Development of node architecture and emergence of molecular organizer characteristics in the pig embryo.

BACKGROUND: The avian node is the equivalent of the amphibian Spemann's organizer, as indicated by its ability to induce a secondary axis, cellular contribution, and gene expression, whereas the node of the mouse, which displays limited inductive capacities, was suggested to be a part of spatially distributed signaling. Furthermore, the structural identity of the mouse node is subject of controversy, while little is known about equivalent structures in other mammals. RESULTS: We analyzed the node and emerging organizer in the pig using morphology and the expression of selected organizer genes prior to and during gastrulation. The node was defined according to the "four-quarter model" based on comparative consideration. The node of the pig displays a multilayered, dense structure that includes columnar epithelium, bottle-like cells in the dorsal part, and mesenchymal cells ventrally. Expression of goosecoid (gsc), chordin, and brachyury, together with morphology, reveal the consecutive emergence of three distinct domains: the gastrulation precursor domain, the presumptive node, and the mature node. Additionally, gsc displays a ventral expression domain prior to epiblast epithelialization. CONCLUSION: Our study defines the morphological and molecular context of the emerging organizer equivalent in the pig and suggests a sequential development of its function.

The evolutionary history of Brachyury genes in Hydrozoa involves duplications, divergence, and neofunctionalization.

Brachyury, a member of T-box gene family, is widely known for its major role in mesoderm specification in bilaterians. It is also present in non-bilaterian metazoans, such as cnidarians, where it acts as a component of an axial patterning system. In this study, we present a phylogenetic analysis of Brachyury genes within phylum Cnidaria, investigate differential expression and address a functional framework of Brachyury paralogs in hydrozoan Dynamena pumila. Our analysis indicates two duplication events of Brachyury within the cnidarian lineage. The first duplication likely appeared in the medusozoan ancestor, resulting in two copies in medusozoans, while the second duplication arose in the hydrozoan ancestor, resulting in three copies in hydrozoans. Brachyury1 and 2 display a conservative expression pattern marking the oral pole of the body axis in D. pumila. On the contrary, Brachyury3 expression was detected in scattered presumably nerve cells of the D. pumila larva. Pharmacological modulations indicated that Brachyury3 is not under regulation of cWnt signaling in contrast to the other two Brachyury genes. Divergence in expression patterns and regulation suggest neofunctionalization of Brachyury3 in hydrozoans.

Abnormal left-right organizer and laterality defects in Xenopus embryos after formin inhibitor SMIFH2 treatment.

Left-right symmetry breaking in most studied vertebrates makes use of so-called leftward flow, a mechanism which was studied in detail especially in mouse and Xenopus laevis embryos and is based on rotation of monocilia on specialized epithelial surface designated as left-right organizer or laterality coordinator. However, it has been argued that prior to emergence of leftward flow an additional mechanism operates during early cleavage stages in Xenopus embryo which is based on cytoskeletal processes. Evidence in favour of this early mechanism was supported by left-right abnormalities after chemical inhibition of cytoskeletal protein formin. Here we analyzed temporal dimension of this effect in detail and found that reported abnormalities arise only after treatment at gastrula-neurula stages, i.e. just prior to and during the operation of left-right organizer. Moreover, molecular and morphological analysis of the left-right organizer reveals its abnormal development. Our results strongly indicate that left-right abnormalities reported after formin inhibition cannot serve as support of models based on early symmetry breaking event in Xenopus embryo.

Nodal asymmetry and hedgehog signaling during vertebrate left-right symmetry breaking.

