions that in mammals also there are earlier signs of axis formation by using correlative low and high-resolution light microscopy on tissues from rabbit embryos at 6.3 and 6.5 days post-conception, i.e. immediately before and after primitive streak formation. A series of semithin sections were cut from resin-embedded embryonic discs that had been photographed previously at low power. In embryos at 6.5-days post-conception the primitive streak is as long as up to half the diameter of the embryonic disc, extending anteriorly from a thickening, here called the posterior node, at the posterior margin, which contains the first mesoderm cells ingressing from the epiblast. On both sides of the primitive streak there is a triangular area that appears light in surface views of fixed embryos and correlates with stretches of low-columnar simple epithelium in an otherwise high-columnar pseudostratified epiblast. Within the anterior margin, which has a sharper contour than the rest of the circumference of the embryonic disc, there is a narrow, crescent-shaped dark zone caused by increased cellular height and number in both epiblast and hypoblast. These characteristics of the anterior margin are also found at 6.3 days post-conception, at which stage there is no sign of a primitive streak or a posterior node. The posterior margin, in contrast, is ill-defined in these earlier embryos, or there is a light crescent within the posterior margin, which has the same histological characteristics as the bilateral posterior triangular areas of primitive streak stages. Because the anterior differentiation occurs prior to primitive streak formation and is a sign of both the anterior-posterior and the transverse axes of the embryonic disc, and because some of its histological characteristics are found in primate and human embryos, we propose to name this structure the ‘anterior marginal crescent’ and to add it to the list of transient structures that gradually establish the principal body axes in mammals. The anterior manifestation of body axes in mammals is thus essentially different from axis development in the avian embryo, where differentiation of these axes is first manifest at the posterior margin.