Structural robustness of mammalian transcription factor networks reveals plasticity across development.

Network biology aims to understand cell behavior through the analysis of underlying complex biomolecular networks. Inference of condition-specific interaction networks from epigenomic data enables the characterization of the structural plasticity that regulatory networks can acquire in different tissues of the same organism. From this perspective, uncovering specific patterns of variation by comparing network structure among tissues could provide insights into systems-level mechanisms underlying cell behavior. Following this idea, here we propose an empirical framework to analyze mammalian tissue-specific networks, focusing on characterizing and contrasting their structure and behavior in response to perturbations. We structurally represent the state of the cell/tissue by condition specific transcription factor networks generated using DNase-seq chromatin accessibility data, and we profile their systems behavior in terms of the structural robustness against random and directed perturbations. Using this framework, we unveil the structural heterogeneity existing among tissues at different levels of differentiation. We uncover a novel and conserved systems property of regulatory networks underlying embryonic stem cells (ESCs): in contrast to terminally differentiated tissues, the promiscuous regulatory connectivity of ESCs produces a globally homogeneous network resulting in increased structural robustness. We show that this property is associated with a more permissive, less restrictive chromatin accesibility state in ESCs. Possible biological consequences of this property are discussed.

Dynamic network modelling to understand flowering transition and floral patterning.

Differentiation and morphogenetic processes during plant development are particularly robust. At the cellular level, however, plants also show great plasticity in response to environmental conditions, and can even reverse apparently terminal differentiated states with remarkable ease. Can we understand and predict both robust and plastic systemic responses as a general consequence of the non-trivial interplay between intracellular regulatory networks, extrinsic environmental signalling, and tissue-level mechanical constraints? Flower development has become an ideal model system to study these general questions of developmental biology, which are especially relevant to understanding stem cell patterning in plants, animals, and human disease. Decades of detailed study of molecular developmental genetics, as well as novel experimental techniques for in vivo assays in both wild-type and mutant plants, enable the postulation and testing of experimentally grounded mathematical and computational network dynamical models. Research in our group aims to explain the emergence of robust transitions that occur at the shoot apical meristem, as well as flower development, as the result of the collective action of key molecular components in regulatory networks subjected to intra-organismal signalling and extracellular constraints. Here we present a brief overview of recent work from our group, and that of others, focusing on the use of simple dynamical models to address cell-fate specification and cell-state stochastic dynamics during flowering transition and cell-state transitions at the shoot apical meristem of Arabidopsis thaliana. We also focus on how our work fits within the general field of plant developmental modelling, which is being developed by many others.

Descriptive vs. mechanistic network models in plant development in the post-genomic era.

Network modeling is now a widespread practice in systems biology, as well as in integrative genomics, and it constitutes a rich and diverse scientific research field. A conceptually clear understanding of the reasoning behind the main existing modeling approaches, and their associated technical terminologies, is required to avoid confusions and accelerate the transition towards an undeniable necessary more quantitative, multidisciplinary approach to biology. Herein, we focus on two main network-based modeling approaches that are commonly used depending on the information available and the intended goals: inference-based methods and system dynamics approaches. As far as data-based network inference methods are concerned, they enable the discovery of potential functional influences among molecular components. On the other hand, experimentally grounded network dynamical models have been shown to be perfectly suited for the mechanistic study of developmental processes. How do these two perspectives relate to each other? In this chapter, we describe and compare both approaches and then apply them to a given specific developmental module. Along with the step-by-step practical implementation of each approach, we also focus on discussing their respective goals, utility, assumptions, and associated limitations. We use the gene regulatory network (GRN) involved in Arabidopsis thaliana Root Stem Cell Niche patterning as our illustrative example. We show that descriptive models based on functional genomics data can provide important background information consistent with experimentally supported functional relationships integrated in mechanistic GRN models. The rationale of analysis and modeling can be applied to any other well-characterized functional developmental module in multicellular organisms, like plants and animals.

Recent long-distance transgene flow into wild populations conforms to historical patterns of gene flow in cotton (Gossypium hirsutum) at its centre of origin.

