Elizabeth D. Hay - US grants

The effect of extracellular matrix (laminin, fibronectin, collagen, proteoglycans) on the differentiation of embryonic corneal epithelium (avian) is being studied. We are especially interested in the effect of these molecules on the configuration of the basal epithelial surface and the organization of the basal epithelial cytoskeleton. We are using heavy meromyosin fragments (S1) to analyse the orientation of actin filaments in cells before and after exposure to matrix molecules. We are using immunohistochemistry and biochemical techniques to follow the differentiation of the epithelium. The effect of matrix molecules on fibroblast shape and migration is also being studied, using similar methods and including Nomarski optics. Avian fibroblasts from cornea, skin, heart, and limb will be compared.

The research proposed in this grant is expected to contribute to understanding mechanisms of differentiation of eye tissues, with particular emphasis on cornea and lens. We describe a gene program for epithelial- mesenchymal transformation (EMT) in normal development that we believe is turned on abnormally in certain optic pathologies. This gene program may be turned on in part for motility (as in primary mesenchyme) or in whole to create true fibroblasts (as in secondary mesenchyme). The outline of the work is as follows. I.Epithelial-mesenchymal transformations contributing to normal eye development. The cephalic neural crest arises by epithelial-mesenchymal transformation from the cranial neural folds. We will use beta- galactosidase expressing, replication incompetent retroviruses to label cells to find out if one group of crest cells (more like secondary mesenchyme) unique to the mouse or chicken head gives rise to corneal fibroblasts and another (more like trunk crest) to corneal nerves and conjuntival melanocytes. Also, we will learn the mesenchyme of origin of the corneal endothelium. Using immunohistochemistry and in situ hybridization, we will look for expression of genes for molecules that might cause EMT: CAMs (cell adhesion molecules), SAMs (substrate adhesion molecules), protooncogenes. II.Tissue phenotype transitions by corneal fibroblasts and epithelia. We expect to be able to transform differentiated corneal fibroblasts to epithelioid cells by transfection with cDNAs for E-cadherin, alpha6beta1 integrin, and syndecan, and to transform differentiated corneal epithelial and endothelial cells to fibroblasts by transfection with c-src and c-mos DNAs, and antibodies to cadherins and syndecan or their antisense DNA. We will examine the transformed cells to assay whether parts or all of the tissue phenotype is turned on or off by a given gene. III.Epithelial-mesenchymal transformation from lens and cornea in collagen gels. We will examine by the retrovirus-label method the developmental potentials of the mesenchymal cells that form from lens and corneal epithelia in gels. Since they look like secondary mesenchyme, we expect they will form cartilage. CAMs, SAMs, and protooncogenes will be examined in the lens epithelium before and after it is induced to give rise to mesenchyme in collagen gels. We will use subtractive hybridization to search for the mesenchymal master gene(s) that is turned on during collagen-induced transformation of lens to fibroblasts and compare its sequence(s) with those of CAMs, SAMs, protooncogenes, Homeobox genes, growth factors, and other candidates to be master genes. IV. Epithelial-mesenchymal transformation in eye pathologies. We describe relevant CAMs, protooncogenes, and master genes in preinvasive and invasive conjunctival tumors, and metaplasias of retinal pigmented and nonpigmented epithelia. We investigate the interesting possibility that the gene program for metastasis is an abnormally reactivated embryonic program for epithelial-mesenchymal transformation.

