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1.  [No title available] 
PMCID: PMC3944086  PMID: 24281837
2.  [No title available] 
PMCID: PMC3973152  PMID: 24288358
3.  On the cutting edge of organ renewal: identification, regulation and evolution of incisor stem cells 
The rodent incisor is one of a number of organs that grow continuously throughout the life of an animal. Continuous growth of the incisor arose as an evolutionary adaptation to compensate for abrasion at the distal end of the tooth. The sustained turnover of cells that deposit the mineralized dental tissues is made possible by epithelial and mesenchymal stem cells residing at the proximal end of the incisor. A complex network of signaling pathways and transcription factors regulates the formation, maintenance, and differentiation of these stem cells during development and throughout adulthood. Research over the past 15 years has led to significant progress in our understanding of this network, which includes FGF, BMP, Notch, and Hh signaling, as well as cell adhesion molecules and microRNAs. This review surveys key historical experiments that laid the foundation of the field and discusses more recent findings that definitively identified the stem cell population, elucidated the regulatory network, and demonstrated possible genetic mechanisms for the evolution of continuously growing teeth.
PMCID: PMC4252016  PMID: 24307456
hypselodont; tissue regeneration; tooth; dental; renewal
4.  Guidelines for the Nomenclature of Genetic Elements in Tunicate Genomes 
Tunicates are invertebrate members of the chordate phylum, and are considered to be the sister group of vertebrates. Tunicates are composed of ascidians, thaliaceans, and appendicularians. With the advent of inexpensive high-throughput sequencing, the number of sequenced tunicate genomes is expected to rise sharply within the coming years. To facilitate comparative genomics within the tunicates, and between tunicates and vertebrates, standardized rules for the nomenclature of tunicate genetic elements need to be established. Here we propose a set of nomenclature rules, consensual within the community, for predicted genes, pseudogenes, transcripts, operons, transcriptional cis-regulatory regions, transposable elements, and transgenic constructs. In addition, the document proposes guidelines for naming transgenic and mutant lines.
PMCID: PMC4308547  PMID: 25220678
tunicates; genome annotation; gene; transposable element; cis-regulatory sequences
5.  The C. elegans nuclear receptor NHR-6 functionally interacts with the JUN-1 transcription factor during spermatheca development 
The NR4A nuclear receptor NHR-6 is an essential regulator of spermatheca organogenesis in C. elegans. In this study, we perform a focused, RNAi-based screen to identify modifiers of partial nhr-6 loss of function. Ninety-eight genes that encode signaling proteins expressed in the spermatheca were screened for enhancement of the nhr-6 RNAi phenotype. We identify the C. elegans gene jun-1, which encodes the homolog of the Jun transcription factor, as a strong enhancer of nhr-6 partial loss of function. We show that nhr-6 and jun-1 function together to regulate development of the spermatheca and are necessary for generating an organ with the normal number of cells. jun-1 is expressed in all cells of the developing spermatheca. We also provide evidence that NHR-6 and JUN-1 can physically interact in a yeast two-hybrid assay. Our results provide in vivo evidence that NR4A nuclear receptor and Jun transcription factor interactions are essential in regulating developmental processes in metazoans.
PMCID: PMC3954850  PMID: 24178943
RNAi interaction screen; NR4A; modifier
6.  RUSH & CRUSH: a rapid and conditional RNA interference method in mice 
RNA interference (RNAi) is a powerful approach to phenocopy mutations in many organisms. Gold standard conventional knock-out mouse technology is labor- and time-intensive, however off-target effects may confound transgenic RNAi approaches. Here we describe a rapid method for conditional and reversible gene silencing in RNAi transgenic mouse models and embryonic stem (ES) cells. RUSH and CRUSH RNAi vectors were designed for reversible or conditional knockdown, respectively, demonstrated using targeted replacement in an engineered ROSA26lacZ ES cell line and wildtype V6.5 ES cells. RUSH was validated by reversible knockdown of Dnmt1 in vitro. Conditional mouse model production using CRUSH was expedited by deriving ES cell lines from Cre transgenic mouse strains (nestin, cTnnT, and Isl1) and generating all-ES Go transgenic founders by tetraploid complementation. A control CRUSHGFP RNAi mouse strain showed quantitative knockdown of GFP fluorescence as observed in compound CRUSHGFP, Ds-Red Cre-reporter transgenic mice, and confirmed by western blotting. The capability to turn RUSH and CRUSH alleles off or on using Cre recombinase enables this method to rapidly address questions of tissue-specificity and cell autonomy of gene function in development.
