Date: December 10, 2005 2:05:15 AM EST

To: ybrun@indiana.edu

Subject: Recommendation Letter for F. Hernan Espinoza

December 9, 2005

Yves Brun

Systems Biology Faculty Search

Department of Biology, Indiana University

Jordan Hall 142

1001 E 3rd Street

Bloomington, IN 47405-7005

Dear Search Committee:

This is a recommendation letter for Dr. Hernan Espinoza, an applicant for a faculty position. Hernan has been a postdoctoral fellow in my lab for the past seven years, during which he initiated and has been carrying out an ambitious plan to map the gene expression program of the developing mouse lung, and has recently completed a heroic genome-wide screen for all the signaling and receiving centers in embryonic mouse lung. Before coming to the lab, Hernan trained in biochemistry and is an accomplished biochemist and molecular biologist from his graduate work at UCSF with David Morgan, where he made several significant advances in understanding CDK cell cycle kinases. He then decided to broaden his training into developmental biology, genetics, and genomics and came here to pursue a project in lung development.

Despite the importance of the lung, surprisingly little is known about the genetic program that controls its growth and development. For over a dozen years, my lab has been dissecting the program of respiratory system development in Drosophila. Hernan, together with an outstanding MD/PhD student, Ross Metzger, embarked on a related but even more challenging project aimed at elucidating the genetic program of mouse lung development. Hernan's plan was to generate a genome-wide view of the gene expression program of the developing lung. Only a handful of genes have well-established functions in lung development, and nearly all of these were first implicated by studies showing that the genes are expressed in spatial patterns coincident with the events they control. This suggested that an expression-based strategy would succeed in identifying other important genes. Hernan took advantage of progress in mouse genomics to design a genome-wide, expression-based screen for lung development genes, concentrating on genes encoding ligands and receptors because of their critical roles in developmental control.

Hernan carried out a bioinformatic analysis to identify all the known and predicted ligand and receptor genes in the mouse genome. He found 350 ligand genes and 600 receptor genes, excluding olfactory and taste receptors. He then obtained cDNA clones for about two-thirds of these genes from public collections and confirmed them by DNA sequencing; he cloned the rest of the genes by PCR. He also worked out techniques for rapid isolation and fixation of embryonic mouse lungs of defined stages, for parallel synthesis of 96 in situ hybridization probes, and for in situ hybridization of whole mount lungs with a custom 96-well staining platform, one probe and one lung per well. He developed software for recording, analyzing, archiving, and presenting the expression patterns. Using these approaches, he isolated thousands of lungs and examined the spatial expression patterns in early lung development of all of the ligand and receptor genes. He found 40 ligand genes expressed in 23 different spatial patterns; these may represent most or all of the localized signaling centers in the early lung. He identified 48 receptor genes expressed in 24 different spatial patterns (15 of which are the same as a ligand pattern); these may represent most or all of the receiving centers. Of the 88 ligand and receptor genes with localized expression, only 13 were previously known. By comparing the expression patterns to each other and to the developmental events that are occurring, and drawing on knowledge of the functions of the signaling pathways in other systems, we are generating specific hypotheses about what aspects of the lung development process the identified signaling pathways control. Some of the patterns are highly specific and suggest roles for the genes in induction and patterning of airway branches and development and patterning of the pulmonary vasculature. Hernan and his collaborators in lab are beginning to test some of these hypotheses by examining the lung phenotype of genes for which a knockout is available. For genes in which the knockout causes early lethality or pleiotropic effects, we are developing methods for highly localized gene inactivation or misexpression in lung.

In addition to the ligand and receptor genes, Hernan has examined the spatial expression patterns of over a thousand other genes from a mouse lung cDNA library. He has found 190 genes expressed in 41 different patterns, 30 of which coincide with a signaling or receptor center and hence are good candidates for downstream genes controlled by the signaling pathways, or upstream genes that control expression of the signaling genes.

Hernan's work is a tour de force for lung development, and for mammalian developmental biology. It provides the first global analysis of signaling and receiving centers in the developing lung, indeed it is the first for any tissue in any organism. It has started to revolutionize the study of lung development, by associating specific signaling pathways with specific developmental events, and by providing the first genome-wide view of the organization of the lung development program. It also has long term potential beyond lung development, for example in aiding diagnosis and treatment of lung disease, by helping identify the signaling processes that have gone awry in the disease state and by providing molecular markers that could help distinguish diseased from normal lung tissue. As we prepare Hernan's work for publication, I have begun presenting it at a few meetings. The response has been incredible. Beyond the awe at the scale of his project and accomplishments, there have been multiple requests to collaborate on specific projects on mouse lung biology as well as requests to collaborate on other organs and aspects of mouse development and biology using Hernan's expertise and the resources he has developed.

Hernan is a highly motivated, hard working scientist, and a skilled experimentalist and computational biologist. There are few with the long term scientific vision to set out on an ambitious project like this, and few if any with the scientific enthusiasm, dedication, and focus to complete it. Hernan is smart and widely read, and he has enjoyed both the intellectual challenge of discovering the lung development program, and the technical challenge inherent in carrying out a genome-scale project. His progress is particularly impressive considering that neither he nor my lab had experience in this area before he began, so he had to develop everything from scratch. His pioneering work helped him obtain a senior fellowship from the Frances Family Foundation to complete the project, and it helped the lab obtain a major NIH grant for the project, which has provided Hernan with supplies and, for the past year and a half, two research assistants to assist him.

On a personal level, Hernan is extremely kind and helpful to all, very community minded and a delight to have in lab. He is an enthusiastic participant in lab discussions and journal clubs, and he is full of innovative ideas that he is glad to share. He has an irrepressible optimism and outlook on science and life, which allow him to get past any obstacles he encounters. He has been a terrific supervisor and mentor to his two research assistants.

I think Hernan's ability to thrive in the two very different research areas of his graduate work and postdoctorate demonstrates his versatility and potential as an independent scientist. He has the ability to identify the key questions and experimental approaches in a field and to tackle them effectively. He is a natural at applying new technologies to important biomedical questions, and I think he would thrive in an interactive department or institute that can build on his expertise and the genomic foundation he has established for the field of lung development and mouse biology. I am confident that he will succeed in his long term plan of elucidating the gene expression program of lung development, and continue to make important contributions to the understanding of lung development and disease throughout his career. In addition, Hernan is an underrepresented minority and would be an outstanding role model and mentor for other Latino scientists.

Sincerely,

Mark Krasnow, M.D., Ph.D.

Professor and Associate Chairman

650-723-7191 Office

650-723-6783 Fax