Mark Krasnow - US grants

Homeotic genes in Drosophila such as Ultrabithorax (Ubx) and Antennapedia (Antp) select and maintain the identity of specific body segments during development, and must therefore be deployed at specific body positions at particular times in development. A regulatory network of about 30 genes, which play early roles on defining the major body axis and partitioning the animal into segments, also govern the initial patterns of expression of the homeotics, and these patterns are subsequently refined by cross- regulatory interactions between the homeotics. Maintenance of the patterns requires the action of another set of genes, the Polycomb group, and perhaps autoregulatory activities of the homeotics. The long term goal of this research is to reconstruct homeotic gene regulation in vitro from purified components, in order to determine the biochemical mechanisms of the processes by which gene expression is initiated, refined, and maintained during development. Several regulatory interactions have been recapitulated in a Drosophila cell culture system. Using a cotransfection assay in which a plasmid which express one gene product is introduced into cultured cells along with a candidate target gene, cross-regulatory and autoregulatory activities of Antp and Ubx proteins have been demonstrated. Also, the regulatory activity of fushi tarazu protein, the product of a gene involved in segmentation, has been demonstrated. Purified Ubx protein binds with high affinity to sequences in and around Antp and Ubx promoters, suggesting that at least some of these regulatory effects may be direct and mediated by protein binding to transcriptional control regions. The goals of this project are to determine the mechanism of regulation of Antp and Ubx expression by an Ubx protein, using a combined in vivo and in vitro approach and to begin to explore other aspects of homeotic gene regulation in the cell culture system. Specifically, we plan to: 1. Identify and characterize in detail the cis-acting sites of regulation of Ubx protein in the Antp and Ubx promoter regions using the cotransfection assay, and determine the relationship of these sites to the in vitro binding sites of the protein and to the regulatory sites of Antp and fushi tarazu proteins. 2. Evaluate the function of the identified regulatory sites in the developing animal. 3. Establish an in vitro system to study regulation of Antp and Ubx expression by Ubx protein, and begin biochemical fractionation of the system. 4. Attempt to reconstruct the combinatorial control of homeotic gene expression by fushi tarazu and other segmentation genes in the cell culture system.

This is a proposal to identify and characterize response pathways in Drosophila cells that are triggered by lack of oxygen (hypoxia). During the previous funding period, Dr. Krasnow has developed a Drosophila model system for studying the development of trachea, an epithelial derived tissue that delivers oxygen to larval cells. He has found that there are two cues that dictate the final pattern of the tracheal structure, which resembles a tree with a trunk and branches. First is the developmental genetic program that specifies the stereotypical primary and secondary branch patterns, and the second cue is lack of oxygen that influences the density of terminal branches. It is this second step that is the main focus of this proposal. During the past funding period, Dr. Krasnow has made a number of important discoveries regarding how cells deprived of oxygen produce signals to attract the terminal branches of trachea. The key signal appears to be the Drosophila homolog of Fibroblast Growth Factor (FGF), called Branchless. The synthesis of FGF increases in cells deprived of oxygen. The rise in FGF levels in turn attracts terminal tracheal branches which express the FGF receptor (named Breathless). He has also isolated Drosophila homologs of the two subunits of Hypoxia Inducible Factor I (tango and similar) as well as a factor (VHL) that regulates HIF-1 protein stability. These are proteins known from the work in mammalian systems to be needed for hypoxic response. In a genetic screen, Dr. Krasnow has also identified a potential response gene, named cropped. He shows that Cropped protein localizes to the nucleus in response to hypoxia. The first aim of this proposal is to determine if the five hypoxic response genes -- tango, similar, dVHL, cropped, branchless -- are members of the same or different pathways. Since they were identified by different means (although they all respond to hypoxia) they don't necessarily have to represent a single and unified response to the lack of oxygen. Cropped and HIF-1 are both transcription factors. Dr. Krasnow will first determine if Cropped transcriptionally regulates HIF-1 or vice versa. This will be done by making mutant clones of one gene and examining the expression of the other. The second question will be to ask if Cropped and HIF-1 induces Branchless FGF, the probable output signal of hypoxic conditions. Mutant clones of cropped or HIF-1 (tango) will be induced and Branchless expression will be determined. The rationale for both of these experiments is that if the lack of function in one gene compromises the expression of another gene, they are members of the same response pathway (and establishes their epistatic relationships). Another plan is to determine the relationship of HIF-1 and Branchless to nitric oxide (NO)-mediated response, another hypoxic response pathway recently described in Pat O'Farrel's lab. The second aim is to carry out comprehensive screens for genes that respond to hypoxia. Microarray chips with 8000 Drosophila cDNAs will be screened with probe cDNAs prepared from different sources (including tissue culture cells, animals of different developmental stages, and different tissues) that have been exposed to hypoxic conditions. Microarray chips will also be used to identify genes regulated by HIF-1 (and Cropped). Three criteria will be used to find genes of interest: first, genes with the same induction profile as the known HIF-1 target; two, genes induced in wildtype animals but not in HIF-1 mutant animals; three, genes induced by the over expression of HIF-1. The last aim is to identify additional hypoxia response genes using a genetic screen. The proposed F1 screen involves generating mitotic clones of mutated chromosome arms in larvae and examining the ability of mutant clones to attract terminal tracheal branches. Mutant clones that fail to attract are presumably unable to respond to hypoxia. A variation of the well established FLP/FRT system that allows examination of clones in living larvae is suggested for this purpose.

