Dynamical modelling of pattern formation during embryonic development

The combination of genetic and molecular biology techniques has uncovered the intricacies of several gene networks controlling developmental processes. In the face of such complex regulatory networks, developmental geneticists cannot rely on reasoning alone; a thorough understanding of the spatio-temporal properties of these networks clearly requires the use of proper computational tools and methods.     

Analysis of pattern formation during embryonic development of Hydractinia echinata

Summary Patterning processes during embryonic development of Hydractinia echinata were analysed for alterations in morphology and physiology as well as for changes at the cellular level by means of treatment with proportioning altering factor (PAF). PAF is an endogenous factor known to change body proportions and to stimulate nerve cell differentiation in hydroids (Plickert 1987, 1989). Applied during early embryogenesis, this factor interferes with the proper establishment of polarity in the embryo. Instead of normal shaped planulae with one single anterior and one single posterior end, larvae with multiple termini develop. Preferentially, supernumerary posterior ends, which give rise to polyp head structures during metamorphosis, form while anterior ends are reduced. The formation of such polycaudal larvae coincide with an increase in the number of interstitial cells and their derivatives at the expense of epithelial cells. Treatment of further advanced embryonic stages causes an increase in length, presumably due to the general stimulation of cell proliferation observed in such embryos. Also, the spatial arrangement of cells (i.e. cells in proliferation and RFamide (Arg-Phe-amide immunopositive nerve cells) is altered by PAF. Larvae that develop from treated embryos display altered physiological properties and are remarkably different from normal planulae with respect to their morphogenetic potential: (1) Larvae lose their capacity to regenerate missing anterior parts; isolated posterior larva fragments form regenerates of a bicaudal phenotype. (2) In accordance with the frequently observed reduction of anterior structures, the capacity to respond to metamorphosis-inducing stimuli decreases. (3) The morphogenetic potential to form basal polyp parts is found to be reduced. In contrast, the potential to form head structures during metamorphosis increases, since primary polyps with supernumerary hypostomes and tentacles metamorphose from treated animals.     

Signals regulating muscle formation in the limb during embryonic development

Classically, somites have been the preparation of choice for the study of muscle development, while the limb bud is the preferred model of axis formation. Nevertheless, the limb bud offers some experimental advantages for muscle studies. This review describes the successive events involved in limb muscle formation during embryonic development, the properties of the key marker molecules and resumes our current knowledge of the signalling pathways involved.     

Gene expression and pattern formation during early embryonic development in amphibians

Temporal and spatial gene expression and inductive interactions control the establishment of the body plan during embryogenesis in invertebrates and vertebrates. The best-studied vertebrate model system is the amphibian embryo. Seventy-five years after the famous organizer experiment of Hans Spemann and Hilde Mangold in 1924 our knowledge of the molecular mechanisms of the multi-step formation of embryonic axis has substantially improved. Although in the 30s and 40s the interest of many laboratories was focussed on neural induction (determination of the central nervous system), only crude factors from so-called heterogeneous inducers (liver, bone marrow, etc.,) could be isolated by the traditional biochemical techniques available at this time. An important breakthrough was the characterization and purification of a mesoderm inducing factor, the so-called vegetalizing factor (homologous to Activin) in highly purified from chicken embryos. Much later after the introduction of molecular techniques Vgl and Activin (both belonging to the TGF-β family) and FGFs could be identified as important factors for mesoderm formation. It was in the 90s that secreted neuralizing factors (chordin, noggin, follistatin and cerberus) could be detected, which are expressed at the dorsal side of the early embryo including the Spemann organizer. In contrast to the classical view, these proteins act as antagonists to factors like BMP-4 localized on the ventral side. Of special interest was the fact that inDrosophila sog, homologous to chordin, determines the ventral side, whiledpp, homologous toBMP-4, participates in the formation of the dorsal side. These data of evolutionary conserved genes in both invertebrates and vertebrates support the view that they are descendents of common ancestors, the urbilateralia, living around 300 million years ago. The expression of those genes coding for secreted proteins is closely related to inductive interactions between cells and germ layers. Recently it was shown that planar signals are not sufficient to generate a specific anterior/posterior pattern during the primary steps of neural induction, i.e., formation of the central nervous system in amphibians.     

