Vivian Irish - US grants

Dr. Irish is receiving a Plant Postdoctoral Fellowship to study the developmental genetics of Arabidopsis at Yale University.

Plant morphogenesis relies on the continued proliferation and differentiation of cells derived from the shoot apical meristem. In response to a variety of signals, cells in the shoot apical meristem convert from vegetative to floral growth. This switch is accompanied by a number of morphological and biochemical changes, resulting in a change in the pattern of organ initiation and the type of organ that is formed. In addition, intercellular signals appear to be required for establishing the floral pattern. Dr. Irish proposes to investigate these processes in Arabidopsis by characterizing some of the key genes required for establishing both the floral pattern of organogenesis and specific tissue identities. She will take two approaches to address these problems. By using both molecular and genetic techniques, she hopes to clarify the pathway(s) involved in establishing the floral pattern by defining new genes required to specify the first steps in floral morphogenesis. In addition she proposes clonal analysis experiments aimed at testing the cellular autonomy of previously characterized mutations, which allow her to define the gene products involved in cell-cell signalling mechanisms required for the development of the floral pattern.

9630716 Irish The dicot flower generally consists of four whorls of floral organs, which arise through the coordinated proliferation of cells in each of the three cell layers of the floral meristem. Cell-cell interactions are important for the development of this floral pattern. These intercellular signaling events potentially include interwhorl, interorgan, and interlayer interactions. This project will investigate the extent to which each of these possible signaling mechanisms participates in establishing the floral pattern. Previous work has shown that expression of the DTA gene in the petal and stamen organ primordia results in ablation of petals and stamens. Grafing experiments, in which Nicotiana plants containing the DTA gene construct are grafted with wild type plants, will be used to examine the effects of ablating only part of the floral organ primordia. The resulting chimeric plants can be used to assess the degree to which remaining tissues can regenerate part or all of the missing patern elements. The results of these experiments will help to define the extent to which different signaling processes play in the specification of the differentiated floral pattern. In addition, the role of the floral homeotic APETALA3 (AP3) gene in cell-cell interactions in the development of the Arabidopsis flower will be defined. Using the heterologous Ac/Ds transposable element system, it will be possible to generate Arabidopsis plants that are mosaics of wild type AP3 and mutant ap3 tissues. These plants will be used to assess the role of the AP3 gene product in intercellular communication. Defining the parameters of where intercellular signals act, in combination with the analyses of the roles of individual gene products in these signaling processes, will lead to a more complete understanding of how meristematic cells coordinate their development to form the differentiated flower.

Based on the similarities in sequence and function of a number of floral homeotic genes, it has been proposed that the molecular mechanisms controlling floral development are similar across dicots, and perhaps across all angiosperm species. Nonetheless, flowers display remarkable morphological diversity, and it is thought that particular floral organ types may have evolved more than once. In particular, petals are thought to have arisen numerous times in the angiosperms. These independent origins of petals imply that the molecular mechanisms specifying petal identity may be different in different species. This proposal seeks to explicitly test the hypothesis that the expression and function of the APETALA3 (AP3) and PISTILLATA (PI) genes which are known to control petal identity in higher eudicot species may have different roles in other angiosperm clades. Three sets of experiments are proposed to begin to understand how the functions of AP3 and PI orthologous genes may have been modulated over evolutionary time. First, expression analyses of AP3 and PI orthologs will be carried out in selected angiosperm species to see if changes in their expression correlate with the independent origins of petals. Second, recent work has identified another gene, TM6, which appears to be an AP3 paralog and may also function in petal development. Loss-of-function phenotypes in TM6 orthologs in Petunia and Nicotiana will be generated and characterized to determine the role of TMd-like genes. Third, recent phylogenetic analyses have identified conserved A.P3 and PI carboxy-terminal motifs; deletion and swapping experiments will be carried out in transgenic Arabidopsis to determine the role(s) of these conserved sequences.