Homeotic genes in Drosophila such as Ultrabithorax (Ubx) and Antennapedia (Antp) select and maintain the identity of specific body segments during development, and must therefore be deployed at specific body positions at particular times in development. A regulatory network of about 30 genes, which play early roles on defining the major body axis and partitioning the animal into segments, also govern the initial patterns of expression of the homeotics, and these patterns are subsequently refined by cross- regulatory interactions between the homeotics. Maintenance of the patterns requires the action of another set of genes, the Polycomb group, and perhaps autoregulatory activities of the homeotics. The long term goal of this research is to reconstruct homeotic gene regulation in vitro from purified components, in order to determine the biochemical mechanisms of the processes by which gene expression is initiated, refined, and maintained during development. Several regulatory interactions have been recapitulated in a Drosophila cell culture system. Using a cotransfection assay in which a plasmid which express one gene product is introduced into cultured cells along with a candidate target gene, cross-regulatory and autoregulatory activities of Antp and Ubx proteins have been demonstrated. Also, the regulatory activity of fushi tarazu protein, the product of a gene involved in segmentation, has been demonstrated. Purified Ubx protein binds with high affinity to sequences in and around Antp and Ubx promoters, suggesting that at least some of these regulatory effects may be direct and mediated by protein binding to transcriptional control regions. The goals of this project are to determine the mechanism of regulation of Antp and Ubx expression by an Ubx protein, using a combined in vivo and in vitro approach and to begin to explore other aspects of homeotic gene regulation in the cell culture system. Specifically, we plan to: 1. Identify and characterize in detail the cis-acting sites of regulation of Ubx protein in the Antp and Ubx promoter regions using the cotransfection assay, and determine the relationship of these sites to the in vitro binding sites of the protein and to the regulatory sites of Antp and fushi tarazu proteins. 2. Evaluate the function of the identified regulatory sites in the developing animal. 3. Establish an in vitro system to study regulation of Antp and Ubx expression by Ubx protein, and begin biochemical fractionation of the system. 4. Attempt to reconstruct the combinatorial control of homeotic gene expression by fushi tarazu and other segmentation genes in the cell culture system.

The Medical Scientist Training Program (MSTP) provides a limited number of medical students with an opportunity to pursue a training program designed to equip them for careers in academic investigative medicine. Individualization of the curricular and research programs of each trainee is the hallmark of the Program. The flexible curriculum at Stanford Medical School allows each student to pursue (in consultation with her/his preceptor and other advisors) a plan of study that will satisfy the requirements for the MD degree and allow performance of doctoral level research leading to the PhD degree. Each trainee pursues an independent research program under the immediate direction of a faculty preceptor and the general supervision of an advisory committee. Additional activities include a seminar program, annual research retreat and non-medical school graduate courses. The individual and organized activities provide the trainees with fundamental biological knowledge and techniques, and serve to focus their attention toward the application of this knowledge to biomedical problems. The training received over the estimated six years of participation in the Program, followed by 1-2 years of postdoctoral training, will prepare the recipients for investigative careers in medicine. Trainees consist of those medical students, selected by the Admissions Committees of the School of Medicine and the MSTP, who are highly motivated for careers in medical science, and who are committed to in- depth training in knowledge of and experimental approaches to medical problems. They generally have extensive undergraduate training in the biological and physical sciences and represent a highly selected and gifted group of potential scientists. Eight to ten trainees per year will be admitted, reaching a steady-state level of 50 in the fifth year of this project period.