Expression pattern of the homeodomain transcription factor Pitx2 during muscle development

Late-stage Pitx2(+/LacZ) mouse embryos stained with x-gal appeared to have blue muscles, suggesting that Pitx2 expression specifically marks some phase of the myogenic progression or muscle anlagen formation. Detailed temporal and spatial analyses were undertaken to determine the extent and onset of Pitx2 expression in muscle. Pitx2 was specifically expressed in the vast majority of muscles of the head and trunk in late embryos and adults. Early Pitx2 expression in the cephalic mesoderm, first branchial arch and somatopleure preceded specification of head muscle. In contrast, Pitx2 expression appeared to follow muscle specification events in the trunk. However, Pitx2 expression was rapidly upregulated in these myogenic structures by E10.5. Upregulation correlated tightly with the apposition of a non-myogenic, Pitx2-expressing, cell cluster lateral to the dermomyotome. This cluster first appeared at the forelimb level at E10.25, gradually elongated in the posterior direction, appeared to aggregate from delaminated cells emanating from the ventrally located somatopleure, and was named the dorsal somatopleure. Immunohistochemistry on appendicular sections after E10.5 demonstrated that Pitx2 neatly marked the areas of muscle anlagen, that Pax3, Lbx1, and the muscle regulatory factors (MRFs) stained only subsets of Pitx2(+) cells within these areas, and that virtually all Pitx2(+) cells in these areas express at least one of these known myogenic markers. Taken together, the results demonstrate that, within muscle anlagen, Pitx2 marks the muscle lineage more completely that any of the known markers, and are consistent with a role for Pitx2 in muscle anlagen formation or maintenance.     

Proximal-distal pattern formation inDrosophila: graded requirement forDistal-less gene activity during limb development

Summary The development of all of the adult limbs inDrosophila depends upon the activity of theDistal-less gene. We report here the phenotypic characterization of a number of hypomorphicDistal-less alleles which indicates that there is a graded requirement forDistal-less activity in the developing limbs. Previous analysis of genetically mosaic animals indicated that cells in the early primordia of the limb imaginal dises possess a graded proximal-distal positional information which depends on the presence of theDistal-less gene for its expression. Taken together these data suggest thatDistal-less may directly encode the graded positional information that is required to organise the proximal-distal axis of the developing limbs.     

Cortical pattern formation during visual hallucinations

We theoretically investigate pattern formation during simple visual hallucinations caused by epileptic activity. To this end we analyze the activator-inhibitor model of Ermentrout and Cowan [1]. In contrast to these authors we focus on a different disease mechanism: According to experimental findings (cf. [2]) we decrease the influence of the inhibitor on the activator. This causes spontaneous pattern formation due to a bifurcation. The model parameters determine whether one or two or four modes become unstable. By means of the center manifold theorem, in all cases the order parameter equation is derived, the stability of the solution is proofed, and the bifurcating activity pattern is calculated explicitely in lowest order. Taking into account terms up to third order in all cases the order parameter equation has a potential. For the two-modes and the four-modes instability this potential causes a winner-takes all dynamics. We integrate the order parameter equation numerically and plot the visual hallucinations which result from the bifurcating cortical activity. The theoretically derived hallucinations correspond to clinically observed visual hallucinations (cf. [3, 4]), which are, for instance, well-known from petit mal epilepsy [5].Finally we investigate the influence of noise on the activity patterns as well as the visual hallucinations.     

Cullin-3 regulates pattern formation, external sensory organ development and cell survival during Drosophila development

