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Designing the Bodyplan: Developmental Mechanisms
Models for the generation of the primary body axes of higher organisms
Max-Planck-Institut für Entwicklungsbiologie, Spemannstr. 35; D-72076 Tübingen
Evolutionary ancestral radial-symmetric animals are proposed to be the key to understand the more evolved bilateral-symmetric body plans. A comparison of gene expression data reveals that the system that was organizing once the body pattern of ancestral radial-symmetric organisms evolved into the system that organizes the AP axis of the brain of higher organisms. The early patterning of the vertebrate gastrula can be regarded as a remnant of this ancestral system. The small hydra organizer located around the gastric opening became in vertebrates a large ring, the marginal zone in amphibians, which acts as organizing region for the anteroposterior (AP) pattern. The trunk is an evolutionary later addition that appears, as the rule, also later during the individual development. The AP organization of the trunk depends on a time-controlled posterior transformation in which an oscillation plays a crucial role. This oscillation also leads to the repetitive nature of the trunk pattern as seen in somites or segments. Also an evolutionary later invention is the midline. Due to the required geometry - long extension along the anteroposterior axis but only a small extension along the dorsoventral axis – midline formation is an intricate patterning problem. Using vertebrates, insects and planarians as example it will be shown that very different solutions were found during evolution. In vertebrates the dorsal organizer initiates and elongates the midline. In insects, an inhibition from a dorsal organizer confines the midline to the ventral side, a view that also provides a rational for the well-known DV-VD reversal between insects and vertebrates. In planarians, a DV border has obtained organizing functions and is the precondition to form the anterior and posterior terminal structures. The different modes of midline formation (and mesoderm initiation) were presumably points of no return in the separation of the major phyla. Computer simulations will illustrate that these molecularly feasible models describe the observed dynamic properties very well and are compatible with many molecular-genetic observations.
Redefining the role of ectoderm in somitogenesis: a player in the formation of the fibronectin matrix of presomitic mesoderm.
The absence of ectoderm impairs somite formation in cultured presomitic mesoderm explants, suggesting that an ectoderm-derived signal is essential for somitogenesis. Here we show that the standard enzymatic treatments used for explant isolation destroy the fibronectin matrix surrounding the anterior presomitic mesoderm which fails to form somites when cultured for 6 hours. In contrast, when explants are isolated with collagenase, their fibronectin matrix is maintained, and they form somites when cultured for 6 hours. The additional presence of ectoderm enhances somite formation, whereas endoderm has no effect. We further show that when pancreatin-isolated presomitic mesoderm explants are cultured in fibronectin-supplemented medium, their ability to form somites is significantly improved. Interestingly, ectoderm is the major producer of fibronectin (Fn1) transcripts, while the non-determined region of the presomitic mesoderm expresses the fibronectin assembly receptor, integrin alpha5 (Itga5). We therefore propose that the major phase of fibronectin matrix assembly occurs in this non-determined region where ectoderm-derived fibronectin is assembled by mesodermal alpha5beta1 integrin. We conclude that a fibronectin matrix is necessary for morphological somite formation and that a major, previously unrecognised, role of ectoderm in somitogenesis is the synthesis of fibronectin.
Role of hoxb13a during zebrafish development
Max Corredor Institute of Biology, University of Leiden email@example.com
Hox genes are a family of transcription factors that have been shown to play a key role in animal development. They are present in genomic clusters in most animals and are expressed in a collinear fashion, providing positional information for antero-posterior segment differentiation. In our previous work on zebrafish hox genomics we reported the presence of hoxb13a in the genome sequence of Danio rerio when other studies had indicated its absence. Furthermore, we proved that the gene is expressed during development and performed an in situ hybridization that revealed its expression at the tip of the tail at the 24 h.p.f. stage. The hoxb13 paralog group is therefore present in all vertebrates but little is known about the role of these genes in development and body patterning. In this communication we present additional information about hoxb13a expression pattern during zebrafish development as well as some insights into its function by the study of MO knock-down embryos. Besides phenotypic data from the morphological level, we will also present an analysis of the changes at the transcriptomal level using custom designed whole genome Agilent arrays.