This grant proposal is about the development of the palate, with special attention to the role of the medial edge epithelium (MEE) and cranial neural crest mesenchyme. Mesenchymal cells arise by epithelial-mesenchymal transformation (BMT). They have the ability to invade collagen gels and other extracellular matrix (ECM), whereas epithelia sit on top of ECM and exhibit apical-basal polarity. Mesenchymal cells migrate through the matrix with front end/back end polarity. They differentiate into the cells that inhabit the ECM interior: fibroblasts, bone, melanocytes, and neurones. We will consider the following aspects of the origin and differentiation of the craniofacial mesenchymes that contribute to palatal morphogenesis. I. Mesenchymal Cell Lineages in the Developing Palate. Using recombinant retroviruses to label specific groups of cells in the embryo, we expect to confirm and extend our earlier study that MEE-derived mesenchyme forms fibroblasts but not bone, and to prove for the first time that head neural crest in the rodent consists of two physically separate cell populations, one of which forms connective tissue and the other, melanocytes and neurones. The significance of this study to students of palate morphogenesis is that it establishes a multiple origin of palate mesenchyme and calls attention to the fact that palate MEE does not die as previous authors thought. It casts a new light on roles the MEE might play in cleft palate. II. Effect of Bone Inducing Molecules on the Differentiation of Mesenchymal Cell Lineages. We will attempt to drive the cells above that do not normally form cartilage/bone to do so by administering BMP to them in vitro. It will be particularly interesting to see if mesenchyme formed from palate epithelium and neurogenic crest cells will undergo chondrogenesis under the influence of this powerful inducing agent. The results could have important impact on our ideas about the ability of "determined" cells (e.g., neurogenic crest) to reawaken apparently closed pathways of differentiation. III. Induction and Inhibition of Epithelial Fusion and EMT in Nonfusing and Fusing Palates. Chicken palates do not normally fuse probably because the MEE stratifies and periderm does not slough to expose the basal epithelia to each other. We will surgically remove the periderm and place chicken palatal shelves in close contact to try to induce formation of a midline seam. TGFbeta3 will be added to promote EMT from this seam and the fate of mesenchymal cells followed. Antibodies and antisense oligonucleotides to TGFbeta1-3 will be added to try to inhibit EMT from MEE of fused rodent palates. The recognition our recent work has given to the fact that MEE transforms to mesenchyme establishes a whole new way of looking at the possible role of TGFbeta's and sloughing of the outer layer of the MEE in palatogenesis. The immediate health relevance is to understanding normal development, but the work could give new insights into causes of congenital anomalies, especially cleft palate.

The research proposed in this grant is expected to contribute to understanding mechanisms of differentiation of eye tissues, with particular emphasis on cornea and lens. We describe a gene program for epithelial- mesenchymal transformation (EMT) in normal development that we believe is turned on abnormally in certain optic pathologies. This gene program may be turned on in part for motility (as in primary mesenchyme) or in whole to create true fibroblasts (as in secondary mesenchyme). The outline of the work is as follows. I.Epithelial-mesenchymal transformations contributing to normal eye development. The cephalic neural crest arises by epithelial-mesenchymal transformation from the cranial neural folds. We will use beta- galactosidase expressing, replication incompetent retroviruses to label cells to find out if one group of crest cells (more like secondary mesenchyme) unique to the mouse or chicken head gives rise to corneal fibroblasts and another (more like trunk crest) to corneal nerves and conjuntival melanocytes. Also, we will learn the mesenchyme of origin of the corneal endothelium. Using immunohistochemistry and in situ hybridization, we will look for expression of genes for molecules that might cause EMT: CAMs (cell adhesion molecules), SAMs (substrate adhesion molecules), protooncogenes. II.Tissue phenotype transitions by corneal fibroblasts and epithelia. We expect to be able to transform differentiated corneal fibroblasts to epithelioid cells by transfection with cDNAs for E-cadherin, alpha6beta1 integrin, and syndecan, and to transform differentiated corneal epithelial and endothelial cells to fibroblasts by transfection with c-src and c-mos DNAs, and antibodies to cadherins and syndecan or their antisense DNA. We will examine the transformed cells to assay whether parts or all of the tissue phenotype is turned on or off by a given gene. III.Epithelial-mesenchymal transformation from lens and cornea in collagen gels. We will examine by the retrovirus-label method the developmental potentials of the mesenchymal cells that form from lens and corneal epithelia in gels. Since they look like secondary mesenchyme, we expect they will form cartilage. CAMs, SAMs, and protooncogenes will be examined in the lens epithelium before and after it is induced to give rise to mesenchyme in collagen gels. We will use subtractive hybridization to search for the mesenchymal master gene(s) that is turned on during collagen-induced transformation of lens to fibroblasts and compare its sequence(s) with those of CAMs, SAMs, protooncogenes, Homeobox genes, growth factors, and other candidates to be master genes. IV. Epithelial-mesenchymal transformation in eye pathologies. We describe relevant CAMs, protooncogenes, and master genes in preinvasive and invasive conjunctival tumors, and metaplasias of retinal pigmented and nonpigmented epithelia. We investigate the interesting possibility that the gene program for metastasis is an abnormally reactivated embryonic program for epithelial-mesenchymal transformation.

