Spatial Analysis of Expression Patterns Predicts Genetic Interactions at the Mid-Hindbrain Boundary

The isthmic organizer mediating differentiation of mid- and hindbrain during vertebrate development is characterized by a well-defined pattern of locally restricted gene expression domains around the mid-hindbrain boundary (MHB). This pattern is established and maintained by a regulatory network between several transcription and secreted factors that is not yet understood in full detail. In this contribution we show that a Boolean analysis of the characteristic spatial gene expression patterns at the murine MHB reveals key regulatory interactions in this network. Our analysis employs techniques from computational logic for the minimization of Boolean functions. This approach allows us to predict also the interplay of the various regulatory interactions. In particular, we predict a maintaining, rather than inducing, effect of Fgf8 on Wnt1 expression, an issue that remained unclear from published data. Using mouse anterior neural plate/tube explant cultures, we provide experimental evidence that Fgf8 in fact only maintains but does not induce ectopic Wnt1 expression in these explants. In combination with previously validated interactions, this finding allows for the construction of a regulatory network between key transcription and secreted factors at the MHB. Analyses of Boolean, differential equation and reaction-diffusion models of this network confirm that it is indeed able to explain the stable maintenance of the MHB as well as time-courses of expression patterns both under wild-type and various knock-out conditions. In conclusion, we demonstrate that similar to temporal also spatial expression patterns can be used to gain information about the structure of regulatory networks. We show, in particular, that the spatial gene expression patterns around the MHB help us to understand the maintenance of this boundary on a systems level

Odefy -- From discrete to continuous models

<p>Abstract</p> <p>Background</p> <p>Phenomenological information about regulatory interactions is frequently available and can be readily converted to Boolean models. Fully quantitative models, on the other hand, provide detailed insights into the precise dynamics of the underlying system. In order to connect discrete and continuous modeling approaches, methods for the conversion of Boolean systems into systems of ordinary differential equations have been developed recently. As biological interaction networks have steadily grown in size and complexity, a fully automated framework for the conversion process is desirable.</p> <p>Results</p> <p>We present <it>Odefy</it>, a MATLAB- and Octave-compatible toolbox for the automated transformation of Boolean models into systems of ordinary differential equations. Models can be created from sets of Boolean equations or graph representations of Boolean networks. Alternatively, the user can import Boolean models from the CellNetAnalyzer toolbox, GINSim and the PBN toolbox. The Boolean models are transformed to systems of ordinary differential equations by multivariate polynomial interpolation and optional application of sigmoidal Hill functions. Our toolbox contains basic simulation and visualization functionalities for both, the Boolean as well as the continuous models. For further analyses, models can be exported to SQUAD, GNA, MATLAB script files, the SB toolbox, SBML and R script files. Odefy contains a user-friendly graphical user interface for convenient access to the simulation and exporting functionalities. We illustrate the validity of our transformation approach as well as the usage and benefit of the Odefy toolbox for two biological systems: a mutual inhibitory switch known from stem cell differentiation and a regulatory network giving rise to a specific spatial expression pattern at the mid-hindbrain boundary.</p> <p>Conclusions</p> <p>Odefy provides an easy-to-use toolbox for the automatic conversion of Boolean models to systems of ordinary differential equations. It can be efficiently connected to a variety of input and output formats for further analysis and investigations. The toolbox is open-source and can be downloaded at <url>http://cmb.helmholtz-muenchen.de/odefy</url>.</p

