Skeletal Muscle's Role in Prenatal Inter-organ Communication: A Phenogenomic Study with Qualitative Citation Analysis.

Gene targeting in mice allows for a complete elimination of skeletal (striated or voluntary) musculature in the body, from the beginning of its development, resulting in our ability to study the consequences of this ablation on other organs. Here I focus on the relationship between the muscle and lung, motor neurons, skeleton, and special senses. Since the inception of my independent laboratory, in 2000, with my team, we published more than 30 papers (and a book chapter), nearly 400 pages of data, on these specific relationships. Here I trace, using Web of Science, nearly 600 citations of this work, to understand its impact. The current report contains a summary of our work and its impact, NCBI's Gene Expression Omnibus accession numbers of all our microarray data, and three clear future directions doable by anyone using our publicly available data. Together, this effort furthers our understanding of inter-organ communication during prenatal development.

Mechanics of Lung Development.

We summarize how skeletal muscle and lung developmental biology fields have been bridged to benefit from mouse genetic engineering technologies and to explore the role of fetal breathing-like movements (FBMs) in lung development, by using skeletal muscle-specific mutant mice. It has been known for a long time that FBMs are essential for the lung to develop properly. However, the cellular and molecular mechanisms transducing the mechanical forces of muscular activity into specific genetic programs that propel lung morphogenesis (development of the shape, form and size of the lung, its airways, and gas exchange surface) as well as its differentiation (acquisition of specialized cell structural and functional features from their progenitor cells) are only starting to be revealed. This chapter is a brief synopsis of the cumulative findings from that ongoing quest. An update on and the rationale for our recent International Mouse Phenotyping Consortium (IMPC) search is also provided.

Roles of Skeletal Muscle in Development: A Bioinformatics and Systems Biology Overview.

The ability to assess various cellular events consequent to perturbations, such as genetic mutations, disease states and therapies, has been recently revolutionized by technological advances in multiple "omics" fields. The resulting deluge of information has enabled and necessitated the development of tools required to both process and interpret the data. While of tremendous value to basic researchers, the amount and complexity of the data has made it extremely difficult to manually draw inference and identify factors key to the study objectives. The challenges of data reduction and interpretation are being met by the development of increasingly complex tools that integrate disparate knowledge bases and synthesize coherent models based on current biological understanding. This chapter presents an example of how genomics data can be integrated with biological network analyses to gain further insight into the developmental consequences of genetic perturbations. State of the art methods for conducting similar studies are discussed along with modern methods used to analyze and interpret the data.

Differences in Immunohistochemical and Ultrastructural Features between Podocytes and Parietal Epithelial Cells (PECs) Are Observed in Developing, Healthy Postnatal, and Pathologically Changed Human Kidneys.

During human kidney development, cells of the proximal nephron gradually differentiate into podocytes and parietal epithelial cells (PECs). Podocytes are terminally differentiated cells that play a key role in both normal and pathological kidney function. Therefore, the potential of podocytes to regenerate or be replaced by other cell populations (PECs) is of great interest for the possible treatment of kidney diseases. In the present study, we analyzed the proliferation and differentiation capabilities of podocytes and PECs, changes in the expression pattern of nestin, and several early proteins including WNT4, Notch2, and Snail, as well as Ki-67, in tissues of developing, postnatal, and pathologically changed human kidneys by using immunohistochemistry and electron microscopy. Developing PECs showed a higher proliferation rate than podocytes, whereas nestin expression characterized only podocytes and pathologically changed kidneys. In the developing kidneys, WNT4 and Notch2 expression increased moderately in podocytes and strongly in PECs, whereas Snail increased only in PECs in the later fetal period. During human kidney development, WNT4, Notch2, and Snail are involved in early nephrogenesis control. In kidneys affected by congenital nephrotic syndrome of the Finnish type (CNF) and focal segmental glomerulosclerosis (FSGS), WNT4 decreased in both cell populations, whereas Notch2 decreased in FSGS. In contrast, Snail increased both in CNF and FSGS, whereas Notch2 increased only in CNF. Electron microscopy revealed cytoplasmic processes spanning the urinary space between the podocytes and PECs in developing and healthy postnatal kidneys, whereas the CNF and FSGS kidneys were characterized by numerous cellular bridges containing cells with strong expression of nestin and all analyzed proteins. Our results indicate that the mechanisms of gene control in nephrogenesis are reactivated under pathological conditions. These mechanisms could have a role in restoring glomerular integrity by potentially inducing the regeneration of podocytes from PECs.