Distinct funtions for ERK1 and ERK2 in gastrulation cell migration processes during zebrafish embryogenesis
Gabby Krens, Institute of
The functions of the various modern mitogen-activated protein kinases (MAPKs) in embryogenesis are still poorly understood. We annotated, cloned and determined the expression patterns of zebrafish mapk gene family, containing the ERK, JNK and p38 subfamilies. For ERK1 and ERK2 MAPK, we defined specific functions in early embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in severe phenotypes. Cell tracing experiments revealed differential functions for ERK1 and ERK2 MAPK during gastrulation cell-migration processes. Knockdown of ERK1 affected convergence cell movements in a greater extend than knockdown of ERK2. In contrast ERK2 knockdown severely reduced extension of the body axis during gastrulation. Further analysis of knockdown embryos demonstrated that ERK2 protein but not ERK1 protein, is specifically activated in the margin at the onset of epiboly. Absence of active ERK2 in the margin blocked initiation of epiboly cell migration processes during zebrafish development, leading to an arrest of embryogenesis.
We have performed Agilent array based transcriptome analysis of ERK1MO and ERK2MO injected embryos to determine ERK1 and ERK2 specific downstream targets and to get more insight in their specific roles during developmental processes. The results show distinct gene expression profiles expression after knock-down or ERK1 and ERK2 proving their separate functions in embryogenesis.
Spatio-temporal gene expression analysis of 14-3-3 family proteins in zebrafish, using 3D reconstruction and the 3D atlas of zebrafish development
Monique Welten/ Aimy Sels,
The 14-3-3 proteins comprise a family of acidic, dimeric proteins present in all eukaryotes. The 14-3-3 family members are involved in numerous cellular processes such as metabolism, signaling, cell cycle, differentiation, and apoptosis. In insects and vertebrates, 14-3-3 proteins are found to be involved in brain development.
Given their numerous roles in cellular processes, it is not surprising that 14-3-3 family members play a role in human cancer and neurological disorders. Recent studies in humans, rat and mice show altered 14-3-3 protein expression brain tissue and cerebrospinal fluid, in neurological disorders such as Creutzfeldt-Jakob disease, scrapie , Alzheimer’s disease and epilepsy.
In order to employ zebrafish as a model to study cancer and neurological disorders in human, it is extremely important to characterize the role of zebrafish 14-3-3 proteins during development. Besser et al (2006) already investigated the expression patterns of eleven 14-3-3 isofroms in developing zebrafish; however, the anatomical localization of 14-3-3 gene expression patterns was not yet studied in detail, by means of tissue sectioning.
To further characterize the gene expression patterns and their spatial relationships, we applied fluorescent in situ hybridization (Welten et al. 2006), confocal laser scanning microscopy and 3D reconstruction. To confirm our findings, samples stained for in situ hybridization were sectioned and processed for 3D reconstruction. In our study we present a spatio-temporal gene expression analysis of two 14-3-3 isoforms during zebrafish development, using 3D reconstruction software and the 3D atlas of zebrafish development as a reference.
Notochord singnaling effects on somite segmentation
Tatiana P. Resende, Raquel P. Andrade and Isabel Palmeirim
and Health Sciences Research Institute (ICVS), School of Health Sciences,
Somites are metameric structures that bud off from the rostral presomitic mesoderm (PSM) with a periodicity of 90 minutes during chick embryo development. This strict temporal control is exerted by the Somitogenesis Clock, first evidenced by cyclic hairy1 expression in chick embryo PSM, also with a 90 minute periodicity.
The notochord is an early embryonic structure responsible for supplying structural support to the developing organism and functioning as a signaling source during axial organs formation. It was previously shown that removal of the chick embryo notochord leads to the absence of floor plate and to problems in somite development.