Over 95% of the currently cultivated cotton was domesticated from Gossypium hirsutum, which originated and diversified in Mexico. Demographic and genetic studies of this species at its centre of origin and diversification are lacking, although they are critical for cotton conservation and breeding. We investigated the actual and potential distribution of wild cotton populations, as well as the contribution of historical and recent gene flow in shaping cotton genetic diversity and structure. We evaluated historical gene flow using chloroplast microsatellites and recent gene flow through the assessment of transgene presence in wild cotton populations, exploiting the fact that genetically modified cotton has been planted in the North of Mexico since 1996. Assessment of geographic structure through Bayesian spatial analysis, BAPS and Genetic Algorithm for Rule-set Production (GARP), suggests that G. hirsutum seems to conform to a metapopulation scheme, with eight distinct metapopulations. Despite evidence for long-distance gene flow, genetic variation among the metapopulations of G. hirsutum is high (He = 0.894 ± 0.01). We identified 46 different haplotypes, 78% of which are unique to a particular metapopulation, in contrast to a single haplotype detected in cotton cultivars. Recent gene flow was also detected (m = 66/270 = 0.24), with four out of eight metapopulations having transgenes. We discuss the implications of the data presented here with respect to the conservation and future breeding of cotton populations and genetic diversity at its centre of crop origin.

Transgenes in Mexican maize: molecular evidence and methodological considerations for GMO detection in landrace populations.

A possible consequence of planting genetically modified organisms (GMOs) in centres of crop origin is unintended gene flow into traditional landraces. In 2001, a study reported the presence of the transgenic 35S promoter in maize landraces sampled in 2000 from the Sierra Juarez of Oaxaca, Mexico. Analysis of a large sample taken from the same region in 2003 and 2004 could not confirm the existence of transgenes, thereby casting doubt on the earlier results. These two studies were based on different sampling and analytical procedures and are thus hard to compare. Here, we present new molecular data for this region that confirm the presence of transgenes in three of 23 localities sampled in 2001. Transgene sequences were not detected in samples taken in 2002 from nine localities, while directed samples taken in 2004 from two of the positive 2001 localities were again found to contain transgenic sequences. These findings suggest the persistence or re-introduction of transgenes up until 2004 in this area. We address variability in recombinant sequence detection by analyzing the consistency of current molecular assays. We also present theoretical results on the limitations of estimating the probability of transgene detection in samples taken from landraces. The inclusion of a limited number of female gametes and, more importantly, aggregated transgene distributions may significantly lower detection probabilities. Our analytical and sampling considerations help explain discrepancies among different detection efforts, including the one presented here, and provide considerations for the establishment of monitoring protocols to detect the presence of transgenes among structured populations of landraces.

Phenotypic evolution is restrained by complex developmental processes.

The paper, "Evolution and development of inflorescence architectures" by Przemyslaw Prusinkiewicz, Yvette Erasmus, Brendan Lane, Lawrence D. Harder and Enrico Coen [Science, 316, 1452-1456 (2007)], sets to accomplish a longstanding goal: to explain, for the first time, how and to what extent developmental constraints restrict phenotypic evolution. Prusinkiewicz and collaborators provide a relatively simple model that accounts for the variety of patterns of inflorescence architecture found among angiosperms, in which only a few of all possible types are observed.

A cDNA for nuclear-encoded chloroplast translational initiation factor 2 from a higher plant is able to complement an infB Escherichia coli null mutant.

Formation of the initiation translation complex containing the three initiation factors, IF1, IF2, and IF3, tRNA(fMet), and GTP constitutes the earliest event in the protein synthesis. IF2, a GTP-binding protein, is the principal factor involved in selecting and binding fMet-tRNA(fMet) to the 30 S ribosomal subunit. Although some chloroplast initiation translational factors have been identified and purified from algae, none of these factors have been characterized from plants. In this work, we report the molecular characterization of a nuclear-encoded chloroplastic IF2 gene from common bean (PvIF2cp). We show that the PvIF2cp gene encodes a protein containing a chloroplast translocation signal peptide, able to target a green fluorescent protein fusion protein to chloroplasts. A high accumulation of PvIF2cp transcript was found in photosynthetic tissues, whereas low mRNA levels were detected in etiolated plants and in nonphotosynthetic organs. Additional data indicate that the PvIF2cp transcript accumulation is modulated by light. The PvIF2cp gene encodes a functional factor, since the PvIF2cp conserved region, containing the G-domain and the C-terminal end, complements an Escherichia coli infB null mutation. Phylogenetic analysis using the PvIF2cp conserved region suggests that the PvIF2cp gene originated via endosymbiotic gene transfer to the nucleus and that it may be a useful marker for phylogeny reconstruction.

Conversion of leaves into petals in Arabidopsis.