Ubiquitin-mediated proteolysis regulates the steady-state abundance of proteins and controls cellular homoeostasis by abrupt elimination of key effector proteins. A multienzyme system targets proteins for destruction through the covalent attachment of a multiubiquitin chain. The specificity and timing of protein ubiquitination is controlled by ubiquitin ligases, such as the Skp1-Cullin-F box protein complex. Cullins are major components of SCF complexes, and have been implicated in degradation of key regulatory molecules including Cyclin E, beta-catenin and Cubitus interruptus. Here, we describe the genetic identification and molecular characterisation of the Drosophila Cullin-3 homologue. Perturbation of Cullin-3 function has pleiotropic effects during development, including defects in external sensory organ development, pattern formation and cell growth and survival. Loss or overexpression of Cullin-3 causes an increase or decrease, respectively, in external sensory organ formation, implicating Cullin-3 function in regulating the commitment of cells to the neural fate. We also find that Cullin-3 function modulates Hedgehog signalling by regulating the stability of full-length Cubitus interruptus (Ci155). Loss of Cullin-3 function in eye discs but not other imaginal discs promotes cell-autonomous accumulation of Ci155. Conversely, overexpression of Cullin-3 results in a cell-autonomous stabilisation of Ci155 in wing, haltere and leg, but not eye, imaginal discs suggesting tissue-specific regulation of Cullin-3 function. The diverse nature of Cullin-3 phenotypes highlights the importance of targeted proteolysis during Drosophila development.     

Key aspects of phrenic motoneuron and diaphragm muscle development during the perinatal period.

At the time of birth, respiratory muscles must be activated to sustain ventilation. The perinatal development of respiratory motor units (comprising an individual motoneuron and the muscle fibers it innervates) shows remarkable features that enable mammals to transition from in utero conditions to the air environment in which the remainder of their life will occur. In addition, significant postnatal maturation is necessary to provide for the range of motor behaviors necessary during breathing, swallowing, and speech. As the main inspiratory muscle, the diaphragm muscle (and the phrenic motoneurons that innervate it) plays a key role in accomplishing these behaviors. Considerable diversity exists across diaphragm motor units, but the determinant factors for this diversity are unknown. In recent years, the mechanisms underlying the development of respiratory motor units have received great attention, and this knowledge may provide the opportunity to design appropriate interventions for the treatment of respiratory disease not only in the perinatal period but likely also in the adult.     

Morphogen pattern formation and development in growth

The dependence of the spatial concentration profiles of morphogens on a characteristic dimension is obtained by continuation techniques for Gierer and Meinhardt's activator-inhibitor model of morphogenesis. The study of the behaviour of the system during growth, where the linear and exponential increase of the characteristic dimension is considered, revealed that more complex patterns of morphogen spatial concentrations appear regularly in a reproducible way.     

Evolutionary aspects of calmodulin

Calmodulin (CaM) is a major cellular sensor of calcium signaling, interacts with numerous proteins associated with cellular second messenger systems (e.g., cyclic AMP, nitric oxide), and is associated with neurosecretory activity. An identical CaM protein consisting of four helix-loop-helix regions that arose by gene duplication is encoded by three nonallelic mammalian genes that are some of the most highly conserved genes known. Differential tissue and cellular expression of each CaM suggest unique functions that promote strong selective preservation of these replicate, yet distinct, CaM genes in mammals. Each gene displays the same exon-intron arrangement but is characterized by distinct promoter elements and by unique 5'- and 3'-untranslated regions that are highly conserved among human, rat, and mouse. These distinct untranslated regions may permit regulation of CaM levels at discrete cellular sites during differentiation and in highly specialized cell types such as neurons.     

Mechanisms of pattern formation in development and evolution

We present a classification of developmental mechanisms that have been shown experimentally to generate pattern and form in metazoan organisms. We propose that all such mechanisms can be organized into three basic categories and that two of these may act as composite mechanisms in two different ways. The simple categories are cell autonomous mechanisms in which cells enter into specific arrangements ('patterns') without interacting, inductive mechanisms in which cell communication leads to changes in pattern by reciprocal or hierarchical alteration of cell phenotypes ('states') and morphogenetic mechanisms in which pattern changes by means of cell interactions that do not change cell states. The latter two types of mechanism can be combined either morphostatically, in which case inductive mechanisms act first, followed by the morphogenetic mechanism, or morphodynamically, in which case both types of mechanisms interact continuously to modify each other's dynamics. We propose that this previously unexplored distinction in the operation of composite developmental mechanisms provides insight into the dynamics of many developmental processes. In particular, morphostatic and morphodynamic mechanisms respond to small changes in their genetic and microenvironmental components in dramatically different ways. We suggest that these differences in 'variational properties' lead to morphostatic and morphodynamic mechanisms being represented to different extents in early and late stages of development and to their contributing in distinct ways to morphological transitions in evolution.