In the present work we studied the role of notochord on PSM segmentation. Chick embryo tissue explants with or without notochord were cultured for different time periods. The rate and number of somites formed under these conditions was evaluated, as well as molecular clock gene expression. The relevance of notochord signaling for PSM segmentation will be discussed.
Long distance regulation of SHH in the limb
Elisabeth Lodder, Marianne Hoogeveen, Ben Oostra, Esther de Graaff
Sonic hedgehog (SHH) is a well-known morphogen in embryonic development. It plays a key role in the specification of the anterior-posterior axis in the limb. Normally, shh is expressed in a group of cells named the Zone of Polarising Activity (ZPA) on the posterior side of the limb bud. Ectopic anterior expression of shh induces the formation of extra digits on the anterior side of the limb.
We have recently identified a limb specific, cis-acting regulatory element, that is responsible for the normal shh expression in the ZPA. Interestingly, the ZPA Regulatory Sequence (ZRS), resides 1 Mb upstream of SHH in an intron of another gene[i]. The ZRS is both necessary and sufficient for driving shh expression in the ZPA. Point mutations in the ZRS lead to ectopic anterior expression of shh indicating that a correct ZRS is also required for anterior repression of SHH. However the mechanism through which this element regulates shh expression remains unclear.
Currently we’re employing several methods to elucidate the mechanism behind the long distance regulation of shh in the limb: knock out mice and mutation analysis to determine the minimal region of the ZRS, 3D-FISH and cryo-FISH are employed to detect looping of the ZRS to the SHH promotor area, Y1H is used to find proteins binding to the ZRS.
Hox, Cdx and the genetics of axial extension and patterning in the mouse embryo
Deschamps, Hubrecht Laboratory, Netherlands Institute
for Developmental Biology,
Tissue generation and patterning are intimately linked during vertebrate embryogenesis. Posterior elongation of the axis to generate the trunk and tail depends on genes belonging to the same genetic network as the genes specifying positional identity of nascent structures. Hox and ParaHox genes are among the players in both these aspects of morphogenesis. Whereas the Hox genes have so far been mostly associated with the acquisition of antero-posterior information in axial and paraxial structures, Cdx loss of function predominantly leads to an arrest of axial elongation and defects in posterior embryonic tissues, in a dosage-dependent way. Whether the Cdx mutations achieve their effects exclusively by misregulating Hox genes is not clear yet. The cellular basis for axial elongation impaired in Cdx mutants may involve a putative stem cell population located between the node and the anterior primitive streak, and later at the corresponding place in the tailbud (Cambray and Wilson, 2002). The gene network downstream of the Cdx transcription factors during axial elongation is only beginning to be unveiled.
plan of plants controlled by polar-auxin transport
Ramiro E. Magno
Faculty of Biology, University of
Athanasius F. M. Marée, Theoretical Biology
and Bioinformatics Group Faculty of Biology,
Modeling body axis formation as a dynamic, out of equilibrium, multilevel process
Biology and Bioinformatics Group,
Groupe Développement et Neuropharmacologie
Ecole normale supérieure, Paris, France Alain.Prochiantz@ens.fr
Homeogenes code for homeoprotein transcription factors that have fundamental roles in development and throughout adulthood. They are key players in genetic networks that lay out the body plan and regulate morphological and physiological phenomena at the cellular and multi-cellular levels. Surprisingly, homeoproteins share activities that extend beyond transcription to include translation regulation and intercellular transfer. Homeoproteins non-cell autonomous activity may be at the origin of homeogenetic extension, boundary formation in developing organisms, and signaling both during development and in the adult. We shall review these novel activities and frame them within the context of the nervous system. Three models will be developed that illustrate how homeoprotein transfer is used at three distinct times during the development of the visual system. Data will be presented that illustrate how Pax6, Engrailed and Otx2 transduction play a role in the development of the eye anlagen (Pax6), in the establishment of retino-tectal maps (Engrailed) and in the opening of the critical period for binocular vision (Otx2).