PMCID: PMC3985430  PMID: 24166816
RNAi; ES cells; transgenesis; mouse models
7.  The role of pygopus in the differentiation of intra-cardiac valves in Drosophila 
Cardiac valves serve an important function; they support unidirectional blood flow and prevent blood regurgitation. Wnt signaling plays an important role in the formation of mouse cardiac valves and cardiac valve proliferation in Zebrafish, but identification of the specific signaling components involved has not been addressed systematically. Of the components involved in Wnt signal transduction, pygopus (pygo), first identified as a core component of Wnt signaling in Drosophila, has yet to be investigated with respect to valve development and differentiation. Here, we take advantage of the Drosophila heart model to study the role of pygo in formation of valves between the cardiac chambers. We found that cardiac-specific pygo knockdown in the Drosophila heart causes dilation in the region of these cardiac valves, and their characteristic dense mesh of myofibrils does not form and resembles that of neighboring cardiomyocytes. In contrast, heart-specific knockdown of the transcription factors, arm/β-Cat, lgs/BCL9 or pan/TCF, which mediate canonical Wnt signal transduction, shows a much weaker valve differentiation defect. Double-heterozygous combinations of mutants for pygo and the Wnt-signaling components have no additional effect on heart function compared to pygo heterozygotes alone. These results are consistent with the idea that pygo functions independently of canonical Wnt signaling in the differentiation of the adult inter-chamber cardiac valves.
PMCID: PMC4142678  PMID: 24265259
intra-cardiac valves; pygo; heart; Wnt; cardiomyocyte
8.  Conditional Gene Recombination by Adenovirus-Driven Cre in the Mouse Uterus 
Cre-mediated conditional gene targeting has been shown to be successful in many cell and tissue types. However, gene recombination in the uterus with heterogeneous cell types by Cre activation is not yet well established. Using recombinant adenoviruses expressing a functional Cre (ADV-Cre) and ROSA26 reporter mice, we show here that ADV-Cre infused intraluminally in a small volume (10 μl) conditionally excises the loxP site, resulting in lacZ expression in uterine luminal epithelial cells without significantly affecting pregnancy. In contrast, a similar intraluminal infusion of ADV-Cre in a larger volume (50 μl) damages the normal architecture and integrity of the luminal epithelium, inducing gene recombination in the underneath stromal cells, with disruption of pregnancy. Further, decidualizing stromal cells at the implantation sites can be targeted by ADV-Cre after intravenous administration on days 5–6. This route of administration also elicits Cre activity in other tissues, including the liver, spleen, ovary, and, more remarkably, in the adrenal cortex. These findings demonstrate the feasibility of achieving conditional expression or deletion of specific genes in uterine cells at desired times and physiological states.
PMCID: PMC4267753  PMID: 16416422
conditional gene targeting; adenovirus; Cre; uterus
9.  Nkx2-5 Lineage Tracing Visualizes the Distribution of Second Heart Field-Derived Aortic Smooth Muscle 
Genesis (New York, N.Y. : 2000)  2013;51(12):862-869.
During embryonic development, smooth muscle within the ascending aorta arises from two distinct sources: second heart field progenitors and the neural crest. It has recently been hypothesized that the boundary between smooth muscle from these distinct origins may be particularly susceptible to acute aortic dissection. While the contribution of second heart field progenitors to the ascending aorta is well established, detailed mapping of the anatomical distribution of second heart field-derived smooth muscle at this smooth muscle boundary has yet to be observed using a committed cardiac progenitor Cre-lineage. Using Nkx2-5-Cre knockin mice, the anatomical distribution of second heart field derived aortic smooth muscle was mapped in detail. Specifically, Nkx2-5-Cre-labeled cells constitute the entirety of the smooth muscle layer at the aortic base and then become restricted to the adventitial side of the ascending aortic media. This distribution pattern is present by E12.5 in the embryo and persists throughout embryonic development. These data reveal previously unappreciated details regarding the anatomical distribution of second heart field-derived smooth muscle within the aorta as well as the non-cardiomyocyte fates labeled by the Nkx2-5-Cre lineage.
