Whole-rat conditional gene knockout via genome editing.

Animal models with genetic modifications under temporal and/or spatial control are invaluable to functional genomics and medical research. Here we report the generation of tissue-specific knockout rats via microinjection of zinc-finger nucleases (ZFNs) into fertilized eggs. We generated rats with loxP-flanked (floxed) alleles and a tyrosine hydroxylase promoter-driven cre allele and demonstrated Cre-dependent gene disruption in vivo. Pronuclear microinjection of ZFNs, shown by our data to be an efficient and rapid method for creating conditional knockout rats, should also be applicable in other species.

Standardized In Vitro Models of Human Adipose Tissue Reveal Metabolic Flexibility in Brown Adipocyte Thermogenesis.

Functional human brown and white adipose tissue (BAT and WAT) are vital for thermoregulation and nutritional homeostasis, while obesity and other stressors lead, respectively, to cold intolerance and metabolic disease. Understanding BAT and WAT physiology and dysfunction necessitates clinical trials complemented by mechanistic experiments at the cellular level. These require standardized in vitro models, currently lacking, that establish references for gene expression and function. We generated and characterized a pair of immortalized, clonal human brown (hBA) and white (hWA) preadipocytes derived from the perirenal and subcutaneous depots, respectively, of a 40-year-old male individual. Cells were immortalized with hTERT and confirmed to be of a mesenchymal, nonhematopoietic lineage based on fluorescence-activated cell sorting and DNA barcoding. Functional assessments showed that the hWA and hBA phenocopied primary adipocytes in terms of adrenergic signaling, lipolysis, and thermogenesis. Compared to hWA, hBA were metabolically distinct, with higher rates of glucose uptake and lactate metabolism, and greater basal, maximal, and nonmitochondrial respiration, providing a mechanistic explanation for the association between obesity and BAT dysfunction. The hBA also responded to the stress of maximal respiration by using both endogenous and exogenous fatty acids. In contrast to certain mouse models, hBA adrenergic thermogenesis was mediated by several mechanisms, not principally via uncoupling protein 1 (UCP1). Transcriptomics via RNA-seq were consistent with the functional studies and established a molecular signature for each cell type before and after differentiation. These standardized cells are anticipated to become a common resource for future physiological, pharmacological, and genetic studies of human adipocytes.

WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature.

Macrophages have a critical role in inflammatory and immune responses through their ability to recognize and engulf apoptotic cells. Here we show that macrophages initiate a cell-death programme in target cells by activating the canonical WNT pathway. We show in mice that macrophage WNT7b is a short-range paracrine signal required for WNT-pathway responses and programmed cell death in the vascular endothelial cells of the temporary hyaloid vessels of the developing eye. These findings indicate that macrophages can use WNT ligands to influence cell-fate decisions--including cell death--in adjacent cells, and raise the possibility that they do so in many different cellular contexts.

Live-cell imaging and analysis reveal cell phenotypic transition dynamics inherently missing in snapshot data.

Recent advances in single-cell techniques catalyze an emerging field of studying how cells convert from one phenotype to another, in a step-by-step process. Two grand technical challenges, however, impede further development of the field. Fixed cell-based approaches can provide snapshots of high-dimensional expression profiles but have fundamental limits on revealing temporal information, and fluorescence-based live-cell imaging approaches provide temporal information but are technically challenging for multiplex long-term imaging. We first developed a live-cell imaging platform that tracks cellular status change through combining endogenous fluorescent labeling that minimizes perturbation to cell physiology and/or live-cell imaging of high-dimensional cell morphological and texture features. With our platform and an A549 VIM-RFP epithelial-to-mesenchymal transition (EMT) reporter cell line, live-cell trajectories reveal parallel paths of EMT missing from snapshot data due to cell-cell dynamic heterogeneity. Our results emphasize the necessity of extracting dynamical information of phenotypic transitions from multiplex live-cell imaging.

Wnt7b activates canonical signaling in epithelial and vascular smooth muscle cells through interactions with Fzd1, Fzd10, and LRP5.

Wnt7b is a Wnt ligand that has been demonstrated to play critical roles in several developmental processes, including lung airway and vascular development and chorion-allantois fusion during placental development. Wnt signaling involves the binding of Wnt ligands to cell surface receptors of the frizzled family and coreceptors of the LRP5/6 family. However, little is known of the ligand-receptor specificity exhibited by different Wnts, Fzds, and LRPs in Wnt signaling. Expression analysis of Fzds and LRP5/6 in the developing lung and vasculature showed that Fzd1, -4, -7, and -10 and LRP5/6 are expressed in tissue-specific patterns during lung development. Fzd1, -4, and -7 are expressed primarily in the developing lung mesenchyme, and Fzd10 is expressed in airway epithelium. LRP5 and LRP6 are expressed in airway epithelium during lung development, whereas LRP5 but not LRP6 expression is observed in the muscular component of large blood vessels, including the aorta. Cell transfection studies demonstrate that Wnt7b can activate the canonical Wnt pathway but not the noncanonical Wnt pathway in a cell-specific manner. Biochemical analysis demonstrates that Wnt7b can bind to Fzd1 and -10 on the cell surface and cooperatively activate canonical Wnt signaling with these receptors in the presence of LRP5. Together, these data demonstrate that Wnt7b signals through Fzd1 and -10 and LRP5 and implicate these Wnt coreceptors in the regulation of lung airway and vascular development.

Foxp2 and Foxp1 cooperatively regulate lung and esophagus development.