Development of visceral left-right asymmetry in bilateria is based on initial symmetry breaking followed by subsequent asymmetric molecular patterning. An important step is the left-sided expression of transcription factor pitx2 which is mediated by asymmetric expression of the nodal morphogen in the left lateral plate mesoderm of vertebrates. Processes leading to emergence of the asymmetric nodal domain differ depending on the mode of symmetry breaking. In Xenopus laevis and mouse embryos, the leftward fluid flow on the ventral surface of the left-right organizer leads through intermediate steps to enhanced activity of the nodal protein on the left side of the organizer and subsequent asymmetric nodal induction in the lateral plate mesoderm. In the chick embryo, asymmetric morphogenesis of axial organs leads to paraxial nodal asymmetry during the late gastrulation stage. Although it was shown that hedgehog signaling is required for initiation of the nodal expression, the mechanism of its asymmetry remains to be clarified. In this study, we established the activation of hedgehog signaling in early chick embryos to further study its role in the initiation of asymmetric nodal expression. Our data reveal that hedgehog signaling is sufficient to induce the nodal expression in competent domains of the chick embryo, while treatment of Xenopus embryos led to moderate nodal inhibition. We discuss the role of symmetry breaking and competence in the initiation of asymmetric gene expression.

Epiblast and trophoblast morphogenesis in the pre-gastrulation blastocyst of the pig. A light- and electron-microscopical study.

The epiblast of the amniote embryo is of paramount importance during early development as it gives rise to all tissues of the embryo proper. In mammals, it emerges through segregation of the hypoblast from the inner cell mass and subsequently undergoes transformation into an epithelial sheet to create the embryonic disc. In rodents and man, the epiblast cell layer is covered by the polar trophoblast which forms the placenta. In mammalian model organisms (rabbit, pig, several non-human primates), however, the placenta is formed by mural trophoblast whereas the polar trophoblast disintegrates prior to gastrulation and thus exposes the epiblast to the microenvironment of the uterine cavity. Both, polar trophoblast disintegration and epiblast epithelialization, thus pose special cell-biological requirements but these are still rather ill-understood when compared to those of gastrulation morphogenesis. This study therefore applied high-resolution light and transmission electron microscopy and three-dimensional (3D) reconstruction to 8- to 10-days-old pig embryos and defines the following steps of epiblast transformation: (1) rosette formation in the center of the ball-shaped epiblast, (2) extracellular cavity formation in the rosette center, (3) epiblast segregation into two subpopulations - addressed here as dorsal and ventral epiblast - separated by a "pro-amniotic" cavity. Ventral epiblast cells form between them a special type of desmosomes with a characteristic dense felt of microfilaments and are destined to generate the definitive epiblast. The dorsal epiblast remains a mass of non-polarized cells and closely associates with the disintegrating polar trophoblast, which shows morphological features of both apoptosis and autophagocytosis. Morphogenesis of the definitive epiblast in the pig may thus exclude a large portion of bona fide epiblast cells from contributing to the embryo proper and establishes contact de novo with the mural trophoblast at the junction between the two newly defined epiblast cell populations.

Initial morphological symmetry breaking in the foregut and development of the omental bursa in human embryos.

Bilaterally symmetrical primordia of visceral organs undergo asymmetrical morphogenesis leading to typical arrangement of visceral organs in the adult. Asymmetrical morphogenesis within the upper abdomen leads, among others, to the formation of the omental bursa dorsally to the rotated stomach. A widespread view of this process assumes kinking of thin mesenteries as a main mechanism. This view is based on a theory proposed already by Johannes Müller in 1830 and was repeatedly criticized, but some of the most plausible alternative views (initially proposed by Swaen in 1897 and Broman in 1904) still remain to be proven. Here, we analyzed serial histological sections of human embryos between stages 12 and 15 at high light microscopical resolution to reveal the succession of events giving rise to the development of the omental bursa and its relation to the emerging stomach asymmetry. Our analysis indicates that morphological symmetry breaking in the upper abdomen occurs within a wide mesenchymal plate called here mesenteric septum and is based on differential behavior of the coelomic epithelium which causes asymmetric paragastric recess formation and, importantly, precedes initial rotation of stomach. Our results thus provide the first histological evidence of breaking the symmetry of the early foregut anlage in the human embryo and pave the way for experimental studies of left-right symmetry breaking in the upper abdomen in experimental model organisms.

Matrix-filled microcavities in the emerging avian left-right organizer.