More than 200 years ago, Goethe proposed that each of the distinct flower organs represents a modified leaf [1]. Support for this hypothesis has come from genetic studies, which have identified genes required for flower organ identity. These genes have been incorporated into the widely accepted ABC model of flower organ identity, a model that appears generally applicable to distantly related eudicots as well as monocot plants. Strikingly, triple mutants lacking the ABC activities produce leaves in place of flower organs, and this finding demonstrates that these genes are required for floral organ identity [2]. However, the ABC genes are not sufficient for floral organ identity since ectopic expression of these genes failed to convert vegetative leaves into flower organs. This finding suggests that one or more additional factors are required [3, 4]. We have recently shown that SEPALLATA (SEP) represents a new class of floral organ identity genes since the loss of SEP activity results in all flower organs developing as sepals [5]. Here we show that the combined action of the SEP genes, together with the A and B genes, is sufficient to convert leaves into petals.

MADS-box gene evolution beyond flowers: expression in pollen, endosperm, guard cells, roots and trichomes.

MADS-box genes encode transcriptional regulators involved in diverse aspects of plant development. Here we describe the cloning and mRNA spatio-temporal expression patterns of five new MADS-box genes from Arabidopsis: AGL16, AGL18, AGL19, AGL27 and AGL31. These genes will probably become important molecular tools for both evolutionary and functional analyses of vegetative structures. We mapped our data and previous expression patterns onto a new MADS-box phylogeny. These analyses suggest that the evolution of the MADS-box family has involved a rapid and simultaneous functional diversification in vegetative as well as reproductive structures. The hypothetical ancestral genes had broader expression patterns than more derived ones, which have been co-opted for putative specialized functions as suggested by their expression patterns. AGL27 and AGL31, which are closely related to the recently described flowering-time gene FLC (previously AGL25), are expressed in most plant tissues. AGL19 is specifically expressed in the outer layers of the root meristem (lateral root cap and epidermis) and in the central cylinder cells of mature roots. AGL18, which is most similar in sequence to the embryo-expressed AGL15 gene, is expressed in the endosperm and in developing male and female gametophytes, suggesting a role for AGL18 that is distinct from previously characterized MADS-box genes. Finally, AGL16 RNA accumulates in leaf guard cells and trichomes. Our new phylogeny reveals seven new monophyletic clades of MADS-box sequences not specific to flowers, suggesting that complex regulatory networks involving several MADS-box genes, similar to those that control flower development, underlie development of vegetative structures.

Genetic regulation of root hair development in Arabidopsis thaliana: a network model.

The root epidermis of Arabidopsis thaliana is formed by alternate files of hair and non-hair cells. Epidermal cells overlying two cortex cells eventually develop a hair, while those overlying only one cortex cell do not. Here we propose a network model that integrates most of the available genetic and molecular data on the regulatory and signaling pathways underlying root epidermal differentiation. The network architecture includes two pathways; one formed by the genes TTG, R homolog, GL2 and CPC, and the other one by the signal transduction proteins ETR1 and CTR1. Both parallel pathways regulate the activity of AXR2 and RHD6, which in turn control the development of root hairs. The regulatory network was simulated as a dynamical system of eight discrete state variables. The distinction between epidermal cells contacting one or two cortical cells was accounted for by fixing the initial states of CPC and ETR1 proteins. The model allows for predictions of mutants and pharmacological effects because it includes the ethylene receptor. The dynamical system reaches one of the six stable states depending upon the initial state of the CPC variable and the ethylene receptor. Two of the stable states describe the activation patterns observed in mature trichoblasts (hair cells) and atrichoblasts (non-hair cells) in the wild-type phenotype and under normal ethylene availability. The other four states correspond to changes in the number of hair cells due to experimentally induced changes in ethylene availability. This model provides a hypothesis on the interactions among genes that encode transcription factors that regulate root hair development and the proteins involved in the ethylene transduction pathway. This is the first effort to use a dynamical system to understand the complex genetic regulatory interactions that rule Arabidopsis primary root development. The advantages of this type of models over static schematic representations are discussed.

An ancestral MADS-box gene duplication occurred before the divergence of plants and animals.