In Arabidopsis, two floral homeotic genes, APETALA3 (AP3) and PISTILLATA (PI), are required for the formation of petals and stamens. AP3 and PI are both expressed in petal and stamen primordia, consistent with their proposed organ identity roles. AP3 and PI both encode MADS-box proteins; the MADS-box is required for DNA binding and has been found in a number of other yeast and mammalian transcription factors. AP3 and PI bind to DNA as an obligate heterodimer, and several lines of evidence suggest that the specificity of AP3/PI action may depend on interactions with other cofactors as well as on protein modification. This proposal aims to investigate how the action of the AP3 and PI proteins is regulated. A modified one-hybrid screen is proposed to identify cofactors that interact with AP3 and PI to modulate their biological specificity. Since both AP3 and PI contain a conserved potential phosphorylation site, it is possible that protein modification may also play a role in modulating AP3/PI action. Immunoprecipitation experiments and transgenic studies will be used to ascertain whether the phosphorylation status of AP3 and/or PI plays a role in modifying their function. Finally, AP3 and PI may act in a non-cell autonomous fashion, indicating that these gene products may play a role in intercellular signaling. Plants mosaic for AP3 (or PI) will be generated using a modified Ac/Ds system. Mosaic patches generated at different times in floral ontogeny will define the temporal parameters of AP3 (or PI) action. In addition, anti-AP3 and anti-PI antibodies will be used to assess whether AP3 and PI proteins actually traffic between cells to effect their non-autonomous functions. Using complementary biochemical, genetic, and developmental approaches to elucidate the mechanisms by which AP3 and PI act will provide a framework for uderstanding how these floral homeotic gene products attain functional specificity. Furthermore, these analyses should be valuable in beginning to define the mechanisms by which plant cells communicate with each other to coordinate their growth and differentiation.

Plant MADS-box genes encode transcriptional regulators that are required for a variety of critical developmental processes, including organ identity, flowering, fruit development, root development, and pattern formation. This family of genes has diversified dramatically during the evolution of vascular plants, and especially within the angiosperms, resulting in the large number of these genes in modern flowering plant species. The problem at hand is to define the roles of these MADS-box genes, as well as to understand the evolutionary changes that resulted in their great diversity of functions. Cloning and sequencing MADS-box genes in a variety of plant species will lay the groundwork to address this question. These sequences will be used in a series of phylogenetic analyses to determine the relationships among these genes. Understanding the diversity of functions encoded by the MADS-box genes, coupled with knowledge of how these processes have been modified through the evolution of vascular plants, will be key in developing targeted strategies for manipulating the growth and development of non-model plant species.

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Irish

Flowers are a defining characteristic of the angiosperms, yet there is wide variation in floral form. Based on results from Arabidopsis and several other higher eudicot species, it has been proposed that, despite the wide variation in floral architecture, the molecular mechanisms controlling floral development are conserved across all angiosperms. This hypothesis, however, has not been critically tested. One key regulatory gene, the floral homeotic APETALA3 (AP3) gene, has been shown to be required for specifying petals and stamens in the higher eudicot Arabidopsis. The experiments described will address the larger question of whether the molecular mechanisms controlling floral development are conserved or have diverged by investigating the functions of AP3-like genes in other angiosperm species. These studies will be carried out in tomato and in opium poppy.

Phylogenetic analyses indicate that a duplication event in the AP3 lineage occurred at the base of the higher eudicots, leading to two AP3-like lineages in these species: the euAP3 lineage and the TM6 lineage. To date, all functional analyses have focussed on the role of the euAP3 lineage genes, of which the Arabidopsis AP3 gene is a member. The roles of the paralogous TM6 lineage genes are currently unknown and will be addressed by carrying out functional studies in tomato, a species for which a number of genetic and molecular tools exist. These investigations will include screening for insertional mutations in the TM6 gene, transgenic manipulation of the TM6 gene, and genetic experiments utilizing a previously identified tomato AP3 insertional mutation. The lower eudicots and basal angiosperms contain only one AP3-like lineage. These genes are more similar in sequence to the higher eudicot TM6 genes and are termed the paleoAP3 lineage genes. The function of a paleoAP3 lineage member will be investigated using opium poppy as a model system. These studies will include cloning and characterizing the opium poppy paleoAP3 gene and determining whether any of the previously identified poppy floral homeotic mutations correspond to lesions in this gene. In addition, expression studies will be carried out in order to characterize the processes controlling floral development in this species.

The results obtained from these studies will be valuable in shedding light on how gene duplication and diversification are related to changes in gene function. They will also provide a basis for developing new and more rigorous hypotheses to explain, at the molecular genetic level, how morphological innovations in floral form have arisen.

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Irish, V.F.