Order and coherence in the fate map of the zebrafish nervous system

The zebrafish is an excellent vertebrate model for the study of the cellular interactions underlying the patterning and the morphogenesis of the nervous system. Here, we report regional fate maps of the zebrafish anterior nervous system at two key stages of neural development: the beginning (6 hours) and the end (10 hours) of gastrulation. Early in gastrulation, we find that the presumptive neurectoderm displays a predictable organization that reflects the future anteroposterior and dorsoventral order of the central nervous system. The precursors of the major brain subdivisions (forebrain, midbrain, hindbrain, neural retina) occupy discernible, though overlapping, domains within the dorsal blastoderm at 6 hours. As gastrulation proceeds, these domains are rearranged such that the basic order of the neural tube is evident at 10 hours. Furthermore, the anteroposterior and dorsoventral order of the progenitors is refined and becomes aligned with the primary axes of the embryo. Time-lapse video microscopy shows that the rearrangement of blastoderm cells during gastrulation is highly ordered. Cells near the dorsal midline at 6 hours, primarily forebrain progenitors, display anterior-directed migration. Cells more laterally positioned, corresponding to midbrain and hindbrain progenitors, converge at the midline prior to anteriorward migration. These results demonstrate a predictable order in the presumptive neurectoderm, suggesting that patterning interactions may be well underway by early gastrulation. The fate maps provide the basis for further analyses of the specification, induction and patterning of the anterior nervous system, as well as for the interpretation of mutant phenotypes and gene-expression patterns

Development of a chordate anterior–posterior axis without classical retinoic acid signaling

AbstractDevelopmental signaling by retinoic acid (RA) is thought to be an innovation essential for the origin of the chordate body plan. The larvacean urochordate Oikopleura dioica maintains a chordate body plan throughout life, and yet its genome appears to lack genes for RA synthesis, degradation, and reception. This suggests the hypothesis that the RA-machinery was lost during larvacean evolution, and predicts that Oikopleura development has become independent of RA-signaling. This prediction raises the problem that the anterior–posterior organization of a chordate body plan can be developed without the classical morphogenetic role of RA. To address this problem, we performed pharmacological treatments and analyses of developmental molecular markers to investigate whether RA acts in anterior–posterior axial patterning in Oikopleura embryos. Results revealed that RA does not cause homeotic posteriorization in Oikopleura as it does in vertebrates and cephalochordates, and showed that a chordate can develop the phylotypic body plan in the absence of the classical morphogenetic role of RA. A comparison of Oikopleura and ascidian evidence suggests that the lack of RA-induced homeotic posteriorization is a shared derived feature of urochordates. We discuss possible relationships of altered roles of RA in urochordate development to genomic events, such as rupture of the Hox-cluster, in the context of a new understanding of chordate phylogeny

Brain diversity develops early: a study on the role of patterning on vertebrate brain evolution

The brain has been one of the central foci in studies of vertebrate evolution. Work in East African cichlids and other emerging fish models like the Mexican cavefish (Astyanax mexicanus) offer new insight on the role of patterning on brain evolution. These fish can be grouped into two major categories according to habitat; for cichlids it is rock-dwelling (known locally as mbuna) and sand-dwelling (non-mbuna) lineage. The brain development of mbuna versus non-mbuna is defined by changes in gene deployment working along the dorsal/ventral (DV) and anterior/posterior (AP) neuraxes, respectively. Comparison of disparate fish ecotypes offer a new perspective of the role of patterning on brain evolution; through the slight and early modification of signal pathways working across 3-D axes, and a subsequent magnifying effect across ontogeny, evolution can generate widespread changes in the brain. To illustrate this patterning model of brain evolution, two comparative studies were done between mbuna and non-mbuna, examining the action of gene pathways that work to pattern the cichlid forebrain. The first study found that non-mbuna cichlids have a more rapid AP expansion of a gene pathway (Wingless) into the presumptive midbrain and diencephalon versus mbuna. These forebrain structures are involved in sight processing and could be of ecological benefit to vision-focused non-mbuna. The second study described a difference within the developing telencephalon. The embryonic telencephalon is split into the pallium, which processes visual signals, and the subpallium, which develops into the olfactory bulbs. Mbuna possess a larger subpallium relative to non-mbuna, which have a larger pallium. This was correlated to a more rapid expansion of another gene pathway (Hedgehog) along the DV axis. The difference in size of the pallial vs. subpallialial comparments between cichlids can be correlated to expanded olfaction in mbuna and vision in non-mbuna adult brains. Overall, East African cichlids are an excellent system to investigate the role of patterning on brain evolution because they allow for the comparison of the earliest patterning events in brain ontogeny between distinct ecotypes. These fish systems link study in brain development to the brain morphology comparisons employed in classic studies of brain evolution.PhDCommittee Chair: Streelman, Jeffery Todd; Committee Member: Condie, Brian; Committee Member: Goodisman, Michael; Committee Member: Hay, Mark; Committee Member: Yi, Sooji

