Microarray analysis of Myf5-/-:MyoD-/- hypoplastic mouse lungs reveals a profile of genes involved in pneumocyte differentiation.

Fetal breathing-like movements (FBMs) are important in normal lung growth and pneumocyte differentiation. In amyogenic mouse embryos (designated as Myf5-/-:MyoD-/-, entirely lacking skeletal musculature and FBMs), type II pneumocytes fail to differentiate into type I pneumocytes, the cells responsible for gas exchange, and the fetuses die from asphyxia at birth. Using oligonucleotide microarrays, we compared gene expression in the lungs of Myf5-/-:MyoD-/- embryos to that in normal lungs at term. Nine genes were found to be up-regulated and 54 down-regulated at least 2-fold in the lungs of double-mutant embryos. Since many down-regulated genes are involved in lymphocyte function, immunohistochemistry was employed to study T- and B-cell maturity in the thymus and spleen. Our findings of normal lymphocyte maturity implied that the down-regulation was specific to the double-mutant lung phenotype and not to its immune system. Immunostaining also revealed altered distribution of transcription and growth factors (SATB1, c-Myb, CTGF) from down-regulated genes whose knockouts are now known to undergo embryonic or neonatal death secondary to respiratory failure. Together, it appears that microarray analysis has identified a profile of genes potentially involved in pneumocyte differentiation and therefore in the mechanisms that may be implicated in the mechanochemical signal transduction pathways underlying FBMs-dependent pulmonary hypoplasia.

Neurotrophins, airway smooth muscle and the fetal breathing-like movements.

Central nervous system and skeletal muscles secrete a group of polypeptide hormones called neurotrophins (NTs). More recent studies show that NTs and their receptors are also expressed in the lung, suggesting a role for NTs in lung development. To examine the role of NTs during normal and diseased lung organogenesis, we employed wild-type and amyogenic mouse embryos (designated as Myf5-/-:MyoD-/-). Amyogenic embryos completely lacked skeletal muscles and were not viable after birth due to the respiratory failure secondary to lung hypoplasia. To examine the importance of lung-secreted NTs during normal and hypoplastic lung organogenesis, immunohistochemistry was employed. Distribution of NTs and their receptors was indistinguishable between normal and hypoplastic lungs. To further examine the importance of non-lung-secreted NTs (e.g., from the skeletal muscle and CNS) in lung organogenesis, in utero injections of two NTs were performed. The exogenously introduced NTs (i.e., non-lung-secreted) did not appear to improve development of the lung in amyogenic embryos. Moreover, immunohistochemistry showed significantly reduced number of airway smooth muscle cells (ASMCs) in hypoplastic lungs of amyogenic embryos, suggesting that the number of ASMCs is primarily regulated by the fetal breathing-like movements (i.e., mechanical factors).

The role of fetal breathing-like movements in lung organogenesis.

In this review the recent findings concerning the role of fetal breathing-like movements (FBMs) on lung organogenesis are discussed. We first review the consequences that the lack of FBMs has on lung organogenesis and then we discuss the possible pathways that may be employed in this process. Specifically, we review the data in support of the notion that FBMs are required for the cell cycle kinetics regulation (i.e., cell proliferation and cell death) via the expression of growth factors, such as platelet derived growth factors (PDGFs) and insulin growth factors (IGFs), and thyroid transcription factor 1 (TTF-1). Moreover, the role of FBMs on biochemical differentiation of Clara cells, type I and type II pneumocytes is reviewed. Interestingly, even though type II pneumocytes are able to synthesize surfactant-associated proteins (SPs), in the complete absence of FBMs, they are unable to compile, store and release the surfactant. Similarly, in spite of the expression of some early differentiation markers, in the absence of FBMs, type I pneumocytes are unable to flatten in order to allow the gas exchange in the lung. In fact, we are currently employing the cDNA microarray analysis in search for the molecules that might be specific for the lacking functions in pneumocytes.