BACKGROUND: Hensen node of the amniote embryo plays a central role in multiple developmental processes, especially in induction and formation of axial organs. In the chick, it is asymmetrical in shape and has recently been considered to represent the left-right organizer. As mechanisms of breaking the initial left-right symmetry of the embryo are still ill-understood, analyzing the node's microarchitecture may provide insights into functional links between symmetry breaking and asymmetric morphology. RESULTS: In the course of a light- and electron-microscopic study addressing this issue we discovered novel intercellular matrix-filled cavities in the node of the chick during gastrulation and during early neurulation stages; measuring up to 45 µm, they are surrounded by densely packed cells and filled with nanoscale fibrils, which immunostaining suggests to consist of the basement membrane-related proteins fibronectin and perlecan. The cavities emerge immediately prior to node formation in the epiblast layer adjacent to the tip of the primitive streak and later, with emerging node asymmetry, they are predominantly located in the right part of the node. Almost identical morphological features of microcavities were found in the duck node. CONCLUSIONS: We address these cavities as "nodal microcavities" and propose their content to be involved in the function of the avian node by mediating morphogen signaling and storage.

On the Enigma of the Human Neurenteric Canal.

Existence and biomedical relevance of the neurenteric canal, a transient midline structure during early neurulation in the human embryo, have been controversially discussed for more than a century by embryologists and clinicians alike. In this study, the authors address the long-standing enigma by high-resolution histology and three-dimensional reconstruction using new and historic histological sections of 5 human 17- to 21-day-old embryos and of 2 marmoset monkey embryos of the species Callithrix jacchus at corresponding stages. The neurenteric canal presents itself as the classical vertical connection between the amniotic cavity and the yolk sac cavity and is lined (a) craniolaterally by a horseshoe-shaped "hinge of involuting notochordal cells" within Hensen's node and (b) caudally by the receding primitive streak epiblast dorsally and by notochordal plate epithelium ventrally, the latter of which covered the (longitudinal) notochordal canal on its ventral side at the preceding stage. Furthermore, asymmetric parachordal nodal expression in Callithrix and morphological asymmetries within the nodes of the other specimens suggest an early non-cilium-dependent left-right symmetry breaking mode previously postulated for other mammals. We conclude that structure and position of the mammalian neurenteric canal support the notion of its homology with the reptilian blastopore as a whole and with a dorsal segment of the blastopore in amphibia. These new features of the neurenteric canal may further clarify the aetiology of foetal malformations such as junctional neurulation defects, neuroendodermal cysts, and the split notochord syndrome.

Divergent axial morphogenesis and early shh expression in vertebrate prospective floor plate.

BACKGROUND: The notochord has organizer properties and is required for floor plate induction and dorsoventral patterning of the neural tube. This activity has been attributed to sonic hedgehog (shh) signaling, which originates in the notochord, forms a gradient, and autoinduces shh expression in the floor plate. However, reported data are inconsistent and the spatiotemporal development of the relevant shh expression domains has not been studied in detail. We therefore studied the expression dynamics of shh in rabbit, chicken and Xenopus laevis embryos (as well as indian hedgehog and desert hedgehog as possible alternative functional candidates in the chicken). RESULTS: Our analysis reveals a markedly divergent pattern within these vertebrates: whereas in the rabbit shh is first expressed in the notochord and its floor plate domain is then induced during subsequent somitogenesis stages, in the chick embryo shh is expressed in the prospective neuroectoderm prior to the notochord formation and, interestingly, prior to mesoderm immigration. Neither indian hedgehog nor desert hedgehog are expressed in these midline structures although mRNA of both genes was detected in other structures of the early chick embryo. In X. laevis, shh is expressed at the beginning of gastrulation in a distinct area dorsal to the dorsal blastopore lip and adjacent to the prospective neuroectoderm, whereas the floor plate expresses shh at the end of gastrulation. CONCLUSIONS: While shh expression patterns in rabbit and X. laevis embryos are roughly compatible with the classical view of "ventral to dorsal induction" of the floor plate, the early shh expression in the chick floor plate challenges this model. Intriguingly, this alternative sequence of domain induction is related to the asymmetrical morphogenesis of the primitive node and other axial organs in the chick. Our results indicate that the floor plate in X. laevis and chick embryos may be initially induced by planar interaction within the ectoderm or epiblast. Furthermore, we propose that the mode of the floor plate induction adapts to the variant topography of interacting tissues during gastrulation and notochord formation and thereby reveals evolutionary plasticity of early embryonic induction.