Making a brain: the role of timing in developmental decisions
for No. 3, mainly
New experiments reveal variations in design and control of the polarized protrusive activity underlying the mediolateral cell intercalation that is thought to drive convergence and extension of the axial and paraxial mesoderm and neural plate of Xenopus. Dorsal axial (notochordal) and paraxial (presomitic) mesodermal cells restrict protrusive activity to their medial and lateral ends, appear to exert traction on one another, elongate mediolaterally, and then intercalate mediolaterally to form a narrower, longer array (convergence and extension). In contrast, neural plate cells show monopolar, medially-directed protrusive activity. In explants of neural plate and dorsal mesoderm, removing the midline notoplate and notochord tissues and abutting the remaining halves together at a "ghost midline" results in loss of the normal monopolar, medially directed protrusive activity and expression of bipolar protrusive activity. Adding lateral ectopic midline (notoplate/notochord) to the lateral edge of such a midlineless explant stimulates the formation of monopolarized cells throughout the explant. Those cells near the ectopic midline direct their protrusive activity toward the ectopic midline; those near the "ghost midline" direct their protrusive activity toward it; those halfway between the ectopic and ghost midlines orient in all directions. A two-signal model and a one-signal model to account for this behavior will be presented. Explants of neural plate migrate directionally, toward explants of midline tissues, and also converge and extend, suggesting that a diffusible, midline-generated chemotactic signal polarizes/orients neural cells during neural convergence and extension.
High resolution confocal imaging suggests a local biomechanical model of cell-substrate/cell-traction, common to both monopolar and bipolar modes of cell intercalation, that accounts for shearing of cells past one another during intercalation and the somewhat paradoxical formation of a stiffened tissue, capable of generating a pushing
Surprisingly, imaging of urodele (Ambystsoma, Taricha) gastrulation in whole embryos and explants suggest that the biomechanical forces generated by intercalation of deep, mesenchymal, somitic mesodermal cells in Xenopus are generated by ingression of superficial, epithelial somitic mesodermal cells along a "bilateral primitive streak" located at the edges of the vegetal endoderm in the urodele. Hoop stresses important in blastopore closure and involution appear to be generated in the urodele by removal cells from a sheet in a massive epithelial-mesenchymal transition (EMT), whereas the same forces are generated in Xenopus by cell intercalation. These results show a previously unrecognized commonality of amphibian and amniote gastrulation and provide insight into the evolution of gastrulation. Eggs of diverse sizes and yolk content gastrulate in dramatically different ways by biomechanical integration of a few basic cell behaviors. Biomechanical "design" is the major context for functional evaluation of genetic perturbations of morphogenically important properties, such as cell motility, adhesion, and polarity, tissue stiffness, and the patterning of these properties.