The airways of the lung develop through a reiterative process of branching morphogenesis that gives rise to the intricate and extensive surface area required for postnatal respiration. The forkhead transcription factors Foxp2 and Foxp1 are expressed in multiple foregut-derived tissues including the lung and intestine. In this report, we show that loss of Foxp2 in mouse leads to defective postnatal lung alveolarization, contributing to postnatal lethality. Using in vitro and in vivo assays, we show that T1alpha, a lung alveolar epithelial type 1 cell-restricted gene crucial for lung development and function, is a direct target of Foxp2 and Foxp1. Remarkably, loss of a single Foxp1 allele in addition to complete loss of Foxp2 results in increased severity of morphological defects in mutant lungs and leads to perinatal loss of all Foxp2(-/-);Foxp1(+/-) mice. Expression of N-myc and Hop, crucial regulators of lung development, is compromised in Foxp2(-/-);Foxp1(+/-) mutants. In addition to the defects in lung development, esophageal muscle development is disrupted in Foxp2(-/-);Foxp1(+/-) embryos, a tissue where Foxp2 and Foxp1 are co-expressed. These data identify Foxp2 and Foxp1 as crucial regulators of lung and esophageal development, underscoring the necessity of these transcription factors in the development of anterior foregut-derived tissues and demonstrating functional cooperativity between members of the Foxp1/2/4 family in tissues where they are co-expressed.

Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung.

Branching morphogenesis in the lung serves as a model for the complex patterning that is reiterated in multiple organs throughout development. Beta-catenin and Wnt signaling mediate critical functions in cell fate specification and differentiation, but specific functions during branching morphogenesis have remained unclear. Here, we show that Wnt/beta-catenin signaling regulates proximal-distal differentiation of airway epithelium. Inhibition of Wnt/beta-catenin signaling, either by expression of Dkk1 or by tissue-specific deletion of beta-catenin, results in disruption of distal airway development and expansion of proximal airways. Wnt/beta-catenin functions upstream of BMP4, FGF signaling, and N-myc. Moreover, we show that beta-catenin and LEF/TCF activate the promoters of BMP4 and N-myc. Thus, Wnt/beta-catenin signaling is a critical upstream regulator of proximal-distal patterning in the lung, in part, through regulation of N-myc, BMP4, and FGF signaling.

Altered ultrasonic vocalization in mice with a disruption in the Foxp2 gene.

Neurobiology of speech and language has previously been studied in the KE family, in which half of the members have severe impairment in both speech and language. The gene responsible for the phenotype was mapped to chromosome 7q31 and identified as the FOXP2 gene, coding for a transcription factor containing a polyglutamine tract and a forkhead DNA-binding domain. Because of linkage studies implicating 7q31 in autism, where language impairment is a component of the disorder, and in specific language impairment, FOXP2 has also been considered as a potential susceptibility locus for the language deficits in autism and/or specific language impairment. In this study, we characterized mice with a disruption in the murine Foxp2 gene. Disruption of both copies of the Foxp2 gene caused severe motor impairment, premature death, and an absence of ultrasonic vocalizations that are elicited when pups are removed from their mothers. Disruption of a single copy of the gene led to modest developmental delay but a significant alteration in ultrasonic vocalization in response to such separation. Learning and memory appear normal in the heterozygous animals. Cerebellar abnormalities were observed in mice with disruptions in Foxp2, with Purkinje cells particularly affected. Our findings support a role for Foxp2 in cerebellar development and in a developmental process that subsumes social communication functions in diverse organisms.

Wnt7b regulates mesenchymal proliferation and vascular development in the lung.

Although the Wnt signaling pathway regulates inductive interactions between epithelial and mesenchymal cells, little is known of the role that this pathway plays during lung development. Wnt7b is expressed in the airway epithelium, suggesting a possible role for Wnt-mediated signaling in the regulation of lung development. To test this hypothesis, we have mutated Wnt7b in the germline of mice by replacement of the first exon with the lacZ-coding region. Wnt7b(lacZ-/-) mice exhibit perinatal death due to respiratory failure. Defects in early mesenchymal proliferation leading to lung hypoplasia are observed in Wnt7b(lacZ-/-) embryos. In addition, Wnt7b(lacZ-/-) embryos and newborn mice exhibit severe defects in the smooth muscle component of the major pulmonary vessels. These defects lead to rupture of the major vessels and hemorrhage in the lungs after birth. These results demonstrate that Wnt7b signaling is required for proper lung mesenchymal growth and vascular development.

The WNT7b promoter is regulated by TTF-1, GATA6, and Foxa2 in lung epithelium.

In this study, we find that WNT7b is the only member of the WNT family of autocrine/paracrine signaling molecules whose expression in the lung is restricted to the airway epithelium during embryonic development. To study the transcriptional mechanisms that underlie this restricted pattern of WNT7b expression, we isolated the proximal 1.0-kb mouse WNT7b promoter and mapped the transcriptional start sites. Transfection of the lung epithelial cell line MLE-15, which expresses WNT7b, shows that the 1.0-kb mouse WNT7b promoter is highly active in lung epithelial cells. This region of the WNT7b promoter contains several DNA binding sites for the important lung-restricted transcription factors TTF-1, GATA6, and Foxa2. Electrophoretic mobility shift assays showed that TTF-1, GATA6, and Foxa2 can bind to a specific subset of their consensus DNA binding sites within the WNT7b promoter. Using cotransfection assays, we demonstrate that TTF-1, GATA6, and Foxa2 can trans-activate the WNT7b promoter in NIH-3T3 cells. Truncation of GATA6 or Foxa2 binding sites reduced the ability of these transcriptional regulators to trans-activate the WNT7b promoter. Finally, the minimal 118-bp region of the mouse WNT7b promoter containing only TTF-1 binding sites was synergistically activated by TTF-1 and GATA6, and we show that TTF-1 and GATA6 physically interact in vivo. Together, these results suggest that WNT7b gene expression in the lung epithelium is regulated in a combinatorial fashion by TTF-1, GATA6, and Foxa2.