An Early Chick Embryo Culture Device for Extended Continuous Observation.

Appropriate mechanical tension of the vitelline membrane as the culture substrate for the early chick embryo is frequently reported to be required for successful in vitro development. Here we describe a modified device, made of anodized aluminum, for in vitro culture which we used for studies of left-right symmetry breaking with emphasis on morphology and gene expression as readouts. The technique allows for easy, high-throughput tissue handling and provides a suitable tension in a stable and easily reproducible manner proven to be suitable for correct molecular left-right patterning and heart looping after long-term culture.

Paraxial Nodal Expression Reveals a Novel Conserved Structure of the Left-Right Organizer in Four Mammalian Species.

Nodal activity in the left lateral plate mesoderm is a conserved sign of irreversible left-right asymmetry at early somite stages of the vertebrate embryo. An earlier, paraxial nodal domain accompanies the emergence and initial extension of the notochord and is either left-sided, as in the chick and pig, or symmetrical, as in the mouse and rabbit; intriguingly, this interspecific dichotomy is mirrored by divergent morphological features of the posterior notochord (also known as the left-right organizer), which is ventrally exposed to the yolk sac cavity and carries motile cilia in the latter 2 species only. By introducing the cattle embryo as a new model organism for early left-right patterning, we present data to establish 2 groups of mammals characterized by both the morphology of the left-right organizer and the dynamics of paraxial nodal expression: presence and absence of a ventrally open surface of the early (plate-like) posterior notochord correlates with a symmetrical (in mice and rabbits) versus an asymmetrical (in pigs and cattle) paraxial nodal expression domain next to the notochordal plate. High-resolution histological analysis reveals that the latter domain defines in all 4 mammals a novel 'parachordal' axial mesoderm compartment, the topography of which changes according to the specific regression of the similarly novel subchordal mesoderm during the initial phases of notochord development. In conclusion, the mammalian axial mesoderm compartment (1) shares critical conserved features despite the marked differences in early notochord morphology and early left-right patterning and (2) provides a dynamic topographical framework for nodal activity as part of the mammalian left-right organizer.

Rho kinase activity controls directional cell movements during primitive streak formation in the rabbit embryo.

During animal gastrulation, the specification of the embryonic axes is accompanied by epithelio-mesenchymal transition (EMT), the first major change in cell shape after fertilization. EMT takes place in disparate topographical arrangements, such as the circular blastopore of amphibians, the straight primitive streak of birds and mammals or in intermediate gastrulation forms of other amniotes such as reptiles. Planar cell movements are prime candidates to arrange specific modes of gastrulation but there is no consensus view on their role in different vertebrate classes. Here, we test the impact of interfering with Rho kinase-mediated cell movements on gastrulation topography in blastocysts of the rabbit, which has a flat embryonic disc typical for most mammals. Time-lapse video microscopy, electron microscopy, gene expression and morphometric analyses of the effect of inhibiting ROCK activity showed - besides normal specification of the organizer region - a dose-dependent disruption of primitive streak formation; this disruption resulted in circular, arc-shaped or intermediate forms, reminiscent of those found in amphibians, fishes and reptiles. Our results reveal a crucial role of ROCK-controlled directional cell movements during rabbit primitive streak formation and highlight the possibility that temporal and spatial modulation of cell movements were instrumental for the evolution of gastrulation forms.

Early left-right asymmetries during axial morphogenesis in the chick embryo.