Expression and localization of DAB1 and Reelin during normal human kidney development.

AIM: To explore the spatial and temporal expression patterns of DAB1 and Reelin in the developing and postnatal healthy human kidneys as potential determinants of kidney development. METHODS: Paraffin-embedded fetal kidney tissue between the 13/14th and 38th developmental weeks (dw) and postnatal tissue at 1.5 and 7 years were stained with DAB1 and Reelin antibodies by double immunofluorescence. RESULTS: During the fetal kidney development and postnatal period, DAB1 and Reelin showed specific spatial expression pattern and diverse fluorescence intensity. During the fetal period, DAB1 was strongly expressed in the distal convoluted tubules (DCT), with strong reactivity, and diversely in the proximal convoluted tubules (PCT) and glomeruli. In the postnatal period, DAB1 expression decreased. The strongest Reelin expression in early fetal stages was observed in the PCT. In the postnatal period, Reelin expression decreased dramatically in all observed structures. These two markers were colocalized during early developmental stages, mostly in PCT, DCT, and podocytes. CONCLUSION: The appearance of DAB1 and Reelin during fetal kidney development confirms their potential significant role in the formation of kidney structure or function. High DAB1 expression in the DCT implies its regulatory role in tubular formation or function maintenance during development. Reelin was highly expressed in human kidneys at early fetal stages, mostly in the PCT, while at later fetal stages and postnatal period its expression decreased.

Immunohistochemical and electronmicroscopic features of mesenchymal-to-epithelial transition in human developing, postnatal and nephrotic podocytes.

Differentiation of human podocytes starts with mesenchymal-to-epithelial transition (MET) of the metanephric mesenchyme into the S-shaped nephrons. During further development, differentiating podocytes regain mesenchyme-like cell characteristics by epithelial-to-mesenchymal transition (EMT), leading to formation of the terminally differentiated, non-dividing cell. Both MET and EMT processes involve changes in content and organization of cytoskeletal and actin filaments, accompanied by the increased glomerular vascularization. Here, we analyze and compare normal human developing, postnatal and nephrotic podocytes and glomeruli, using immunohistochemical and double immunofluorescent methods for detection of markers of cytoskeletal filaments (nestin, cytokeratin 10-CK10, vimentin and α-SMA), vasculogenesis (CD31 and VEGF) and podocyte function (receptor for advanced glycation end products, RAGE). In addition, electron microscopy is used to detect ultrastructural changes of the podocytes. Early metanephric cup mesenchyme expresses all investigated markers except α-SMA, which characterizes only surface mesenchymal cells. In differentiating podocytes and cells of Bowman's capsule (parietal podocytes) nestin decreases, vimentin increases, while CK10 gradually disappears. Increase in α-SMA is associated with blood vessels development, appearance of podocyte pedicles and slit diaphragm and loss of intercellular connections (zonulae adherentes). Increase in CD31 characterizes vascular glomerular tufts development, while decrease in RAGE expression accompanies normal podocyte differentiation. In congenital nephrotic syndrome of the Finnish type, dedifferentiated podocytes display changes in cytoskeletal filaments and depletion of podocyte pedicles, while glomerular vascular supply is diminished. Our data also suggest high potential of metanephric mesenchyme and parietal podocytes in possible regeneration of the damaged podocytes.

Abnormal retinal development in the Btrc null mouse.

Previous microarray analysis revealed beta-transducin repeat containing (Btrc) down-regulation in the retina of mouse embryos specifically lacking cholinergic amacrine cells (CACs) as a result of the absence of skeletal musculature and fetal ocular movements. To investigate the role of Btrc in the determination of retinal cell fate, the present study examined retinal morphology in Btrc-/- mouse fetuses. The Btrc-/- retina showed a normal number of cell layers and number of cells per layer with normal cell proliferation and apoptosis. However, there was a complete absence of CACs and a decrease in tyrosine hydroxylase-expressing amacrine cells. The population of other amacrine cell subtypes was normal, whereas that of the precursor cells was decreased. There was also a reduction in the number of retinal ganglion cells, whereas their progenitors were increased. These findings suggest a role for Btrc in regulating the eventual ratio of resulting differentiated retinal cell types.

Striated-for-smooth muscle replacement in the developing mouse esophagus.