Regulation of Wnt secretion by the putative Wnt sorting receptor MIG-14/Wls and the retromer complex
Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584 CT,
Wnt proteins are members of a conserved family of secreted signaling molecules that regulate many aspects of development. Although there is a detailed picture of the different signaling pathways that are triggered by Wnt, much less is known about how Wnt is produced and secreted. Because Wnt proteins are lipid modified, it is likely that a specialized secretion pathway is involved. Recently, it was shown that a novel Wnt binding protein called Wls is required for Wnt secretion in Drosophila and human cells. Based on its properties, it is likely that Wls functions as a specific sorting receptor for Wnt. The C. elegans ortholog of Wls is mig-14. In agreement with other studies, we found that mig-14 activity is required in Wnt producing cells. We have previously shown that an intracellular protein sorting complex, called the retromer, is required in Wnt producing cells for long-range Wnt signaling. One of the known functions of the retromer is the retrograde transport of specific sorting receptors from late endosomes to the trans-Golgi network (TGN), which rescues the sorting receptors from lysosomal degradation. Because MIG-14 may function as a sorting receptor for Wnt, we investigated whether the retromer complex and MIG-14 functionally interact. We found that mutations in retromer subunit genes strongly enhance the Wnt phenotype of hypomorphic mig-14 alleles. Furthermore, we found a striking reduction in MIG-14 protein levels in the absence of retromer function. We propose a model in which MIG-14 transports Wnt from the TGN to the plasma membrane for secretion. After release of Wnt, MIG-14 is internalized and recycled back to the TGN via a retromer dependent pathway to maintain efficient Wnt secretion. In the absence of retromer function, MIG-14 is degraded and Wnt secretion is reduced. As predicted by this model, we found that Wnt signaling can be restored in retromer mutants by overexpression of MIG-14. Also in agreement with the model is our recent observation that clathrin mediated endocytosis is required in Wnt producing cells. Thus, we found that in the absence of the AP-2 complex or clathrin, MIG-14 accumulates at the plasma membrane. This block in endocytosis disrupts the normal cycling of MIG-14, leading to the observed defect in Wnt signaling activity. We speculate that regulation of MIG-14 sorting and stability may enable Wnt producing cells to fine-tune the rate of Wnt secretion, and as a consequence, the range of Wnt signaling.
Mesodermal interactions affecting the anterior-posterior patterning of the trunk
Determination of regional specificity in the vertebrate central nervous system begins during gastrulation. A recent model describes a three step mechanism. Activation establishes an anterior neural state. Stabilization within the activated territory maintains a forebrain/midbrain state. Transformation posteriorizes other activated areas. For the transformation step two signalling pathways have been controversially discussed. First planar signalling describes how gradients spread from posterior to anterior within one layer of tissue. Second vertical signals originate from internalized mesoderm and spread into adjacent regions of overlying neurectoderm.
We have recently identified the non-organizer mesoderm as source of vertical signals, which in a temporally controlled interaction with the Spemann organizer mesoderm generates positional information. We postulate the time space translator model describing how a temporal sequence of positional values in the mesoderm is translated into a spatial neural pattern. The components of these interactions (non-organizer mesoderm, Spemann organizer mesoderm, morphogenetic movements) are currently under investigation.
Integration of Patterning, Cell Behavior, and Morphogenic Forces During Gastrulation and Body Plan Formation.
Ray Keller, Ana Rolo,
Dave Shook and Paul Skoglund, Department of Biology and the Morphogenesis
and Regenerative Medicine Institute,
The circumblastoporal convergence forces that close the blastopore in amphibians are generated by at least three different mechanisms. These are convergent extension, which is driven by mediolateral cell intercalation, convergent thickening, for which the cell behavior is unknown, and epithelial mesenchymal transition (EMT), which is driven by apical constriction and ingression. In addition to employing different cell motile behaviors, these “morphogenic machines” appear to have molecular, morpho-mechanical specializations. For example, myosin heavy chain IIB being particularly important for convergent extension but not for convergent thickening, and myosin IIA is essential at high levels for convergent thickening. These machines are also used in varying proportions in gastrulation and body axis elongation of different amphibian species (Xenopus, Epipedobates, Taricha, Ambystoma). Despite their differences in employment and in underlying cell motility, these three machines also have similarities. They all generate a similar pattern of circumblastoporal tensile force that closes the blastopore. And although they employ different cell behaviors, these behaviors are progressively expressed along the presumptive (future) anterior-to-posterior and lateral-to-medial tissue axes, suggesting the use of common underlying patterning devices. The progressive nature and the axial orientation of these local force generating cell behaviors is essential for emergence of large scale, specific patterns of force. Although the number of different patterning devices and different cell behaviors appears to be relatively small, the number of ways these processes can be biomechanically integrated is large. Biomechanical integration thus offers a way of using a small number of conserved molecular and cellular processes to generate many different but specific large scale patterns of force appropriate to bring about gastrulation in species with eggs of vastly different architectures. Supported by NIH R37 25594 to R.K and Fundacao Para a Ciencia e a Tecnologia Fellowship to A.R.