The primitive node is the "hub" of early left-right patterning in the chick embryo: (1) it undergoes asymmetrical morphogenesis immediately after its appearance at Stage 4; (2) it is closely linked to the emerging asymmetrical expression of nodal and shh at Stage 5; and (3) its asymmetry is spatiotemporally related to the emerging notochord, the midline barrier maintaining molecular left-right patterning from Stage 6 onward. Here, we study the correlation of node asymmetry to notochord marker expression using high-resolution histology, and we test pharmacological inhibition of shh signaling using cyclopamine at Stages 4 and 5. Just as noggin expression mirrors an intriguing structural continuity between the right node shoulder and the notochord, shh expression in the left node shoulder confirms a similar continuity with the future floor plate. Shh inhibition at Stage 4 or 5 suppressed nodal in both its paraxial or lateral plate mesoderm domains, respectively, and resulted in randomized heart looping. Thus, the "primordial" paraxial nodal asymmetry at Stage 4/5 (1) appears to be dependent on, but not instructed by, shh signaling and (2) may be fixed by asymmetrical roots of the notochord and the floor plate, thereby adding further twists to the node's pivotal role during left-right patterning.

Paraxial left-sided nodal expression and the start of left-right patterning in the early chick embryo.

A common element during early left-right patterning of the vertebrate body is left-sided nodal expression in the early-somite stage lateral plate mesoderm. Leftward cell movements near the node of the gastrulating chick embryo recently offered a plausible mechanism for breaking the presomite-stage molecular symmetry in those vertebrates which lack rotating cilia on the notochord or equivalent tissues. However, the temporal and functional relationships between generation of the known morphological node asymmetry, onset of leftward cell movements and establishment of stable molecular asymmetry in the chick remain unresolved. This study uses high-resolution light microscopy and in situ gene expression analysis to show that intranodal cell rearrangement during the phase of counter-clockwise node torsion at stage 4+ is immediately followed by symmetry loss and rearrangement of shh and fgf8 expression in node epiblast between stages 5- and 5+. Surprisingly, left-sided nodal expression starts at stage 5-, too, but lies in the paraxial mesoderm next to the forming notochordal plate, and can be rendered symmetrical by minimal mechanical disturbance of distant tissue integrity at stage 4. The "premature" paraxial nodal expression together with morphological and molecular asymmetries in, and near, midline compartments occurring at defined substages of early gastrulation help to identify a new narrow time window for early steps in left-right patterning in the chick and support the concept of a causal relationship between a-still enigmatic-chiral (motor) protein, cell movements and incipient left-right asymmetry in the amniote embryo.

Similar developmental patterns in immunolocalisation of stem cell factor and KIT in bovine meso- and metanephros.

The mesonephros is often regarded as a simplified version of the terminal renal organ, the metanephros. Both renal organs result from an epithelio-mesenchymal interaction between the Wolffian duct and the nephrogenic ridge. It appears that the epithelio-mesenchymal interaction makes use of similar signal cascades for both renal organs and that key events required for the development of the metanephros occur at earlier stages. In murine metanephroi, the stem cell factor (SCF)/-KIT-signal transduction pathway has recently been shown to regulate ureteric bud branching and epithelial cell differentiation. We immunohistochemically defined the time-sequence of KIT and SCF presence in both renal organs using bovine embryos/foetuses with crown rump length (CRL) of 1.7-24 cm. In the mesonephroi, epithelial cells with strong KIT staining were scattered in distal tubules, and SCF was expressed in the epithelial wall of corpuscles and proximal tubules. KIT positivity occurred in the metanephroi of embryos prior to SCF; KIT was predominantly localised at the ureteric bud tips in the nephrogenic zone. In foetuses of 13 cm and more CRL, the SCF/KIT profile of developmentally advanced nephrons mirrored the situation in the mesonephros. Epithelial cells with strong KIT staining were scattered in the cortical areas of distal tubules, while SCF was expressed in the epithelial wall of corpuscles and proximal tubules. Our morphological findings agree with a potential role of KIT at the ureteric bud tips and demonstrate a similar expression of KIT and SCF along the areas of developmentally advanced mesonephric and metanephric nephrons.

Characterization of bovine fetal Leydig cells by KIT expression.