Floral organs arise as lateral outgrowths from the florally determined meristem, and differentiate with distinct identities. The specification of these organs as sepals, petals, stamens or carpels depends on the action of several floral homeotic genes which act as master regulators of these developmental pathways. Two such genes, APETALA3 (AP3) and PISTILLATA (PI), encode MADS domain-containing transcription factors that regulate the specification of petal and stamen identity in Arabidopsis. The proposed work will focus on identifying other genes involved in petal and stamen development, with a particular emphasis on identifying several candidate target genes regulated by AP3 and PI. The analysis of such genes can serve as a model for dissecting the genetic hierarchies by which particular organs and tissue types are specified during plant development.

In order to identify a set of target genes regulated in vivo by these transcription factors, Dr. Irish proposes three complementary approaches. Microarray analyses comparing the expression of a set of Arabidopsis ESTs in various mutant backgrounds will be carried out in order to define those genes that are up- or down-regulated in response to AP3 and PI action. This should define a set of reasonably highly expressed petal and/or stamen specific genes that are directly or indirectly regulated by these floral homeotic gene products. Second, chromatin immunoprecipitation will be employed to identify DNA sequences that are bound in vivo by AP3/PI proteins. Sequences identified by this approach are good candidates for being direct targets of AP3/PI regulation. Third, a gain-of-function genetic approach will be employed, using activation tagging, to mutationally identify genes that are involved in the floral developmental pathway. This approach has the advantage that it can result in the recovery of downstream target genes that act in a redundant fashion, which would preclude their recovery by traditional forward genetic screens.

Potentially hundreds of candidate target genes may be recovered by these three approaches. These approaches are not meant to be exhaustive, as it is likely that only abundantly expressed genes are likely to be recovered. Dr. Irish plans to initially focus further analyses on a limited number of candidates, focussingon those recovered by more than one method. Such genes will be examined to determine whether their transcription is directly dependent on AP3/PI function. This will define a set of direct targets that will in turn be valuable in beginning to assess the spectrum of developmental processes controlled by AP3/PI. In turn these analyses will shed light on the question of whether the floral homeotic gene products act at the top of a long regulatory cascade, or whether these gene products act directly to regulate a wide array of downstream targets responsible for various aspects of morphogenesis. Characterizing how such master regulatory genes effect their functions will be crucial for a detailed understanding of how organogenesis and morphogenesis ensue, and should serve as a model for these processes in other plant systems.

The completion of the Arabidopsis genome sequence has led to the very real possibility of translating genome level information from this model system to many non-model species of agronomic interest. However, this can be hampered by inaccurate or incomplete comparisons between species. In particular, it is vital to accurately assess orthology (genes in different species originating from a single gene in the last common ancestor of the species) versus paralogy (genes arising from a gene duplication event). In general, this requires identifying all related gene family members in the species being compared. Because gene functions are often conserved between orthologs, the assessment of orthology versus paralogy has important implications for hypothesizing gene functions in crop species where traditional forward or reverse genetic techniques are not easily employed

This SGER proposal seeks to develop a redundant oligonucleotide hybridization and PCR-based method to comprehensively identify all members of a gene family in a target crop species for which only BAC resources exist. Having this information will enable robust phylogenetic comparisons of such gene families between species, and identify genes that are likely to confer useful traits. This proposal focuses on identifying all members of the large type 2 MADS box gene family from Lycopersicon esculentum (tomato) to carry out detailed studies of gene family diversification. If successful, the strategy described in this proposal should be generally applicable to most genes families from virtually any species for which a high quality BAC library exists.

A major objective of post-genomics research is to unravel the transcriptional networks that control complex morphological changes. The research described in this proposal will provide a comprehensive approach to dissecting the transcriptional circuitry involved in Arabidopsis petal organogenesis. Previous work has resulted in the identification of transcription factors that coordinately control many aspects of petal development as well as a number of candidate genes involved in various aspects of petal organogenesis. Several complementary approaches, including molecular, biochemical, genetic and computational analyses will be used to characterize the transcriptional regulatory networks controlling petal organogenesis. Experiments will be carried out in order to define the extent to which the petal identity pathway intersects genetically with the petal outgrowth pathway; coordination of growth and specification is a necessary prerequisite for normal organogenesis. Computational approaches will be used to elucidate conserved sequence features and the intersection of identified regulatory networks. Petals are ideally suited for such analyses, since petals are simple laminar organs that are dispensable for growth and reproduction. Since petals can serve as a simple model for studying other, more complex plant organs, the work described here should provide a paradigm for understanding the dynamics of organogenesis in more intricate systems. Part of the described work will be carried out by undergraduates from minority and underrepresented backgrounds; this will expose promising young students to scientific research in the sciences. Training such students in the methodologies and approaches of basic research will facilitate their involvement in future scientific endeavors.