DESCRIPTION (provided by applicant): The lung is composed of two interlocking branched networks of tubes, the airways and pulmonary blood vessels, which converge in the alveoli where blood is reoxygenated. Defects in the structure of these networks or their association can cause or contribute to serious pulmonary disease, but very little is known about how these complex branched structures arise during development or how their structures are coordinated. The lung begins development as a pair of air tubes (bronchi) which are surrounded by a primitive network of blood vessels. Subsequently, millions of airway branches sprout along with a similar number of arteries and veins. A small number of genes and signaling pathways that function in the initial airway branching events have been identified, but the genetic programs that control pulmonary vascular development and coordinate its development with that of the airways have not been elucidated in the humans, mouse, or other animals. The long term goal of this grant is to elucidate the genetic program that controls the formation and branching of the pulmonary blood vessels and coordinates their development with that of the airways. The specific aims are: (1) To define the patterns and cellular mechanisms of the branching events in pulmonary vascular development and their relationship to, and dependence on, the airways and other lung tissues in the developing mouse lung; (2) To map spatial patterns of gene expression on a genome-wide scale in the developing mouse lung, including analysis of their relative expression patterns and the relationship of these patterns to the morphological and cellular events defined in Aim 1. Genes analyzed will include all known signaling molecules, receptors, and genes induced by signaling pathways, and genes implicated in blood vessel development; (3) To establish a method for misexpressing and inactivating lung development genes in highly localized regions and at different positions in the embryonic mouse lung, and to use the method to evaluate the function of two genes potentially involved in the coordination of vascular and airway development.

DESCRIPTION (provided by applicant): The long term objectives of this project is to develop a multidisciplinary career development program that will equip new MD and PhD investigators with the knowledge and skills to identify mutations that cause or predispose to lung diseases and to elucidate the roles of the affected genes in the etiology and pathogenesis of the diseases. This Program will emphasize the genetics and genomics of lung development, and in particular genes that encode components of signaling pathways that control important steps in the lung development program, and how defects in such genes and pathways can cause or contribute to lung disease. The program will train Scholars in state-of-the-art technologies in mouse as well as human genetics and genomics to allow them to move back and forth between a tractable model genetic organism [mouse) where they can more easily identify new components of pathways and determine their functions in ung development and disease, and the more challenging but clinically important human genetic and genomic studies. The Program has three major components. The first is a one-year core curriculum of graduate classes in genetics and genomics that will provide Scholars with the necessary background and conceptual and technical framework for research in this field. The second is a didactic research curriculum that supplements the coursework during the first year and provides practical and ethical information and training for carrying out this type of research to Scholars. The third part is a mentored research project carried out in the second and third years using a genetic or genomic approach in mouse or in human patient samples to identify or characterize genes in a signaling pathway and their roles in lung development and disease. Upon completion of the Program, Scholars will be equipped with the tools to carry out on their own genetic and genomic experiments aimed at identifying disease genes and their roles in pulmonary diseases and obtaining independent funding for such research. Relevance: This program will provide new investigators with the knowledge and skills to discover genes that cause or contribute to lung disease and thereby lead to new ways of diagnosing and potentially treating patients with those diseases. (End of Abstract)