The origin of fetal Leydig cells (FLC) and whether they share a common lineage with adult Leydig cells (ALC) is still under debate, and a marker to reliably track and isolate fetal Leydig precursor cells remains to be identified. We analyzed KIT positive (KIT+) cells in gonads from bovine fetuses with crown-rump-length (CRL) 2.5-85 cm by immunohistochemistry, and found that KIT expression was gender-specific. In female gonads, expression was mainly associated with epithelial cell cords, which extended from the surface epithelium towards the KIT-negative inner stroma. In male gonads of fetuses, after CRL 2.9 cm, KIT expression was strikingly strong in interstitial cells (IC). Only a few KIT+ cells were detected in the epithelial cell cords and in the stromal layer under the surface epithelium after CRL 3.5 cm. In the male fetuses, KIT expression in IC was a continuous and characteristic feature until full term. At all developmental stages KIT+ areas alternated with anti-Müllerian hormone-positive areas. Platelet-derived growth factor receptor alpha production was initiated after the expression of KIT at CRL 4.5 cm. Detection of cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein in KIT+ IC identified them as FLC. KIT+ cells, isolated from testes by magnetic-activated cell sorting, retained their steroidogenic capacity in vitro. Together, these findings show that KIT+ IC of fetal testis correspond to FLC, which can be successfully cultivated for advanced studies.

Expression of adipokines in preimplantation rabbit and mice embryos.

Recent studies point to a role for adipokines in reproduction. Leptin is involved in embryo metabolism and may participate in embryo-maternal crosstalk. Little is known about potential roles of other adipokines in reproduction. We therefore studied the expression of adiponectin and pathway members during the pre- and periimplantation period in rabbits and mice. Adiponectin protein is localized in glandular epithelium of the rabbit endometrium on day 6 and 8 p.c. and in mouse endometrium on day 3.5 and 5 p.c. Rabbit, but not mice blastocysts express adiponectin mRNA. Adiponectin receptors one and two, adiponectin paralogues and PPARs were found in both species. Both, trophoblast and embryoblast were adiponectin positive. Real time PCR for adipoR1 and adipoR2 in rabbit blastocysts of different gastrulation stages at day 6 p.c. revealed a specific switch in expression: Expression was high in the trophoblast in early stages and in the embryoblast shortly prior to implantation. In conclusion, during the pre- and periimplantation period, members of the adiponectin pathway are expressed in endometrium and blastocysts, with a specific expression pattern in the embryonic disk of the gastrulating rabbit blastocyst, giving support to a role of the adipokine network in blastocyst differentiation and embryo-maternal interactions.

The role and limits of a gradient based explanation of morphogenesis: a theoretical consideration.

Development of an organism is a multi-dimensional process leading to the generation of complex species-specific structures. This specificity suggests machine-like organisation. The uneven distribution (gradient) of soluble substances (morphogens) and specific receptor-ligand interactions are known to cause differential gene expression. Therefore gradients of morphogens are used as a causal explanation of developmental processes. However each attempt to describe development causally should take into account both the local fine organisation and global robustness of morphogenesis. The classical view of the role of morphogens will be critically considered and possible alternative proposed. The core idea of my proposal is that the main function of morphogenetic substances could be a context dependent modification of cell behaviour. Both history and different features of morphogenetic fields create the framework for the activity of morphogenes.

Functional anatomy: a taxonomic proposal.

It is argued that medical science requires a classificatory system that (a) puts functions in the taxonomic center and (b) does justice ontologically to the difference between the processes which are the realizations of functions and the objects which are their bearers. We propose formulae for constructing such a system and describe some of its benefits. The arguments are general enough to be of interest to all the life sciences.

What is a role of the morphogens gradients in development?

The core idea of the gradient theory, a paradigm for developmental biology, is that biological morphogensis is based on a gradient-like distribution of a certain substance and its subsequent interpretation. This is an attempt at systematic criticism of this theory: I will argue that the experimental results do not support its core idea. Crucial in this context are the global reactions of an embryonic tissue upon chemical, genetic and mechanical manipulation and the possibility of regulation of the global and local events including redistribution of morphogens and de novo formation of the gene expression pattern. In addition to this, the possibility of other mechanisms should be explored.