Vivian F. Irish
Proposal # IOS-0817744
Dissecting gene regulatory networks controlling petal organogenesis


The formation of an organ depends on the coordination of a variety of cellular processes, including cell division, tissue organization, and patterning of the organ as a whole. Plant organogenesis is not well understood, despite the importance of plants as sources of food, and increasingly, fuel. This project focuses on analyzing the regulation of petal organogenesis in Arabidopsis as a model for understanding how other, more complex tissues and organs are generated. Several complementary approaches will be taken to identify new genes involved in petal organogenesis. These will include microarray-based approaches to identify genes required for the initial stages of petal organogenesis; genetic strategies to identify quantitative trait loci controlling petal shape and size; and molecular approaches to identify microRNA-dependent processes controlling petal organogenesis. Genetic and molecular interactions between newly discovered and previously identified genes will define pathways essential for petal organogenesis. These results will be used to develop a genome-level regulatory network that functions in controlling different steps in petal organogenesis. In turn, characterizing the petal organogenesis network will be valuable in developing a systems level understanding of plant organogenesis, and the emergent properties of such a system. This interdisciplinary approach will provide a unique perspective as to how the spatial and temporal aspects of cell division, cell type differentiation, and the coordination of growth and development result in the formation of a particular organ type. Furthermore, this comprehensive characterization will also be important for generating new tools to understand and manipulate organogenesis in other, economically important plant species. A component of this project is to provide a variety of training opportunities for a postdoctoral associate, a graduate student, and undergraduates that will foster critical analytical skills in the next generation of researchers.

DESCRIPTION (provided by applicant): The proposed program will provide training of thirteen predoctoral students in developmental biology using all available modern approaches. This training program emphasizes six interdisciplinary areas: 1) organ system development;2) development of model organisms;3) model genetic systems (including Caenorhabitis, Drosophila, mouse, zebrafish, and Arabidopsis);4) morphogenesis, cell shape and cell movement;5) nucleic acid studies;and 6) developmental evolution. These development biology research areas bear on many aspects of molecular and cellular biology, genetics, and genomics. We believe that our integrated approach, using developmental genetic model systems in combination with multiple approaches, is particularly relevant. This is in light of the highly conserved nature of developmental regulatory systems, as well as the revolution of technologies useful for development biology research. The program is the only University-wide program in development biology, involving faculty from 3 departments in the Yale College of Arts and Sciences (FAS) and 6 departments/programs from the Yale School of Medicine. There are a total of 42 trainers in the program. Trainees are admitted to the Yale Graduate School by a Combined Program in the Biological and Biomedical Sciences (BBS). This admissions and training program has resulted in a steady increase in the size of the applicant pool, the yield, and the quality of pre-doctoral matriculants. The BBS also provides matriculants maximum flexibility in fashioning a personalized course of graduate studies leading to the PhD degree. Students may be appointed to the training program during their first, but more likely their second year. At all stages of training, the trainees are mentored by an advisor and an advisory committee, although, the composition of the advisors may change during progress toward graduation, especially after they advance to PhD candidacy. Trainees who enter this program will ordinarily hold at least a B.A. or B.S. degree in biology, biochemistry, chemistry, physics or related sciences. They will be selected on the basis of performance in undergraduate studies, letters of recommendation, GRE scores, and interviews. Students with strong potential for research will be sought. We will rely heavily on letters of recommendation and personnel interviews where feasible. After receiving the PhD degree, most of our trainees will spend two or more years in postdoctoral research, after which they take up positions as faculty members in medical schools, universities or colleges, or as research staff in medical centers, scientific research institutes, or biotech firms. The record of graduate students going on to run academic laboratories in development biology is historically strong and continues to be so. Graduate study in basic developmental research is today exciting and intellectually rewarding. The research is also satisfying and appealing, since it contributes increasingly to the solution of the human scourges such as birth defects, chronic diseases such as Alzheimer's and oncologies, and inadequate food production. For these reasons, the subject attracts top caliber students.

