HOXA5 Participates in Brown Adipose Tissue and Epaxial Skeletal Muscle Patterning and in Brown Adipocyte Differentiation.

Brown adipose tissue (BAT) plays critical thermogenic, metabolic and endocrine roles in mammals, and aberrant BAT function is associated with metabolic disorders including obesity and diabetes. The major BAT depots are clustered at the neck and forelimb levels, and arise largely within the dermomyotome of somites, from a common progenitor with skeletal muscle. However, many aspects of BAT embryonic development are not well understood. Hoxa5 patterns other tissues at the cervical and brachial levels, including skeletal, neural and respiratory structures. Here, we show that Hoxa5 also positively regulates BAT development, while negatively regulating formation of epaxial skeletal muscle. HOXA5 protein is expressed in embryonic preadipocytes and adipocytes as early as embryonic day 12.5. Hoxa5 null mutant embryos and rare, surviving adults show subtly reduced iBAT and sBAT formation, as well as aberrant marker expression, lower adipocyte density and altered lipid droplet morphology. Conversely, the epaxial muscles that arise from a common dermomyotome progenitor are expanded in Hoxa5 mutants. Conditional deletion of Hoxa5 with Myf5/Cre can reproduce both BAT and epaxial muscle phenotypes, indicating that HOXA5 is necessary within Myf5-positive cells for proper BAT and epaxial muscle development. However, recombinase-based lineage tracing shows that Hoxa5 does not act cell-autonomously to repress skeletal muscle fate. Interestingly, Hoxa5-dependent regulation of adipose-associated transcripts is conserved in lung and diaphragm, suggesting a shared molecular role for Hoxa5 in multiple tissues. Together, these findings establish a role for Hoxa5 in embryonic BAT development.

Deletion of Yy1 in mouse lung epithelium unveils molecular mechanisms governing pleuropulmonary blastoma pathogenesis.

Pleuropulmonary blastoma (PPB) is a very rare pediatric lung disease. It can progress from abnormal epithelial cysts to an aggressive sarcoma with poor survival. PPB is difficult to diagnose as it can be confounded with other cystic lung disorders, such as congenital pulmonary airway malformation (CPAM). PPB is associated with mutations in DICER1 that perturb the microRNA (miRNA) profile in lung. How DICER1 and miRNAs act during PPB pathogenesis remains unsolved. Lung epithelial deletion of the Yin Yang1 (Yy1) gene in mice causes a phenotype mimicking the cystic form of PPB and affects the expression of key regulators of lung development. Similar changes in expression were observed in PPB but not in CPAM lung biopsies, revealing a distinctive PPB molecular signature. Deregulation of molecules promoting epithelial-mesenchymal transition (EMT) was detected in PPB specimens, suggesting that EMT might participate in tumor progression. Changes in miRNA expression also occurred in PPB lung biopsies. miR-125a-3p, a candidate to regulate YY1 expression and lung branching, was abnormally highly expressed in PPB samples. Together, these findings support the concept that reduced expression of YY1, due to the abnormal miRNA profile resulting from DICER1 mutations, contributes to PPB development via its impact on the expression of key lung developmental genes.This article has an associated First Person interview with the joint first authors of the paper.

Mek1Y130C mice recapitulate aspects of human cardio-facio-cutaneous syndrome.