DESCRIPTION (provided by applicant): Identifying and characterizing lung progenitor cells and their signaling niches is crucial for understanding how a healthy lung is built and maintained, how alteration of development and maintenance pathways cause or contribute to lung disease, and how disease can be prevented and damaged lung tissue restored or replaced. Identifying and characterizing lung progenitor cells and their niches has been hampered by the complex three-dimensional structure of the lung and the lack of tools to mark, follow the fate, and manipulate gene expression in individual lung cells in vivo. Here we propose to develop such tools, by adapting for use in mouse lung the systematic genetic approaches and single cell resolution genetic tools ("clonal analysis") that have been used over the past decade to elucidate progenitor and stem cells and their signaling niches in the model organism Drosophila. We combine this in vivo approach with a high throughput in vitro approach that takes advantage of recent advances in genomics and microfluidics to characterize individual lung progenitor cells and their developmental responses to the signals identified in the in vivo experiments. This combined approach is general and applicable to progenitor cells throughout the lung and other mouse tissues, although we focus on the poorly characterized progenitor cells in lung mesenchyme. The specific aims are: To establish a general multi-color in vivo cell marking method to follow the fate of individual mouse lung cells, and to use this method to identify the number and types of progenitors cells in the mesenchyme of developing and mature mouse lung. To use high throughput in situ hybridization studies to localize all of the signaling and receiving centers in progenitor cell niches in the mouse lung; To determine the in vivo functions of the identified signaling and receiving centers in lung progenitor cell biology by constructing a library of transgenic mouse strains that can be used to create ectopic signaling centers and clonally inactivate each signaling pathway in the mouse lung; To establish a high throughput, microfluidic approach to isolate, propagate, profile, and assess the developmental potential of identified lung progenitor cells under defined culture conditions, and to use this to determine the number of molecularly and functionally distinct progenitor cell populations in lung mesenchyme; To establish methods for directing development of lung progenitor cells along specific lineages using the high throughput microfluidic approach to expose isolated, identified progenitor cells systematically to different concentrations, gradients, temporal patterns, and combinations of signaling molecules identified in the in vivo experiments.

DESCRIPTION (provided by applicant): Identifying and characterizing lung progenitor cells and their signaling niches is crucial for understanding how a healthy lung is built and maintained, how alteration of development and maintenance pathways cause or contribute to lung disease, and how disease can be prevented and damaged lung tissue restored or replaced. Identifying and characterizing lung progenitor cells and their niches has been hampered by the complex three-dimensional structure of the lung and the lack of tools to mark, follow the fate, and manipulate gene expression in individual lung cells in vivo. Here we propose to develop such tools, by adapting for use in mouse lung the systematic genetic approaches and single cell resolution genetic tools ("clonal analysis") that have been used over the past decade to elucidate progenitor and stem cells and their signaling niches in the model organism Drosophila. To facilitate this, we will develop a new microscopy procedure (multidimensional microscopic molecular profiling) that allows the levels of dozens of molecular markers to be quantified at subcellular resolution on a single tissue section, the equivalent of multi-dimensional FACS analysis for intact tissues. We combine these in vivo approaches with a high throughput in vitro approach that takes advantage of recent advances in genomics and microfluidics to characterize individual lung progenitor cells and their developmental responses to the signals identified in the in vivo experiments. This combined approach is general and applicable to progenitor cells throughout the lung and other mouse tissues, although we focus on the poorly characterized progenitor cells in lung mesenchyme.

Small cell lung carcinoma (SCLC) is one of the deadliest cancers, a ?recalcitrant? cancer for which there is no effective treatment except when the disease is diagnosed early. However, only a small fraction of patients are diagnosed early in disease. The greatest challenge to early diagnosis is that SCLC tumor cells typically acquire an exceptional mutation burden and metastasize early, so for most patients disease has spread beyond the lung at the time of diagnosis. The key to developing effective early diagnosis and treatment methods is to elucidate the earliest molecular and cellular events of tumor initiation to uncover ones that can be detected by screening during the premalignant phase of the disease. The goal of this proposal is to define the early, premalignant molecular and cellular events of SCLC, so that they can be detected early and destroyed before they become a deadly, untreatable disease. SCLC is a neuroendocrine cancer. The prominent cell of origin is pulmonary neuroendocrine (NE) cells, neurosensory and neurosecretory epithelial cells that sense and respond to the environment in the lung. Recently, a minor subpopulation of NE cells was found to have stem cell activity, proliferating, dispersing, and replenishing the surrounding bronchial epithelium following severe airway injury. Loss of tumor suppressors Rb and p53 constitutively activates the stem cell program within days of loss of the tumor suppressors, even in the absence of injury. In this proposal, a combination of genetics, cell culture, and single-cell genomics is used to systematically interrogate these stem cells at cellular resolution, both in healthy lungs and in the early, premalignant stage of SCLC. The goal is to define the molecular events immediately following loss of Rb and p53 that constitutively activate the stem cell program and initiate their transformation into cancer stem cells that spread, mutate, and escape immune destruction, and to identify the signals they secrete that might allow the tumors to be detected before they become deadly.