The esophagus is a muscular tube which transports swallowed content from the oral cavity and the pharynx to the stomach. Early in mouse development, an entire layer of the esophagus, the muscularis externa, consists of differentiated smooth muscle cells. Starting shortly after mid-gestation till about two weeks after birth, the muscularis externa almost entirely consists of striated muscle. This proximal-to-distal replacement of smooth muscle by the striated muscle depends on a number of factors. To identify the nature of the hypothetical "proximal" (mainly striated muscle originating) and "distal" (mainly smooth muscle originating) signals that govern the striated-for-smooth muscle replacement, we compared the esophagus of Myf5:MyoD null fetuses completely lacking striated muscle to the normal control using cDNA microarray analysis, followed by a comprehensive database search. Here we provide an insight into the nature of "proximal" and "distal" signals that govern the striated-for-smooth muscle replacement in the esophagus.

Immunohistochemical expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the normal human kidney development.

AIM: Present study analyses the co-localisation of RIP5 with FGFR1, FGFR2 and HIP2 in the developing kidney, as RIP5 is a major determinant of urinary tract development, downstream of FGF-signaling. METHODS: Paraffin embedded human kidney tissues of 16 conceptuses between the 6th-22th developmental week were analysed using double-immunofluorescence method with RIP5/FGFR1/FGFR2 and HIP2 markers. Quantification of positive cells were performed using Kruskal-Wallis test. RESULTS: In the 6th week of kidney development RIP5 (89.6%) and HIP2 (39.6%) are strongly expressed in the metanephric mesenchyme. FGFR1 shows moderate/strong expression in the developing nephrons (87.3%) and collecting ducts (70.5%) (p < 0.05). RIP5/FGFR1 co-localized at the marginal zone and the ureteric bud with predominant FGFR1 expression. FGFR2 (26.1%) shows similar expression pattern as FGFR1 (70.5%) in the same kidney structures. RIP5/FGFR2 co-localized at the marginal zone and the collecting ducts (predominant expression of FGFR2). HIP2 is strongly expressed in collecting ducts (96.7%), and co-localized with RIP5. In 10th week, RIP5 expression decrease (74.2%), while the pattern of expression of RIP5 and FGFR1 in collecting ducts (33.4% and 91.9%) and developing nephrons (21.9% and 32.4%) (p < 0.05) is similar to that in the 6th developmental week. Ureter is moderately expressing RIP5 while FGFR1 is strongly expressed in the ureteric wall. FGFR2 is strongly expressed in the collecting ducts (84.3%) and ureter. HIP2 have 81.1% positive cells in the collecting duct. RIP5/FGFR1 co-localize in collecting ducts and Henley's loop. CONCLUSIONS: The expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the human kidney development might indicate their important roles in metanephric development and ureteric muscle layer differentiation through FGF signaling pathways.

Role of skeletal muscle in ear development.

The current paper is a continuation of our work described in Rot and Kablar, 2010. Here, we show lists of 10 up- and 87 down-regulated genes obtained by a cDNA microarray analysis that compared developing Myf5-/-:Myod-/- (and Mrf4-/-) petrous part of the temporal bone, containing middle and inner ear, to the control, at embryonic day 18.5. Myf5-/-:Myod-/- fetuses entirely lack skeletal myoblasts and muscles. They are unable to move their head, which interferes with the perception of angular acceleration. Previously, we showed that the inner ear areas most affected in Myf5-/-:Myod-/- fetuses were the vestibular cristae ampullaris, sensitive to angular acceleration. Our finding that the type I hair cells were absent in the mutants' cristae was further used here to identify a profile of genes specific to the lacking cell type. Microarrays followed by a detailed consultation of web-accessible mouse databases allowed us to identify 6 candidate genes with a possible role in the development of the inner ear sensory organs: Actc1, Pgam2, Ldb3, Eno3, Hspb7 and Smpx. Additionally, we searched for human homologues of the candidate genes since a number of syndromes in humans have associated inner ear abnormalities. Mutations in one of our candidate genes, Smpx, have been reported as the cause of X-linked deafness in humans. Our current study suggests an epigenetic role that mechanical, and potentially other, stimuli originating from muscle, play in organogenesis, and offers an approach to finding novel genes responsible for altered inner ear phenotypes.

Role of skeletal muscle in motor neuron development.

