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Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease disrupting lung health throughout the life of an individual and that is increasing in incidence. The TGF-[beta] superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that TGFbr2 is critical for alveolar epithelial (AT1) cell fate maintenance and function. Loss of TGFbr2 in AT1 cells during late lung development leads to AT1-AT2 cell reprogramming and altered pulmonary architecture, which persists into adulthood. Restriction of fetal lung stretch and associated AT1 cell spreading through a model of oligohydramnios enhances AT1-AT2 reprogramming. Transcriptomic and proteomic analyses reveal the necessity of TGFbr2 expression in AT1 cells for extracellular matrix production. Moreover, TGF-[beta] signaling regulates integrin transcription to alter AT1 cell morphology, which further impacts ECM expression through changes in mechanotransduction. These data reveal the cell intrinsic necessity of TGF-[beta] signaling in maintaining AT1 cell fate and reveal this cell lineage as a major orchestrator of the alveolar matrisome., Introduction Bronchopulmonary dysplasia (BPD) is a lung disease that disproportionately affects infants born at less than 28 weeks gestation, the threshold for extreme prematurity (1). Despite multiple medical advances including [...]

Abstract: The Cre/loxP system has become an important tool for the conditional gene knockout and conditional gene expression in genetically engineered mice. The applications of this system depend on transgenic [...]

To determine the origin of adult tissue-resident macrophages, a mouse lineage tracing study has revealed that these cells derive from erythro-myeloid progenitors in the yolk sac that are distinct from fetal and adult haematopoietic stem cells. The origin of adult myeloid cells The developmental origin of tissue-resident macrophage progenitors and their contribution to macrophages in fetal and adult organs relative to bone marrow macrophages are still unclear. Using lineage tracing, Elisa Gomez Perdiguero et al. identify a population of yolk-sac-derived progenitors -- distinct from fetal and adult haematopoetic stem cells -- that gives rise to erythrocytes, macrophages, granulocytes and monocytes in the young mouse fetus, and to the vast majority of adult tissue-resident macrophages. Most haematopoietic cells renew from adult haematopoietic stem cells (HSCs).sup.1,2,3, however, macrophages in adult tissues can self-maintain independently of HSCs.sup.4,5,6,7. Progenitors with macrophage potential in vitro have been described in the yolk sac before emergence of HSCs.sup.8,9,10,11,12,13, and fetal macrophages.sup.13,14,15 can develop independently of Myb.sup.4, a transcription factor required for HSC.sup.16, and can persist in adult tissues.sup.4,17,18. Nevertheless, the origin of adult macrophages and the qualitative and quantitative contributions of HSC and putative non-HSC-derived progenitors are still unclear.sup.19. Here we show in mice that the vast majority of adult tissue-resident macrophages in liver (Kupffer cells), brain (microglia), epidermis (Langerhans cells) and lung (alveolar macrophages) originate from a Tie2.sup.+ (also known as Tek) cellular pathway generating Csf1r.sup.+ erythro-myeloid progenitors (EMPs) distinct from HSCs. EMPs develop in the yolk sac at embryonic day (E) 8.5, migrate and colonize the nascent fetal liver before E10.5, and give rise to fetal erythrocytes, macrophages, granulocytes and monocytes until at least E16.5. Subsequently, HSC-derived cells replace erythrocytes, granulocytes and monocytes. Kupffer cells, microglia and Langerhans cells are only marginally replaced in one-year-old mice, whereas alveolar macrophages may be progressively replaced in ageing mice. Our fate-mapping experiments identify, in the fetal liver, a sequence of yolk sac EMP-derived and HSC-derived haematopoiesis, and identify yolk sac EMPs as a common origin for tissue macrophages., Author(s): Elisa Gomez Perdiguero [sup.1] , Kay Klapproth [sup.2] , Christian Schulz [sup.1] , Katrin Busch [sup.2] , Emanuele Azzoni [sup.3] , Lucile Crozet [sup.1] , Hannah Garner [sup.1] , [...]

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases., Author(s): Feng Yue [1, 2]; Yong Cheng [3]; Alessandra Breschi [4]; Jeff Vierstra [5]; Weisheng Wu [6]; Tyrone Ryba [7]; Richard Sandstrom [5]; Zhihai Ma [3]; Carrie Davis [8]; Benjamin [...]