The RAS/MAPK signaling pathway is one of the most investigated pathways, owing to its established role in numerous cellular processes and implication in cancer. Germline mutations in genes encoding members of the RAS/MAPK pathway also cause severe developmental syndromes collectively known as RASopathies. These syndromes share overlapping characteristics, including craniofacial dysmorphology, cardiac malformations, cutaneous abnormalities and developmental delay. Cardio-facio-cutaneous syndrome (CFC) is a rare RASopathy associated with mutations in BRAF, KRAS, MEK1 (MAP2K1) and MEK2 (MAP2K2). MEK1 and MEK2 mutations are found in ∼25% of the CFC patients and the MEK1Y130C substitution is the most common one. However, little is known about the origins and mechanisms responsible for the development of CFC. To our knowledge, no mouse model carrying RASopathy-linked Mek1 or Mek2 gene mutations has been reported. To investigate the molecular and developmental consequences of the Mek1Y130C mutation, we generated a mouse line carrying this mutation. Analysis of mice from a Mek1 allelic series revealed that the Mek1Y130C allele expresses both wild-type and Y130C mutant forms of MEK1. However, despite reduced levels of MEK1 protein and the lower abundance of MEK1 Y130C protein than wild type, Mek1Y130C mutants showed increased ERK (MAPK) protein activation in response to growth factors, supporting a role for MEK1 Y130C in hyperactivation of the RAS/MAPK pathway, leading to CFC. Mek1Y130C mutant mice exhibited pulmonary artery stenosis, cranial dysmorphia and neurological anomalies, including increased numbers of GFAP+ astrocytes and Olig2+ oligodendrocytes in regions of the cerebral cortex. These data indicate that the Mek1Y130C mutation recapitulates major aspects of CFC, providing a new animal model to investigate the physiopathology of this RASopathy. This article has an associated First Person interview with the first author of the paper.

HOXA5 protein expression and genetic fate mapping show lineage restriction in the developing musculoskeletal system.

HOX proteins act during development to regulate musculoskeletal morphology. HOXA5 patterns skeletal structures surrounding the cervical-thoracic transition including the vertebrae, ribs, sternum and forelimb girdle. However, the tissue types in which it acts to pattern the skeleton, and the ultimate fates of embryonic cells that activate Hoxa5 expression are unknown. A detailed characterization of HOXA5 expression by immunofluorescence was combined with Cre/LoxP genetic lineage tracing to map the fate of Hoxa5 expressing cells in axial musculoskeletal tissues and in their precursors, the somites and lateral plate mesoderm. HOXA5 protein expression is dynamic and spatially restricted in derivatives of both the lateral plate mesoderm and somites, including a subset of the lateral sclerotome, suggesting a local role in regulating early skeletal patterning. HOXA5 expression persists from somite stages through late development in differentiating skeletal and connective tissues, pointing to a continuous and direct role in skeletal patterning. In contrast, HOXA5 expression is excluded from the skeletal muscle and muscle satellite cell lineages. Furthermore, the descendants of Hoxa5-expressing cells, even after HOXA5 expression has extinguished, never contribute to these lineages. Together, these findings suggest cell autonomous roles for HOXA5 in skeletal development, as well as non-cell autonomous functions in muscle through expression in surrounding connective tissues. They also support the notion that different Hox genes display diverse tissue specificities and locations to achieve their patterning activity.

Lung cancer susceptibility genetic variants modulate HOXB2 expression in the lung.

The HOX genes are transcription factors that are expressed in coordinated spatiotemporal patterns to ensure normal development. Ectopic expression may instead lead to the development and progression of tumors. Genetic polymorphisms in the regions of four HOX gene clusters were tested for association with lung cancer in 420 cases and 3,151 controls. The effect of these variants on lung gene expression (expression quantitative trait loci, eQTL) was tested in a discovery set of 409 non-tumor lung samples and validated in two lung eQTL replication sets (n = 287 and 342). The expression levels of HOXB2 were evaluated at the mRNA and protein levels by quantitative real-time PCR and immunohistochemistry in paired tumor and non-tumor lung tissue samples. The most significant SNP associated with lung cancer in the HOXB cluster was rs10853100 located upstream of the HOXB cluster. HOXB2 was the top eQTL-regulated gene with several polymorphisms associated with its mRNA expression levels in lung tissue. This includes the lung cancer SNP rs10853100 that was significantly associated with HOXB2 expression (P=3.39E-7). In the lung eQTL discovery and replication sets, the lung cancer risk allele (T) for rs10853100 was associated with lower HOXB2 expression levels. In paired normal-tumor samples, HOXB2 mRNA and protein levels were significantly reduced in tumors when compared to non-tumor lung tissues. Genetic variants in the HOXB cluster may confer susceptibility to lung cancer by modulating the expression of HOXB2 in the lung.