Anterior posterior axis formation in the trunk region of Xenopus laevis
1Institute of Biology
2Departement of Biochemistry,
We propose a new model that describes the mechanism that patterns the trunk in the frog Xenopus laevis. It incorporates the temporal colineair sequence of Hox genes in the mesoderm, the role of morphogenetic movements, and the role of the Spemann organiser. This model describes that AP identities arise in the non-organiser mesoderm in a domain defined by the presence of Xbra and BMP signaling. Due to morphogenetic movements, mesodermal cells with particular AP identities leave this domain at different times and move nearer to the organiser. Under influence of the organiser, their AP identities also appear in an adjacent neurectodermal domain. Stripes with different AP identities are thereby created within the neurectoderm along the AP axis, converting the original time sequence into a spatial sequence along the AP axis.
Axes in the Limb
Patterning along a secondary body axis, such as a limb, shares some
similarities with patterning along the primary body axis. In both cases,
there are positional fields superimposed on a repeating or segmental
pattern of structures. The segmental nature of the limb is seen very
clearly in the dolphin flipper, where there is a repeating series of
almost identical bones. However the dolphin is exceptional, and in most
amniote limbs, Hox and other genes contribute to a strong
individualisation (in terms of shape and size) of the different limb
bones. We have conducted expression profiling and functional experiments
on the limb in a range of archosaur embryos in order to examine the
evolution and development of the limb axis. We find evidence that, in
some clades with reduced number of digits, there has been a shift in the
primary axis of the digits, as revealed by sox9 and sonic expression.
This supports the idea that dramatic changes in morphology can be
achieved through rather small, early shifts in the spatial register
between different patterning mechanisms.
Retinoic acid and the control of the body plan
Malcolm Maden, MRC Centre for Developmental Neurobiology, King’s College London, UK
Since the original observation that in the absence of retinoic acid a segment of the developing CNS, the posterior hindbrain, fails to develop many other studies have noted that certain mesodermal organs as well as regions of endoderm also do not develop. These observations can be brought together to suggest that retinoic acid is responsible for specifying a segmental region of the body involving all three germ layers. This data will be reviewed and summarised. The possible sources of retinoic acid and whether it exists in the form of a gradient to organise this part of the body plan will be discussed.
Left/right asymmetry for the main body axis
A hox timing mechanism for AP patterning
We manipulated dorsoventrality in the early embryo.Lithium chloride (LiCl) hyperactivates early Wnt signalling, causing hyperdorsalisation. LiCl prevents development of the axis. It prevents both early and late Hox gene expression. Conversely, early UV irradiation (UV) ventralises the embryo by preventing zygotic cortical rotation. UV blocks axis formation. It also blocks late hox expression. As well as (neur) ectodermal hox expression. UV failed to block the early temporally collinear NOM hox gene expression. Recombining a UV gastrula with an organizer is known to rescue axis formation. It also evidently rescues axial expression of hox genes, both in neurectoderm and in axial mesoderm.
These observations suggested a timing mechanism. We (SW) tested this idea . Ventralised (UV) embryos were aged different times before implantating an organizer (SO). This affected the AP pattern produced. Transplanting SO into a late UV gastrula generated only posterior axis. As the UV gastrula became progressively younger, more and more anterior parts of the axis were added. In contrast, ageing the SO had no effect.
We conclude that: temporally collinear hox expression in NOM s required for AP patterning. The NOM hox sequence interacts with SO. As NOM comes in range of SO signals via gastrulation movements, these stop its progression through the hox sequence and cause transfer of frozen hox codes from (now involuted) NOM to overlying (neur) ectoderm. This generates an anterior (early interaction) to posterior (late interaction) sequence of hox codes along the axis.