More than 130 million people worldwide chronically infected with hepatitis C virus (HCV) are at risk of developing severe liver disease. Antiviral treatments are only partially effective against HCV infection, and a vaccine is not available. Development of more efficient therapies has been hampered by the lack of a small animal model. Building on the observation that CD81 and occludin (OCLN) comprise the minimal set of human factors required to render mouse cells permissive to HCV entry (1), we previously showed that transient expression of these two human genes is sufficient to allow viral uptake into fully immunocompetent inbred mice (2). Here we demonstrate that transgenic mice stably expressing human CD81 and OCLN also support HCV entry, but innate and adaptive immune responses restrict HCV infection in vivo. Blunting antiviral immunity in genetically humanized mice infected with HCV results in measurable viraemia over several weeks. In mice lacking the essential cellular cofactor cyclophilin A (CypA), HCV RNA replication is markedly diminished, providing genetic evidence that this process is faithfully recapitulated. Using a cell-based fluorescent reporter activated by the NS3-4A protease we visualize HCV infection in single hepatocytes invivo. Persistently infected mice produce denovoinfectious particles, which can be inhibited with directly acting antiviral drug treatment, thereby providing evidence for the completion of the entire HCV life cycle in inbred mice. This genetically humanized mouse model opens new opportunities to dissect genetically HCV infection invivoand provides an important preclinical platform for testing and prioritizing drug candidates and may also have utility for evaluating vaccine efficacy., The narrow species tropism of HCV is incompletely understood. Mouse cells do not support viral entry and inefficiently replicate HCV RNA, but do support virion assembly and release. HCV uses [...]

Nato 3, a Basic Helix-Loop-Helix Protein, is Expressed in the Floor Plate of the Developing Neural Tube at all Axial Levels in the Gallus gallus and Mus musculus Embryo. Sarala [...]

Structural variation is widespread in mammalian genomes (1,2) and is an important cause of disease (3), but just how abundant and important structural variants (SVs) are in shaping phenotypic variation remains unclear (4,5). Without knowing how many SVs there are, and how they arise, it is difficult to discover what they do. Combining experimental with automated analyses, we identified 711,920 SVs at 281,243 sites in the genomes of thirteen classical and four wild-derived inbred mouse strains. The majority of SVs are less than 1 kilobase in size and 98% are deletions or insertions. The breakpoints of 160,000 SVs were mapped to base pair resolution, allowing us to infer that insertion of retrotransposons causes more than half of SVs. Yet, despite their prevalence, SVs are less likely than other sequence variants to cause gene expression or quantitative phenotypic variation. We identified 24 SVs that disrupt coding exons, acting as rare variants of large effect on gene function. One-third of the genes so affected have immunological functions., The pre-eminent organism for modelling the relationship between phenotype and genotype, including SVs, is the mouse, but our catalogue of SVs in this animal is incomplete (6) and most of [...]

We report genome sequences of 17 inbred strains of laboratory mice and identify almost ten times more variants than previously known. We use these genomes to explore the phylogenetic history of the laboratory mouse and to examine the functional consequences of allele-specific variation on transcript abundance, revealing that at least 12% of transcripts show a significant tissue-specific expression bias. By identifying candidate functional variants at 718 quantitative trait loci we show that the molecular nature of functional variants and their position relative to genes vary according to the effect size of the locus. These sequences provide a starting point for a new era in the functional analysis of a key model organism., Until the end of the 20th century the molecular basis for morphological, physiological, biochemical and behavioural variation in laboratory mice remained largely obscure (1-3). At the beginning of the 21st [...]

It is known from paleontology studies that two premolars have been lost during mouse evolution. During mouse mandible development, two bud-like structures transiently form that may represent rudimentary precursors of the lost premolars. However, the interpretation of these structures and their significance for mouse molar development are highly controversial because of a lack of molecular data. Here, we searched for typical tooth signaling centers in these two bud-like structures, and followed their fate using molecular markers, 3D reconstructions, and lineage tracing in vitro. Transient signaling centers were indeed found to be located at the tips of both the anterior and posterior rudimentary buds. These centers expressed a similar set of molecular markers as the 'primary enamel knot' (pEK), the signaling center of the first molar (M1). These two transient signaling centers were sequentially patterned before and anterior to the M1 pEK. We also determined the dynamics of the M1 pEK, which, slightly later during development, spread up to the field formerly occupied by the posterior transient signaling center. It can be concluded that two rudimentary tooth buds initiate the sequential development of the mouse molars and these have previously been mistaken for early stages of M1 development. Although neither rudiment progresses to form an adult tooth, the posterior one merges with the adjacent M1, which may explain the anterior enlargement of the M1 during mouse family evolution. This study highlights how rudiments of lost structures can stay integrated and participate in morphogenesis of functional organs and help in understanding their evolution, as Darwin suspected long ago. rudiment | signaling center | tooth evolution | SHH | molar development doi/ 10.1073/pnas.1002784107