Crosstalk of Intercellular Signaling Pathways in the Generation of Midbrain Dopaminergic Neurons In Vivo and from Stem Cells

Dopamine-synthesizing neurons located in the mammalian ventral midbrain are at the center stage of biomedical research due to their involvement in severe human neuropsychiatric and neurodegenerative disorders, most prominently Parkinson’s Disease (PD). The induction of midbrain dopaminergic (mDA) neurons depends on two important signaling centers of the mammalian embryo: the ventral midline or floor plate (FP) of the neural tube, and the isthmic organizer (IsO) at the mid-/hindbrain boundary (MHB). Cells located within and close to the FP secrete sonic hedgehog (SHH), and members of the wingless-type MMTV integration site family (WNT1/5A), as well as bone morphogenetic protein (BMP) family. The IsO cells secrete WNT1 and the fibroblast growth factor 8 (FGF8). Accordingly, the FGF8, SHH, WNT, and BMP signaling pathways play crucial roles during the development of the mDA neurons in the mammalian embryo. Moreover, these morphogens are essential for the generation of stem cell-derived mDA neurons, which are critical for the modeling, drug screening, and cell replacement therapy of PD. This review summarizes our current knowledge about the functions and crosstalk of these signaling pathways in mammalian mDA neuron development in vivo and their applications in stem cell-based paradigms for the efficient derivation of these neurons in vitro

From Discrete to Continuous Gene Regulation Models – A Tutorial Using the Odefy Toolbox

no abstrac

Spatial mechanisms of gene regulation in metazoan embryos

The basic characteristics of embryonic process throughout Metazoa are considered with focus on those aspects that provide insight into how cell specification occurs in the initial stages of development. There appear to be three major types of embryogenesis: Type 1, a general form characteristic of most invertebrate taxa of today, in which lineage plays an important role in the spatial organization of the early embryo, and cell specification occurs in situ, by both autonomous and conditional mechanisms; Type 2, the vertebrate form of embryogenesis, which proceeds by mechanisms that are essentially independent of cell lineage, in which diffusible morphogens and extensive early cell migration are particularly important; Type 3, the form exemplified by long germ band insects in which several different regulatory mechanisms are used to generate precise patterns of nuclear gene expression prior to cellularization. Evolutionary implications of the phylogenetic distribution of these types of embryogenesis are considered. Regionally expressed homeodomain regulators are utilized in all three types of embryo, in similar ways in later and postembryonic development, but in different ways in early embryonic development. A specific downstream molecular function for this class of regulator is proposed, based on evidence obtained in vertebrate systems. This provides a route by which to approach the comparative regulatory strategies underlying the three major types of embryogenesis

ELUCIDATING THE ROLE OF NIDOGEN IN THE FUSION OF THE CHOROID FISSURE

In the developing embryo, the timely fusion of opposing epithelial sheets into one uniform layer denotes the completion of several developmental events. Failure of this epithelial sheet fusion event (ESF) within the choroid fissure (CF) is associated with the congenital disorder Ocular Coloboma, and is one of the leading causes of pediatric blindness. A requirement for a highly coordinated dismantling of the basement membrane (BM) to allow for fusion to occur is undoubted, however the underlying mechanisms of this process are poorly understood. Due to its BM crosslinking capabilities, I have hypothesized that the regulation of nidogen plays a crucial role in the disassembly of the BM prior to ESF. Whole mount in situ hybridization for all four BM components has revealed that expression of nidogen decreases prior to that of other BM components. Additionally, preliminary IHC data has revealed nidogen and collagenIV deposition within the CF. Further, knock-down of nidogen1a and 1b, or the expression of dominant negative nidogen1b resulted in gross morphological, as well as BM organization defects in developing eyes. Together, these data suggest that nidogen plays a role in regulating the integrity of the BM of the eye and may play a role in its disassembly prior to ESF