Changes in genes encoding transcriptional regulators can alter development and are important components of the molecular mechanisms of morphological evolution. MADS-box genes encode transcriptional regulators of diverse and important biological functions. In plants, MADS-box genes regulate flower, fruit, leaf, and root development. Recent sequencing efforts in Arabidopsis have allowed a nearly complete sampling of the MADS-box gene family from a single plant, something that was lacking in previous phylogenetic studies. To test the long-suspected parallel between the evolution of the MADS-box gene family and the evolution of plant form, a polarized gene phylogeny is necessary. Here we suggest that a gene duplication ancestral to the divergence of plants and animals gave rise to two main lineages of MADS-box genes: TypeI and TypeII. We locate the root of the eukaryotic MADS-box gene family between these two lineages. A novel monophyletic group of plant MADS domains (AGL34 like) seems to be more closely related to previously identified animal SRF-like MADS domains to form TypeI lineage. Most other plant sequences form a clear monophyletic group with animal MEF2-like domains to form TypeII lineage. Only plant TypeII members have a K domain that is downstream of the MADS domain in most plant members previously identified. This suggests that the K domain evolved after the duplication that gave rise to the two lineages. Finally, a group of intermediate plant sequences could be the result of recombination events. These analyses may guide the search for MADS-box sequences in basal eukaryotes and the phylogenetic placement of new genes from other plant species.

Genetic control of flower morphogenesis in Arabidopsis thaliana: a logical analysis.

MOTIVATION: A large number of molecular mechanisms at the basis of gene regulation have been described during the last few decades. It is now becoming possible to address questions dealing with both the structure and the dynamics of genetic regulatory networks, at least in the case of some of the best-characterized organisms. Most recent attempts to address these questions deal with microbial or animal model systems. In contrast, we analyze here a gene network involved in the control of the morphogenesis of flowers in a model plant, Arabidopsis thaliana. RESULTS: The genetic control of flower morphogenesis in Arabidopsis involves a large number of genes, of which 10 are considered here. The network topology has been derived from published genetic and molecular data, mainly relying on mRNA expression patterns under wild-type and mutant backgrounds. Using a 'generalized logical formalism', we provide a qualitative model and derive the parameter constraints accounting for the different patterns of gene expression found in the four floral organs of Arabidopsis (sepals, petals, stamens and carpels), plus a 'non-floral' state. This model also allows the simulation or the prediction of various mutant phenotypes. On the basis of our model analysis, we predict the existence of a sixth stable pattern of gene expression, yet to be characterized experimentally. Moreover, our dynamical analysis leads to the prediction of at least one more regulator of the gene LFY, likely to be involved in the transition from the non-flowering state to the flowering pathways. Finally, this work, together with other theoretical and experimental considerations, leads us to propose some general conclusions about the structure of gene networks controlling development.

High population differentiation and genetic variation in the endangered Mexican pine Pinus Rzedowskii (Pinaceae).

Pinus rzedowskii is an endangered pine species from Michoacán (central México), which has been previously reported from only three localities. Classified within the subgenus Strobus, it exhibits intermediate morphological characters between subgenera Strobus and Pinus. We analyzed genetic aspects that could shed light on the evolution and conservation of this species. The genetic structure of nine populations was examined using 14 isozyme loci. Pinus rzedowskii has a relatively high level of genetic variation with 46.8% of the loci assayed being polymorphic, a total of 35 alleles, and a mean heterozygosity per population of 0.219. We calculated Wright's F(ST) statistic to estimate gene flow indirectly and to evaluate whether or not there was genetic structuring among populations. We found a marked differentiation among populations (F(ST) = 0.175) and significant inbreeding (F(IS) = 0.247). No pattern of isolation by distance was found. We also constructed a dendrogram based on a genetic distance matrix to obtain an overview of the possible historical relationships among populations. Finally, we found a convex relationship between the genetic distance among populations and the number of ancestral lineages, suggesting that demographically this species has not been at risk recently. Although endangered, with small and fragmented populations, P. rzedowskii shows higher levels of genetic variation than other conifer species with larger populations or similar conservation status.

Phylogenetics of Pinus (Pinaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences.