Respiratory consequences of targeted losses of Hoxa5 gene function in mice.

Fetal development of the respiratory tract and diaphragm requires strict coordination between genetically controlled signals and mechanical forces produced by the neural network that generates breathing. HOXA5, which is expressed in the mesenchyme of the trachea, lung and diaphragm, and in phrenic motor neurons, is a key transcription factor regulating lung development and function. Consequently, most Hoxa5-/- mutants die at birth from respiratory failure. However, the extensive effect of the null mutation makes it difficult to identify the origins of respiratory dysfunction in newborns. To address the physiological impact of Hoxa5 tissue-specific roles, we used conditional gene targeting with the Dermo1Cre and Olig2Cre mouse lines to produce specific Hoxa5 deletions in the mesenchyme and motor neurons, respectively. Hoxa5 expression in the mesenchyme is critical for trachea development, whereas its expression in phrenic motor neurons is essential for diaphragm formation. Breathing measurements in adult mice with whole-body plethysmography demonstrated that, at rest, only the motor neuron deletion affects respiration, resulting in higher breathing frequency and decreased tidal volume. But subsequent exposure to a moderate hypoxic challenge (FiO2 =0.12; 10 min) revealed that both mutant mice hyperventilate more than controls. Hoxa5flox/flox;Dermo1+/Cre mice showed augmented tidal volume while Hoxa5flox/flox;Olig2+/Cre mice had the largest increase in breathing frequency. No significant differences were observed between medulla-spinal cord preparations from E18.5 control and Hoxa5flox/flox;Olig2+/Cre mouse embryos that could support a role for Hoxa5 in fetal inspiratory motor command. According to our data, Hoxa5 expression in the mesenchyme and phrenic motor neurons controls distinct aspects of respiratory development.

HOXA5 plays tissue-specific roles in the developing respiratory system.

Hoxa5 is essential for development of several organs and tissues. In the respiratory system, loss of Hoxa5 function causes neonatal death due to respiratory distress. Expression of HOXA5 protein in mesenchyme of the respiratory tract and in phrenic motor neurons of the central nervous system led us to address the individual contribution of these Hoxa5 expression domains using a conditional gene targeting approach. Hoxa5 does not play a cell-autonomous role in lung epithelium, consistent with lack of HOXA5 expression in this cell layer. In contrast, ablation of Hoxa5 in mesenchyme perturbed trachea development, lung epithelial cell differentiation and lung growth. Further, deletion of Hoxa5 in motor neurons resulted in abnormal diaphragm innervation and musculature, and lung hypoplasia. It also reproduced the neonatal lethality observed in null mutants, indicating that the defective diaphragm is the main cause of impaired survival at birth. Thus, Hoxa5 possesses tissue-specific functions that differentially contribute to the morphogenesis of the respiratory tract.

Lung development requires an active ERK/MAPK pathway in the lung mesenchyme.