Urothelium that lines almost the entire urinary tract performs important functions and is prone to assaults by urinary microbials, metabolites, and carcinogens. To improve our understanding of urothelial physiology and disease pathogenesis, we sought to develop two novel transgenic systems, one that would allow inducible and urothelium-specific gene expression, and another that would allow inducible and urothelium-specific knockout. Toward this end, we combined the ability of the mouse uroplakin II promoter (mUPII) to drive urothelium-specific gene expression with a versatile tetracycline-mediated inducible system. We found that, when constructed under the control of mUPII, only a modified, reverse tetracycline trans-activator (rtTA-M2), but not its original version (rtTA), could efficiently trans-activate reporter gene expression in mouse urothelium on doxycycline (Dox) induction. The mUPII/rtTA-M2-inducible system retained its strict urothelial specificity, had no background activity in the absence of Dox, and responded rapidly to Dox administration. Using a reporter gene whose expression was secondarily controlled by histone remodeling, we were able to identify, colocalize with 5-bromo-2-deoxyuridine incorporation, and semiquantify newly divided urothelial cells. Finally, we established that, when combined with a Cre recombinase under the control of the tetracycline operon, the mUPII-driven rtTA-M2 could inducibly inactivate any gene of interest in mouse urothelium. The establishment of these two new transgenic mouse systems enables the manipulation of gene expression and/or inactivation in adult mouse urothelium at any given time, thus minimizing potential compensatory effects due to gene overexpression or loss and allowing more accurate modeling of urothelial diseases than previously reported constitutive systems. uroplakin; transgenic; conditional; tetracycline doi: 10.1152/ajprenal.00185.2010.

Skeletal muscle atrophy and weakness are major contributors to frailty and impact significantly on quality of life of older people. Muscle aging is characterized by a loss of maximum tetanic force ([P.sub.o]) generation, primarily due to muscle atrophy, to which mitochondrial dysfunction is hypothesized to contribute. We hypothesized that lifelong overexpression of the mitochondrial heat shock protein (HSP) HSP10 in muscle of mice would protect against development of these deficits. [P.sub.o] generation by extensor digitorum longus muscles of adult and old wild-type and HSP10-overexpressing mice was determined in situ. Muscles were subjected to damaging lengthening contractions, and force generation was remeasured at 3 h or 28 days to examine susceptibility to, and recovery from, damage, respectively. Muscles of old wild-type mice had a 23% deficit in [P.sub.o] generation and a 10% deficit in muscle cross-sectional area compared with muscles of adult wild-type mice. Overexpression of HSP10 prevented this age-related fall in [P.sub.o] generation and reduction in cross-sectional area observed in muscles of old wild-type mice. Additionally, overexpression of HSP10 protected against contraction-induced damage independent of age but did not improve recovery if damage occurred. Preservation of muscle force generation and CSA by HSP10 overexpression was associated with protection against the age-related accumulation of protein carbonyls. Data demonstrate that development of age-related muscle weakness may not be inevitable and show, for the first time, that lifelong overexpression of an HSP prevents the age-related loss of [P.sub.o] generation. These findings support the hypothesis that mitochondrial dysfunction is involved in the development of age-related muscle deficits. aging; heat shock protein; Cpnl0; Hspel; atrophy doi: 10.1152/ajpregu.00334.2009.