Vivian F. Irish
IOS-1020843
Evolution of regulatory pathways controlling stem cell proliferation

Stem cells undergo self-renewal as well as producing daughter cells that go on to differentiate into multiple cell types. Thorns represent a unique and previously unexplored opportunity to examine how stem cell proliferation is controlled in plants. Thorns arise from populations of stem cells that, instead of maintaining a stem cell fate, lose their proliferative capacity and terminally differentiate. This project will examine the underlying basis for the arrest of stem cell proliferation in thorns. The first set of goals focus on analyzing known components of the stem cell identity pathway, and determining if these pathways have been utilized during thorn development in Citrus sinensis (sweet orange) and Ulex europaeus (gorse). The second set of goals focus on using high throughput transcriptome analyses to identify novel genes required for restricting stem cell proliferation in Citrus sinensis thorns. Together, these approaches should identify new genes and processes that are critical in the transition from stem cell proliferation to differentiation. By comparing the processes controlling stem cell arrest in thorns to those regulating stem cell proliferation and arrest in other systems, the conserved, as well as divergent, pathways that control stem cell activity can be elucidated. This information will also be valuable in comparing plant stem cell regulation with the processes controlling mammalian stem cell maintenance and differentiation. In addition, this project will serve to train minority and disadvantaged undergraduate students in molecular genetics and developmental biology, thus broadening the participation of underrepresented groups in scientific research.

Stem cells undergo self-renewal to produce more stem cells, as well as to produce daughters that go on to produce multiple specialized cell types. In plants, cells of the shoot apical meristem (SAM) act as stem cells, giving rise to more stem cells as well as specialized cell types contributing to the leaves, branches and fruit. This study investigates the basis for thorn development in Citrus as a means to understand how the termination of stem cell proliferation is controlled. Thorns arise from SAM cells that fail to self-renew, and instead terminally differentiate, providing a unique opportunity to explore how SAM cell proliferation is controlled. Citrus is an excellent study system to explore this question as many Citrus species possess thorns, and multiple genetic and genomic tools exist for work with Citrus. In this study, candidate genes from Citrus that have been implicated in regulating stem cell proliferation will be examined for their roles in thorn development using expression analyses and employing newly developed transgenic approaches. In a complementary set of experiments, key genes involved in the arrest of stem cell proliferation will be identified via genome-wide comparisons of the transcriptomes of thorned vs. thornless Citrus species. These analyses will begin to define the processes important for stem cell arrest. They will also elucidate conserved and novel mechanisms of stem cell maintenance and differentiation. Citrus plant growth, fruit yield and harvest costs are all affected by thorniness, so understanding how to manipulate thorn production will greatly impact the economics of this valuable fruit crop. A postdoctoral associate, a graduate student and three undergraduates will receive training as a part of this project. In addition, a display of Citrus biology will be mounted at the Marsh Botanical Gardens, New
Haven, CT, aimed at promoting public awareness and understanding of science.

The study of surface patterning of plant cells is critical to understanding their function. These range from helping pollinating insects grip petals to decreasing the wettability of plant cell surfaces. In addition, understanding how this cell architecture is achieved can provide new approaches in nanotechnology and the design of novel bioinspired materials. This project combines genetics, biochemistry, and mathematical approaches to investigate how cell wall associated pectin molecules affect plant cell shape and surface pattern and contribute to the mechanical properties of plant cells. Through this project, undergraduates from underrepresented backgrounds, a graduate student and a postdoctoral associate will receive interdisciplinary training at the interface of experimental biology and mathematical modeling. In addition, the results from this project will be communicated to the public through outreach programs aimed at high school students and at the New Haven area community.

Recent work from the PI's lab has shown that mutations in the gene encoding Rhamnose Biosynthesis 1 (RHM1) affect pectin composition and show striking patterning defects in cell morphology, resulting in cells with a left-handed helical twist and a reduction in anisotropic growth. While pectins have been shown to play important roles in regulating cell expansion, this is the first described helical mutant that affects pectin biosynthesis, and so provides an exciting new inroad into elucidating the potential roles of rhamnose-containing pectins in modulating plant cell shape. Through a combination of genetic and biochemical approaches with micro-mechanical testing and mathematical modeling, the role of rhamnose in controlling cell architecture will be elucidated. The results obtained through this project will have the potential to shed new light on how pectin composition can affect cell wall patterning and growth, providing an exciting link between enzyme activity, cell wall architecture, and supracellular patterning events. The combination of experimental approaches with testable mathematical models will lay the groundwork for understanding how the pectin gel matrix can feed back on cellular and subcellular processes necessary to regulate cell growth and form.