DESCRIPTION (adapted from the Investigator's abstract): The broad objective of this application is to understand the tissue transitions involved in the development of the palate and craniofacial mesenchyme. Transformation of the medial edge epithelia (MEE) to mesenchyme in the paired palatal shelves from avian and rodent models will be examined. This represents a model for embryonic tissue remodeling and has potential importance to better understand failure of the palatal shelves to fuse in humans. The Principal Investigator's laboratory was the first to propose and demonstrate that the MEE undergoes epithelial-mesenchymal transformation (EMT) after fusing to form a midline seam, and that the resulting mesenchymal cells become part of the palate connective tissue, thus establishing confluence of the palatal stroma. It is likely that EMT is involved in other craniofacial processes where epithelial fuse, consequently studies are proposed to examine the formation of the upper lip. Understanding the cellular mechanism of palatal EMT will result in new approaches to defining the causes of congenital facial anomalies, such as cleft palate and other facial clefts that may result from failure of the EMT process. The specific aims are to study (1) the role of cell-cell contacts between opposing palatal shelves in initiation of EMT in the MEE; (2) the role of cell-matrix interactions and TGF-beta3 in the emigration phase of EMT; and (3) the role of defective EMT in cleft palate and in cleft lip. It is expected that beta-catenin and plakoglobin associated with the newly formed MEE junctions are the signaling molecules involved in initiating palatal EMT. Also it is expected that matrix-stimulated tyrosine kinases interact with growth factors at the plasmalemma level to promote emigration. Knowledge of the basic cell biology will lead to more effective treatment of human facial clefting.

DESCRIPTION (provided by applicant): Epithelio-Mesenchymal Transition (EMT) has been long recognized as an essential, highly regulated, and often employed mechanism in embryological development, used whenever epithelial cells need to translocate. Many eminent embryologists work in this well-established area. Early indications over the past 2 decades that EMT may also play an important role in carcinoma metastasis have recently matured, with high-level reports documenting the progression-like consequences of EMT in various cancer systems. This has heralded a growing acceptance of EMT in the cancer field, overcoming initial skepticism. Similarly, the role of EMT in other pathologies, including renal fibrosis and cataracts, is becoming clear. However, much is still to be learned about the regulation, roles, and critical targetable features of EMT. Many of the approaches developed and refined for carcinoma research can also be well-utilized by developmental biologists and pathologists in their EMT studies, and vice versa. In particular, acknowledgment of the critical role of microenvironmental cues in regulating both carcinoma and developmental EMT pave the way for the prioritization of studies in this area. Facilitated interaction between developmental and cancer biologists and pathologists is essential to maximize the impact and benefit of the combined wisdom accumulated in these domains. The proposed meeting is groundbreaking in that it aims to do just that. An outstanding International Organizing Committee has been recruited for this timely meeting, an attractive and isolated venue has been chosen, dates set, and sponsorship requests initiated. Karger, who published 2 issues of Acta Anat. on this topic several years ago, has agreed to publish the proceedings. The meeting will feature 3 solid days of symposia, an afternoon devoted to roundtable discussion group, and two other integration sessions. Gender balance has been prioritized and largely achieved, and a number of talks will be selected from submitted abstracts where emphasis again will be placed on women and minorities. Our goal is for this conference to bring together a diverse international group of academic and industrial scientists with interests ranging from basic molecular, cellular and physiological mechanisms of EMT action, to physician-scientists interested in EMTs as therapeutic targets. The relatively small size, focused Scientific Sessions, and continuous Poster Session combine to provide an exciting forum for the exchange of ideas and to provide extensive opportunities for participation by a diverse group of scientists interested in EMTs in cancer, development, and a host of pathologies. We request NIH support primarily for travel for emerging USA scientists in this area, and to support the travel of two more senior, distinguished US speakers to ensure that the most relevant and up-to-date information is featured at this pivotal and highly influential meeting.