Hox genes play evolutionarily conserved roles in specifying axial position during embryogenesis. A prevailing paradigm is that changes in Hox gene expression drive evolution of metazoan body plans. Conservation of Hox function across species, and among paralogous Hox genes within a species, supports a model of functional equivalence. In this report, we demonstrate that zebrafish hoxa3a (zfhoxa3a) expressed from the mouse Hoxa3 locus can substitute for mouse Hoxa3 in some tissues, but has distinct or null phenotypes in others. We further show, by using an allele encoding a chimeric protein, that this difference maps primarily to the zfhoxa3a C-terminal domain. Our data imply that the mouse and zebrafish proteins have diverged considerably since their last common ancestor, and that the major difference between them resides in the C-terminal domain. Our data further show that Hox protein function can evolve independently in different cell types or for specific functions. The inability of zfhoxa3a to perform all of the normal roles of mouse Hoxa3 illustrates that Hox orthologues are not always functionally interchangeable. evolution | Hox | vertebrate | thymus | parathyroid doi/ 10.1073/pnas.1005129107

Electrophoretic surveys of hemoglobin (Hb) polymorphism in house mice from South Asia and the Middle East have revealed that two alternative [beta]-globin haplotypes, [Hbb.sup.d] and [Hbb.sup.p], are often present at intermediate frequencies in geographically disparate populations. Both haplotypes harbor two functionally distinct [beta]-globin paralogs, HBB-T1 (which encodes the [beta]-chain subunits of the major Hb isoform) and HBB-T2 (which encodes the [beta]-chains of the minor Hb isoform). The [Hbb.sup.d] and [Hbb.sup.p] haplotypes share identical HBB-T1 alleles, but products of the alternative HBB-T2 alleles ([d.sub.minor] and [p.sub.minor] ) are distinguished by two amino acid substitutions. To investigate the possible adaptive significance of the [Hbb.sup.d]/[Hbb.sup.p] polymorphism we conducted a population genetic analysis of the duplicated [beta]-globin genes of Indian house mice (Mus castaneus) in conjunction with experimental studies of Hb function in inbred strains of mice that carry the alternative [Hbb.sup.d] and [Hbb.sup.p] haplotypes. The main objectives of this study were (i) to characterize patterns of nucleotide polymorphism and linkage disequilibrium in the duplicated [beta]-globin genes of M. castaneus, (ii) to test the hypothesis that the [Hbb.sup.d] and [Hbb.sup.p] haplotypes are maintained as a balanced polymorphism, and (iii) to assess whether allelic differences in the alternative minor Hb isoforms ( [d.sub.minor] and [p.sub.minor] ) are associated with different [O.sub.2]- binding properties. A multilocus analysis of polymorphism and divergence revealed that levels of diversity at the HBB-T2 gene exceeded neutral expectations, and reconstructed haplotype networks for both [beta]-globin paralogs revealed extensive allele sharing with several other closely related species of Mus. However, despite this suggestive evidence for balancing selection, [O.sub.2]-equilibrium curves revealed no discernible functional differences between red cell lysates containing the [d.sub.minor] and [p.sub.minor] Hb isoforms. If the [d.sub.minor] and [p.sub.minor] alleles are maintained as a balanced polymorphism, our results indicate that the associated fitness variance is not directly related to respiratory functions of Hb.

Endogenous trypsin inhibitors are synthesized, stored, and secreted by pancreatic acinar cells. It is believed that they play a protective role in the pancreas by inhibiting trypsin within the cell should trypsinogen become prematurely activated. Rodent trypsin inhibitors are highly homologous to human serine protease inhibitor Kazal-type 1 (SPINK1). The mouse has one pancreatic trypsin inhibitor known as SPINK3, and the rat has two trypsin inhibitors commonly known as pancreatic secretory trypsin inhibitors I and II (PSTI-I and -II). Rat PSTI-I is a 61-amino acid protein that shares 65% sequence identity with mouse SPINK3. It was recently demonstrated that mice with genetic deletion of the Spink3 gene ([Spink3.sup.-/-]) do not survive beyond 15 days and lack normal pancreata because of pancreatic autophagy. We have shown that targeted transgenic expression of the rat Psti1 gene to acinar cells in mice [TgN(Psti1)] protects mice against caerulein-induced pancreatitis. To determine whether the autophagic phenotype and lethality in [Spink3.sup.-/-] mice were due to lack of pancreatic trypsin inhibitor, we conducted breeding studies with [Spink.sup.3+/-] heterozygous mice and TgN(Psti1) mice. We observed that, whereas [Spink3.sup.+/+], [Spink3.sup.+/-], and [Spink3-/-]/TgN(Pstil) mice had similar survival rates, no [Spink3.sup.-/-] mice survived longer than 1 wk. The level of expression of SPINK3 protein in acini was reduced in heterozygote mice compared with wild-type mice. Furthermore, endogenous trypsin inhibitor capacity was reduced in the pancreas of heterozygote mice compared with wild-type or knockout mice rescued with the rat Psti1 gene. Surprisingly, the lesser amount of SPINK3 present in the pancreata of heterozygote mice did not predispose animals to increased susceptibility to caerulein-induced acute pancreatitis. We propose that a threshold level of expression is sufficient to protect against pancreatitis. doi: 10.1152/ajpgi.00431.2009 PSTI-I; pancreatitis; autophagy