BACKGROUND: Reciprocal epithelial-mesenchymal communications are critical throughout lung development, dictating branching morphogenesis and cell specification. Numerous signaling molecules are involved in these interactions, but the way epithelial-mesenchymal crosstalk is coordinated remains unclear. The ERK/MAPK pathway transduces several important signals in lung formation. Epithelial inactivation of both Mek genes, encoding ERK/MAPK kinases, causes lung agenesis and death. Conversely, Mek mutation in mesenchyme results in lung hypoplasia, trachea cartilage malformations, kyphosis, omphalocele, and death. Considering the negative impact of kyphosis and omphalocele on intrathoracic space and, consequently, on lung growth, the exact role of ERK/MAPK pathway in lung mesenchyme remains unresolved. RESULTS: To address the role of the ERK/MAPK pathway in lung mesenchyme in absence of kyphosis and omphalocele, we used the Tbx4Cre deleter mouse line, which acts specifically in lung mesenchyme. These Mek mutants did not develop kyphosis and omphalocele but they presented lung hypoplasia, tracheal defects, and neonatal death. Tracheal cartilage anomalies suggested a role for the ERK/MAPK pathway in the control of chondrocyte hypertrophy. Moreover, expression data indicated potential interactions between the ERK/MAPK and canonical Wnt pathways during lung formation. CONCLUSIONS: Lung development necessitates a functional ERK/MAPK pathway in the lung mesenchymal layer in order to coordinate efficient epithelial-mesenchymal interactions. Developmental Dynamics 246:72-82, 2017. © 2016 Wiley Periodicals, Inc.

Hoxa5: A Key Player in Development and Disease.

A critical position in the developmental hierarchy is occupied by the Hox genes, which encode transcription factors. Hox genes are crucial in specifying regional identity along the embryonic axes and in regulating morphogenesis. In mouse, targeted mutations of Hox genes cause skeletal transformations and organ defects that can impair viability. Here, we present the current knowledge about the Hoxa5 gene, a paradigm for the function and the regulation of Hox genes. The phenotypic survey of Hoxa5-/- mice has unveiled its critical role in the regional specification of the skeleton and in organogenesis. Most Hoxa5-/- mice die at birth from respiratory distress due to tracheal and lung dysmorphogenesis and impaired diaphragm innervation. The severity of the phenotype establishes that Hoxa5 plays a predominant role in lung organogenesis versus other Hox genes. Hoxa5 also governs digestive tract morphogenesis, thyroid and mammary glands development, and ovary homeostasis. Deregulated Hoxa5 expression is reported in cancers, indicating Hoxa5 involvement in tumor predisposition and progression. The dynamic Hoxa5 expression profile is under the transcriptional control of multiple cis-acting sequences and trans-acting regulators. It is also modulated by epigenetic mechanisms, implicating chromatin modifications and microRNAs. Finally, lncRNAs originating from alternative splicing and distal promoters encompass the Hoxa5 locus.

Epithelial inactivation of Yy1 abrogates lung branching morphogenesis.

Yin Yang 1 (YY1) is a multifunctional zinc-finger-containing transcription factor that plays crucial roles in numerous biological processes by selectively activating or repressing transcription, depending upon promoter contextual differences and specific protein interactions. In mice, Yy1 null mutants die early in gestation whereas Yy1 hypomorphs die at birth from lung defects. We studied how the epithelial-specific inactivation of Yy1 impacts on lung development. The Yy1 mutation in lung epithelium resulted in neonatal death due to respiratory failure. It impaired tracheal cartilage formation, altered cell differentiation, abrogated lung branching and caused airway dilation similar to that seen in human congenital cystic lung diseases. The cystic lung phenotype in Yy1 mutants can be partly explained by the reduced expression of Shh, a transcriptional target of YY1, in lung endoderm, and the subsequent derepression of mesenchymal Fgf10 expression. Accordingly, SHH supplementation partially rescued the lung phenotype in vitro. Analysis of human lung tissues revealed decreased YY1 expression in children with pleuropulmonary blastoma (PPB), a rare pediatric lung tumor arising during fetal development and associated with DICER1 mutations. No evidence for a potential genetic interplay between murine Dicer and Yy1 genes during lung morphogenesis was observed. However, the cystic lung phenotype resulting from the epithelial inactivation of Dicer function mimics the Yy1 lung malformations with similar changes in Shh and Fgf10 expression. Together, our data demonstrate the crucial requirement for YY1 in lung morphogenesis and identify Yy1 mutant mice as a potential model for studying the genetic basis of PPB.