The current paper is a continuation of our work most recently described in Kablar, 2011. Here, we show lists of up- and down-regulated genes obtained by a cDNA microarray analysis that compared developing mouse MyoD-/- limb musculature (MyoD-dependent, innervated by Lateral Motor Column motor neurons) and Myf5-/- back (epaxial) musculature (Myf5-dependent, innervated by Medial Motor Column motor neurons) to the control and to each other, at embryonic day 13.5 which coincides with the robust programmed cell death of motor neurons and the inability of myogenesis to undergo its normal progression in the absence of Myf5 and MyoD that at this embryonic day cannot substitute for each other. We wanted to see if/how the myogenic program couples with the neurotrophic one, and also to separate Lateral from Medial column trophic requirements, potentially relevant to Motor Neuron Diseases with the predilection for the Lateral column. Several follow-up steps revealed that Kif5c, Stxbp1 and Polb, differentially expressed in the MyoD-/- limb muscle, and Ppargc1a, Glrb and Hoxd10, differentially expressed in the Myf5-/- back muscle, are actually regulators of motor neuron numbers. We propose a series of follow-up experiments and various ways to consider our current data.

The role of neurotrophins in the maintenance of the spinal cord motor neurons and the dorsal root ganglia proprioceptive sensory neurons.

The aim of this study was to approach the question of neuronal dependence on neurotrophins during embryonic development in mice in a way other than gene targeting. We employed amyogenic mouse embryos and fetuses that develop without any skeletal myoblasts or skeletal muscle and consequently lose motor and proprioceptive neurons. We hypothesized that if, in spite of the complete inability to maintain motor and proprioceptive neurons, the remaining spinal and dorsal root ganglia tissues of amyogenic fetuses still contain any of the neurotrophins, that particular neurotrophin alone is not sufficient for the maintenance of motor and proprioceptive neurons. Moreover, if the remaining spinal and dorsal root ganglia tissues still contain any of the neurotrophins, that particular neurotrophin alone may be sufficient for the maintenance of the remaining neurons (i.e., mostly non-muscle- and a few muscle-innervating neurons). To test the role of the spinal cord and dorsal root ganglia tissues in the maintenance of its neurons, we performed immunohistochemistry employing double-mutant and control tissues and antibodies against neurotrophins and their receptors. Our data suggested that: (a) during the peak of motor neuron cell death, the spinal cord and dorsal root ganglia distribution of neurotrophins was not altered; (b) the distribution of BDNF, NT-4/5, TrkB and TrkC, and not NT-3, was necessary for the maintenance of the spinal cord motor neurons; (c) the distribution of BDNF, NT-4/5 and TrkC, and not NT-3 and Trk B, was necessary for the maintenance of the DRG proprioceptive neurons; (d) NT-3 was responsible for the maintenance of the remaining neurons and glia in the spinal cord and dorsal root ganglia (possibly via TrkB).

Abnormal development of the diaphragm in mdx:MyoD-/-(9th) embryos leads to pulmonary hypoplasia.

In vitro studies have shown that mechanical factors play an important role in cell cycle kinetics and cell differentiation of the lung through an unknown mechanochemical signal transduction pathway. In this study we evaluated the in vivo role of mechanical factors due to fetal breathing movements (primarily executed by the diaphragm, which is the main respiratory muscle) on lung growth and development by using genetically engineered embryos. Lung growth and development of wild-type, mdx:MyoD+/-(9th) (in which the diaphragm develops normally) and mdx:MyoD-/-(9th) (in which the diaphragm muscle is significantly thinned and not functional) embryos were compared at embryonic day 18.5 using immunohistochemistry, in vivo TUNEL detection and morphometry. No abnormalities in lung organogenesis were observed in mdx:MyoD+/-(9th) term embryos, whereas lung hypoplasia was detected in mdx:MyoD-/-(9th) embryos. In the hypoplastic lung, the number of proliferating lung cells was lower in comparison to the wild-type and mdx:MyoD+/-(9th) embryos, while the gradient of thyroid transcription factor-1 (TTF-1) was not maintained. Surprisingly, no difference was observed in distribution and occurrence of apoptotic lung cells in mdx:MyoD-/-(9th) embryos. Together, it appears that mechanical forces generated by contractile activity of the diaphragm muscle play an important role in normal lung growth and development by affecting cell proliferation and TTF-1 expression.

The Role of Skeletal Muscle in External Ear Development: A Mouse Model Histomorphometric Study.

