Molecular control of pattern formation in the chick lung

Massachusetts General Hospital, Boston MA

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Abstract

Pulmonary malformations are common human congenital anomalies that carry a high morbidity and mortality rate. Pulmonary hypoplasia (PHP), a lung growth and patterning anomaly, accounts for the majority of neonatal mortality in otherwise salvageable cases of congenital diaphragmatic hernia. There is very little known about the molecular aspects of PHP. In this program project we exploit the fact that molecules responsible for normal lung development are conserved across animal species from fly to man and hypothesize that alterations in their function or express pattern cause PHP. Our goal is to identify which pattern the lung and may be mutated, absent or misexpressed in human PHP to find novel treatments, prevention or early diagnosis. We propose to identify and characterize these factors in three different model systems: Drosophila, chick, and rodent- and translate our findings to humans using or archives of normal and abnormal human fetal lungs and families with an affected PHP member. In Project I (herein), the avian retroviral expression system will be used to identify factors necessary for normal lung development by analysis of candidate factors in their developmental expression patterns and function by mis-, over-, and mutated expression in the chick embryonic lung in ovo. Project 1 has three specific aims: (1) Analyze the expression pattern of candidate factors in normal lung development in the chick. Candidate factors include those from the published literature implicated as candidates by their expression pattern in the developing lung or by mutations resulting in a pulmonary phenotype: secreted factors (Shh, Bmp4, Fgf10), transcription factors (Hoxa5, Hoxb5, Sox2, Nkx2.1, Gli2, Gli3), and receptors (Fgf42IIIb). In preliminary data we present new factors identified in a chick lung bud screen (1 novel factor, 2 transmembrane receptors, 1 transcription factor) we will test for developmental pulmonary expression. Additionally, we factors to be identified in the Drosophila screen (Project II) will be cloned in the chick and studied for pulmonary developmental expression. Only those candidates with spatial and/or temporally restricted expression in the developing lung will be studied in Specific Aim 2. (2) Analyze the function of candidates in chick pulmonary development. Avian specific retroviral constructs made from wild-type full-length candidate cDNAs as well as directed mutated forms will be used to mis- and/or over-express these factors in the developing avian lung in ovo. Only those factors which can phenocopy PHP in these experiments will be studied in Specific Aim 3. (3) Identify which factors are expressed in human lungs and if they are altered in expression in hypoplastic lungs. Fetal and pediatric normal and abnormal lungs are available from an archived tissue bank and will be collected prospectively. Human homologs will be obtained or cloned to analyze their expression in these tissues. Those candidates that are deemed strong will be analyzed for expression (as well as function and rescue) in rodent models of pulmonary hypoplasia (Project III) and a genetic analysis of PHP families in Project IV. We expect to identify factors which when mutated or aberrantly expressed result in PHP. This information may provide novel treatment regimes and early diagnosis making early intervention possible. We hope to increase the survival rates of infants with isolated congenital diaphragmatic hernia and PHP.