The objective of this study was to investigate the role of MAPKAP kinase 2 (MK2) and heat shock protein (HSP) HSP60 in the pathogenesis of a new model of severe acute pancreatitis (AP). MK2 plays a significant role in the regulation of cytokines. It has been shown that induction and expression of several HSPs can protect against experimental pancreatitis. Interplay between both systems seems of high interest. Mice with a homozygous deletion of the MK2 gene were used. Severe AP was induced by combined intraperitoneal injections of cerulein with lipopolysaccharide (LPS). Severity of AP was assessed by biochemical markers and histology. The serum IL-6 and lung myeloperoxidase (MPO) levels were determined for assessing the extent of systemic inflammatory response. Expression of HSP25, HSP60, HSP70, and HSP90 was analyzed by Western blotting. Repeated injections of cerulein alone or cerulein plus LPS (Cer+LPS) resulted in local inflammatory responses in the pancreas and corresponding systemic inflammatory changes with pronounced severity in the Cer+LPS group. Compared with the C57B1 wild-type mice, the MK2-/- mice presented with significant milder pancreatitis and attenuated responses of serum amylase and trypsinogen activity. Furthermore, serum IL-6 was decreased as well as lung MPO activity. Injection of LPS alone displayed neither pancreatic inflammatory responses nor alterations of pancreatic enzyme activities but evidently elevated serum IL-6 levels and increased lung MPO activity. In contrast hereto, in the MK2-/-mice, these changes were much milder. Increased expression of HSP25 and HSP60 occurred after induction of AP. Especially, HSP60 was robustly elevated after Cer+LPS treatment, in both MK2-/- and wild-type mice. Thus the homozygous deletion of the MK2 gene ameliorates the severity of acute pancreatitis and accompanying systemic inflammatory reactions in a new model of severe acute pancreatitis. Our data support the hypothesis that MK2 participates in the multifactorial regulation of early inflammatory responses in AP, independently of the regulation of stress proteins like HSP25 and HSP60 and most likely due to its effect on cytokine regulation. MAP kinases; cytokines; heat shock proteins; inflammation doi: 10.1152/ajpgi.00225.2009.

Genetic manipulations of Mn superoxide dismutase (MnSOD), SOD2 expression have demonstrated that altering the level of MnSOD activity is critical for cellular function and life span in invertebrates. In mammals. Sod2 homozygous knockout mice die shortly after birth, and alterations of MnSOD levels are correlated with changes in oxidative damage and in the generation of mitochondrial reactive oxygen species. In this study, we directly tested the effects of overexpressing MnSOD in young (4-6 months) and old (26-28 months) mice on mitochondrial function, levels of oxidative damage or stress, life span, and end-of-life pathology. Our data show that an approximately twofold overexpression of MnSOD throughout life in mice resulted in decreased lipid peroxidation, increased resistance against paraquat-induced oxidative stress, and decreased age-related decline in mitochondrial ATP production. However, this change in MnSOD expression did not alter either life span or age-related pathology. Key Words: Oxidative damage--Mn superoxide dismutase--Pathology--Aging. doi: 10.1093/gerona/glp100

Growth hormone receptor knockout (GHRKO) mice live about 40%-55% longer than their normal (N) littermates. Previous studies of 21-month-old GHRKO and N mice showed major alterations of the hepatic expression of genes involved in insulin signaling. Differences detected at this age may have been caused by the knockout of the growth hormone receptor (GHR) or by differences in biological age between GHRKO and N mice. To address this question, we compared GHRKO and N mice at ages corresponding to the same percentage of median life span to see if the differences of gene expression persisted. Comparison of GHRKO and N mice at ~50% of biological life span showed significant differences in hepatic expression of all 14 analyzed genes. We conclude that these changes are due to disruption of GHR gene and the consequent suppression of growth hormone signaling rather than to differences in 'biological age' between mutant and normal animals sampled at the same chronological age. Key Words: Aging--GHRKO--Insulin. doi: 10.1093/gerona/glp111