BACKGROUND: Mechanical stimuli imparted by skeletal muscles play an important role during embryonic development in vertebrates. Little is known whether skeletal muscles are required for normal external ear development. METHODS: We used Myf5-/-:MyoD-/- (double-mutant) mouse embryos that completely lack skeletal musculature and analyzed the development of the external ear. We concentrated on the external ear because several studies have suggested a muscular cause to various congenital auricular deformities, and middle and inner ear development was previously reported using the same mouse model. Wild-type mouse embryos were used as controls to compare the histomorphometric outcomes. RESULTS: Our findings demonstrated an absence of the external auditory meatus, along with an abnormal auricular appearance, in the double-mutant mouse embryos. Specifically, the auricle did not protrude laterally as noted in the wild-type mouse ears. However, histomorphometric measurements were not significantly different between the wild-type and double-mutant mouse ears. CONCLUSION: Overall, our study showed that the development of the mouse external ear is dependent on the presence of skeletal muscles.

MyoD-lacZ transgenes are early markers in the neural retina, but MyoD function appears to be inhibited in the developing retinal cells.

Recent findings suggest that eye and skeletal muscle development in vertebrates share the same regulatory network. In that network, Pax3 gene is apparently activated through Dach/Eya/Six feedback loop to mediate MyoD-driven myogenesis. The purpose of this study was to investigate previously reported MyoD-lacZ expression in the developing mouse neural retina and to gain insight into the potential role of MyoD in the embryonic retinal cells. The analysis of MD6.0-lacZ and 258/-2.5lacZ transgenic embryos revealed that the retinal temporal expression pattern of the two transgenes resembled their expression pattern in the MyoD-dependent precursor muscle cells. However, MyoD transcripts and protein could not be found in the sites of MyoD-lacZ retinal expression. Furthermore, our immunohistochemical analysis suggests the existence of diverse factors (e.g., Pax6 and Chx10) within the retinal cells that differentially and inappropriately activate the two transgenes. Finally, the retinal phenotype observed in Pax7-/- knock-out mice suggests a role for Pax7 in photoreceptor cell differentiation, retinal lamination and in the etiopathology of retinoblastoma. Taken together, our data suggest that the MyoD gene evolved a different mechanism to achieve its down-regulation within the retina than that of the Myf5 gene.

Information provided by the skeletal muscle and associated neurons is necessary for proper brain development.

Previously, motor cortex of term Myf5(-/-):MyoD(-/-) fetuses (e.g. have ablated skeletal myogenesis and consequent early loss of lower motor and proprioceptive neurons) was found to lack giant pyramidal cells. We further investigated how the absence of the extrinsic stimuli from the lacking structures influences brain development. Apparently normal motor cortex of mutant fetuses was found to have dramatically reduced presence of nestin-expressing processes of neural precursors, calretinin-expressing pyramidal neurons and calbindin-expressing neurons. Consistently, some areas of the extrapyramidal tract had significantly decreased number of differentiated neurons in mutant brains. Surprisingly, we were unable to detect any change in proliferation or cell death in the mutant neuroepithelium. Together, it appears that the information provided by the lacking structures influences the ratios of the differentiated neuronal types and their progenitor cells.

Different regulatory elements within the MyoD promoter control its expression in the brain and inhibit its functional consequences in neurogenesis.

MyoD is a key basic helix-loop-helix (bHLH) transcription factor capable of converting many cells into skeletal muscle. Together with Myf5 it is essential for initiating skeletal myogenesis. In this report, the restricted domains of MyoD-lacZ expression have been revealed in the embryonic mouse brain by the analysis of transgenic mice with reporter genes driven by MyoD regulatory elements. The MD6.0-lacZ transgene was localized in the basal plate of pons, medulla oblongata (i.e. the medial longitudinal fasciculus) and spinal cord of wild-type and mutant mouse embryos at various stages of development, whereas the 258/-2.5lacZ transgene was not detected in the brain. In addition, MyoD RNA and MyoD protein accumulations were monitored in neurons expressing MD6.0-lacZ transgene. Although MyoD was detected in muscle myotomal cells, it was absent in MD6.0-lacZ-expressing neurons. This would account for the lack of myogenic conversion in brain structures and the absence of a neural phenotype in MyoD-/- embryos and mice. Together, these data indicate that within the promoter of MyoD different regulatory elements control its expression and prevent the functional consequences of MyoD in neurogenesis.

Role of skeletal muscle in mandible development.

