Cloning and sequencing of genes that control lung branching morphogenesis

grantor: University of TorontoThe mammalian lung develops from the primitive foregut. The primordium of the respiratory system appears in the developing rat around 11 to 12 days (term = 22 days) with the formation of two buds which undergo dichotomous branching. Although transcription (thyroid transcription factor-1, hepatocyte nuclear factor) and growth factors (platelet-derived growth factor, keratinocyte growth factor and epidermal growth factor) are implicated in regulating lung branching morphogenesis, the exact molecular signals guiding embryonic lung development remain largely unknown. In the present study, we identified genes that are specifically expressed in the embryonic (day 13), but not late fetal (day 21), rat lung using mRNA differential display. After reverse transcription (RT) and polymerase chain reaction (PCR), eight cDNA fragments were shown to be exclusively expressed at day 13 of gestation. Six of them were successfully reamplified, cloned and sequenced. Eleven distinct sequences were obtained, since larger cDNA bands contained more than one DNA species. RT-PCR using cDNA specific primers demonstrated that five of the genes showed a developmental stage-specific expression, i.e. expressed at 12-13 days of gestation but not later during fetal lung development. In addition, mRNA expression of these genes in day 13 fetal lung was greater when compared to other day-13 fetal foregut-derived organs, such as intestine and liver. Functional analysis revealed that an antisense oligonucleotide targeted to gene 4.6 completely blocked branching of embryonic rat lung in vitro. Antisense inhibition of three other genes partially inhibited lung branching. To further identify these genes, a day-13 fetal rat lung cDNA library has been constructed. Localization of the genes will be examined by in situ hybridization. Putative function of the genes in branching morphogenesis will be investigated using transgenic approaches.M.Sc