A 650-bp portion of the nuclear ribosomal DNA internal transcribed spacer region was sequenced in 47 species of Pinus, representing all recognized subsections of the genus, and 2 species of Picea and Cathaya as outgroups. Parsimony analyses of these length variable sequences were conducted using a manual alignment, 13 different automated alignments, elision of the automated alignments, and exclusion of all alignment ambiguous sites. High and moderately supported clades were consistently resolved across the different analyses, while poorly supported clades were inconsistently recovered. Comparison of the topologies highlights taxa of particularly problematic placement including Pinus nelsonii and P. aristata. Within subgenus Pinus, there is moderate support for the monophyly of a narrowly circumscribed subsect. Pinus (=subsect. Sylvestres) and strong support for a clade of North and Central American hard pines. The Himalayan P. roxburghii may be sister species to these "New World hard pines," which have two well-supported subgroups, subsect. Ponderosae and a clade of the remaining five subsections. The position of subsect. Contortae conflicts with its placement in a chloroplast DNA restriction site study. Within subgenus Strobus there is consistent support for the monophyly of a broadly circumscribed subsect. Strobi (including P. krempfii and a polyphyletic subsect. Cembrae) derived from a paraphyletic grade of the remaining soft pines. Relationships among subsects. Gerardianae, Cembroides, and Balfourianae are poorly resolved. Support for the monophyly of subgenus Pinus and subgenus Strobus is not consistently obtained.

Dynamics of the genetic regulatory network for Arabidopsis thaliana flower morphogenesis.

We present a network model and its dynamic analysis for the regulatory relationships among 11 genes that participate in Arabidopsis thaliana flower morphogenesis. The topology of the network and the relative strengths of interactions among these genes were based from published genetic and molecular data, mainly relying on mRNA expression patterns under wild type and mutant backgrounds. The network model is made of binary elements and we used a particular dynamic implementation for the network that we call semi-synchronic. Using this method the network reaches six attractors; four of them correspond to observed patterns of gene expression found in the floral organs of Arabidopsis (sepals, petals, stamens and carpels) as predicted by the ABC model of flower morphogenesis. The fifth state corresponds to cells that are not competent to flowering, and the sixth attractor predicted by the model is never found in wild-type plants, but it could be induced experimentally. We discuss the biological implications and the potential use of this network modeling approach to integrate functional data of regulatory genes of plant development.

Variation in the nuclear ribosomal DNA internal transcribed spacer (ITS) region of Pinus rzedowskii revealed by PCR-RFLP.

In the genus Pinus the internal transcribed spacers (ITS1 and ITS2) and the 5.8s region of the nuclear ribosomal DNA are approximately 3000 bp in length. ITS1 is considerably longer than ITS2 and partial sequences of ITS1 indicate that this region is evolving rapidly and exhibits intraspecific variation. The ITS2 and 5.8s regions are relatively conserved. We surveyed restriction fragment length variability of PCR-amplified fragments (PCR-RFLP) of the ITS region in four populations (86 individuals) of Pinus rzedowskii, a pine endemic to western Michoacán, Mexico. Five of the restriction endonucleases assayed revealed variation, with a total of 13 variants, most of which were length mutations of 300-900 bp. A moderate degree of population differentiation was detected. The average diversity (Shannon's index) of ITS fragment size patterns was 1.19, with 34% of the variation due to differences among populations and 66% due to differences among individuals within populations. The same individuals were assayed for nine polymorphic isozymes, which gave diversity measures similar to those of each restriction endonuclease.

Influence of the pituitary gland on the immune response in young rats.

The response of hypophysectomized (HYPOX) and sham-operated (S-HYPOX) female and male Wistar young rats (8 weeks old) to antigenic stimulation was compared. Humoral antigenic responses against hemocyanin were measured by ELISA. [3H]thymidine incorporation into cultured spleen cells was used to determine proliferative response to concanavalin A (ConA) or antigenic stimulation. Anti-hemocyanin serum titers in the HYPOX animals was about half of that observed in control S-HYPOX rats. Similarly, the cellular proliferative response was significantly decreased in HYPOX animals when compared to S-HYPOX rats; the blastogenic response to hemocyanin in UC rats (which did not receive the antigen injection) was close to zero. S-HYPOX control rats responded to direct ConA stimulation as UC controls. Body weight and the weight of pituitary target organs (adrenal, thyroid, ovary and testes) was about 1/4 of that of controls. Hypophysectomy also resulted in a striking reduction in spleen weight. These results indicate that the pituitary gland is involved in cellular and humoral immune regulation in young rats.

Models of patch dynamics in tropical forests.

Recent interest in the ecology and evolution of metapopulations and conservation of fragmented populations has stimulated the development of models that combine patch and population dynamics in tropical forests. One approach uses matrix models that are actual metapopulation or multi-regional demographic models. Another approach uses computer simulations to model forest succession based on the behavior of individual trees. We review applications of both types of models and suggest new combined modelling approaches.