As a continuation of the previous study on palate development (Rot and Kablar, 2013), here we explore the relationship between the secondary cartilage mandibular condyles (parts of the temporomandibular joint) and the contributions (mechanical and secretory) from the adjacent skeletal musculature. Previous analysis of Myf5-/-:MyoD-/- mouse fetuses lacking skeletal muscle demonstrated the importance of muscle contraction and static loading in mouse skeletogenesis. Among abnormal skeletal features, micrognathia (mandibular hypoplasia) was detected: small, bent and posteriorly displaced mandible. As an example of Waddingtonian epigenetics, we suggest that muscle, in addition to acting via mechanochemical signal transduction pathways, networks and promoters, also exerts secretory stimuli on skeleton. Our goal is to identify candidate molecules at that muscle-mandible interface. By employing Systematic Subtractive Microarray Analysis approach, we compared gene expression between mandibles of amyogenic and wild type mouse fetuses and we identified up- and down-regulated genes. This step was followed by a bioinformatics approach and consultation of web-accessible mouse databases. We searched for individual tissue-specific gene expression and distribution, and for the functional effects of mutations in a particular gene. The database search tools allowed us to generate a set of candidate genes with involvement in mandibular development: Cacna1s, Ckm, Des, Mir300, Myog and Tnnc1. We also performed mouse-to-human translational experiments and found analogies. In the light of our findings we discuss various players in mandibular morphogenesis and make an argument for the need to consider mandibular development as a consequence of reciprocal epigenetic interactions of both skeletal and non-skeletal compartments.

Interplay of proliferation and proapoptotic and antiapoptotic factors is revealed in the early human inner ear development.

HYPOTHESIS: Spatiotemporal interplay of factors controlling proliferation, differentiation and apoptosis within the developing human inner ear is essential for labyrinth morphogenesis and development of vestibular and cochlear functions. BACKGROUND: Studies on the early human inner ear development are scarce and insufficient. METHODS: The immunolocalization of Ki-67, Bcl-2, caspase-3, and IGF-1 was analyzed in 6 human inner ears, 5 to 10 gestational weeks old. Statistical data were analyzed using the Kruskal-Wallis test. RESULTS: During the analyzed period, the otocyst has transformed into cochlear duct and saccule ventrally and semicircular canals and utricle dorsally. Initial differentiation of sensorineural fields characterized organ of Corti, maculae, and cristae ampullares. Intense (50%) and evenly distributed proliferation Ki-67 in the otocyst decreased to 24% to 30% and became spatially restricted within the membranous labyrinth epithelium. Simultaneously, expression of antiapoptotic Bcl-2 protein increased in sensorineural fields of organ of Corti, macula, and crista ampullaris. Throughout the investigated period, apoptotic caspase-3 positive cells were mainly distributed at the luminal and basal surfaces of labyrinth epithelium. An inhibitor of apoptosis IGF-1 co-expressed with Bcl-2 and increased in the sensorineural fields with advancing development. CONCLUSION: The described expression pattern indicates roles for cell proliferation in the growth of the inner ear and Bcl-2 in differentiation of sensorineural fields and protection from apoptosis. Both IGF-1-and caspase-3-mediated apoptosis seem to contribute to proper morphogenesis, differentiation, and innervations of sensorineural fields within the cochlea, semicircular canals, saccule, and utricle. Alterations in spatiotemporal interplay of investigated factors might lead to disturbances of vestibular and cochlear function.

Role of skeletal muscle in lung development.

Skeletal (striated) muscle is one of the four basic tissue types, together with the epithelium, connective and nervous tissues. Lungs, on the other hand, develop from the foregut and among various cell types contain smooth, but not skeletal muscle. Therefore, during earlier stages of development, it is unlikely that skeletal muscle and lung depend on each other. However, during the later stages of development, respiratory muscle, primarily the diaphragm and the intercostal muscles, execute so called fetal breathing-like movements (FBMs), that are essential for lung growth and cell differentiation. In fact, the absence of FBMs results in pulmonary hypoplasia, the most common cause of death in the first week of human neonatal life. Most knowledge on this topic arises from in vivo experiments on larger animals and from various in vitro experiments. In the current era of mouse mutagenesis and functional genomics, it was our goal to develop a mouse model for pulmonary hypoplasia. We employed various genetically engineered mice lacking different groups of respiratory muscles or lacking all the skeletal muscle and established the criteria for pulmonary hypoplasia in mice, and therefore established a mouse model for this disease. We followed up this discovery with systematic subtractive microarray analysis approach and revealed novel functions in lung development and disease for several molecules. We believe that our approach combines elements of both in vivo and in vitro approaches and allows us to study the function of a series of molecules in the context of lung development and disease and, simultaneously, in the context of lung's dependence on skeletal muscle-executed FBMs.