PMCID: PMC3943338  PMID: 24133047
Second Heart Field; Aortic Media; Smooth Muscle Boundary; Outflow Tract; Nkx2-5-Cre; Lineage Tracing
10.  Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis 
Genesis (New York, N.Y. : 2000)  2013;51(12):835-843.
We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers.
PMCID: PMC3947545  PMID: 24123613
tyrosinase; albinism; six3; brain; eye; sgRNA
11.  A Uchl1-Histone2BmCherry:GFP-gpi BAC transgene for imaging neuronal progenitors 
Genesis (New York, N.Y. : 2000)  2013;51(12):852-861.
Uchl1 encodes the protein gene product 9.5 antigen (PGP9.5) that is a widely used to identify migrating neural progenitors in the PNS, mature neurons of the central and peripheral nervous systems, as well as neuroendocrine cells. To facilitate analysis of developing peripheral neurons, we linked regulatory regions of Uchl1 carried within a 160kb bacterial artificial chromosome (BAC) to the dual fluorescent reporter H2BmCherry:GFP-gpi. The Uchl1-H2BmCherry:GFP-gpi transgene exhibits robust expression and allows clear discrimination of individual cells and cellular processes in cranial ganglia, sympathetic chain, the enteric nervous system (ENS), and autonomic ganglia of the urogenital system. The transgene also labels subsets of cell in endocrine tissues where prior in situ hybridization (ISH) studies have previously identified expression of this deubiquinating enzyme. The Uchl1-H2BmCherry:GFP-gpi transgene will be a powerful tool for static and live imaging as well as isolation of viable neural progenitors to investigate processes of autonomic neurogenesis.
PMCID: PMC3953494  PMID: 24123561
Uchl1; PGP9.5; Neural Crest; Neural progenitor; Lower Urinary Tract; Enteric Nervous System; Bacterial Artificial Chromosome (BAC); Neurogenesis; Oncogenesis
12.  Gene regulatory evolution and the origin of macroevolutionary novelties: insights from the neural crest 
Genesis (New York, N.Y. : 2000)  2013;51(7):457-470.
The appearance of novel anatomic structures during evolution is driven by changes to the networks of transcription factors, signaling pathways, and downstream effector genes controlling development. The nature of the changes to these developmental gene regulatory networks (GRNs) is poorly understood. A striking test case is the evolution of the GRN controlling development of the neural crest (NC). NC cells emerge from the neural plate border (NPB) and contribute to multiple adult structures. While all chordates have a NPB, only in vertebrates do NPB cells express all the genes constituting the neural crest GRN (NC-GRN). Interestingly, invertebrate chordates express orthologs of NC-GRN components in other tissues, revealing that during vertebrate evolution new regulatory connections emerged between transcription factors primitively expressed in the NPB and genes primitively expressed in other tissues. Such interactions could have evolved by two mechanisms. First, transcription factors primitively expressed in the NPB may have evolved new DNA and/or cofactor binding properties (protein neofunctionalization). Alternately, cis-regulatory elements driving NPB expression may have evolved near genes primitively expressed in other tissues (cis-regulatory neofunctionalization). Here we discuss how gene duplication can, in principle, promote either form of neofunctionalization. We review recent published examples of interspecies gene-swap, or regulatory-element-swap, experiments that test both models. Such experiments have yielded little evidence to support the importance of protein neofunctionalization in the emergence of the NC-GRN, but do support the importance of novel cis-regulatory elements in this process. The NC-GRN is an excellent model for the study of gene regulatory and macroevolutionary innovation.
PMCID: PMC4249638  PMID: 23712931
Development; Evolution; Transcription; Neural Crest
13.  DNA methylation profiles provide a viable index for porcine pluripotent stem cells 
Genesis (New York, N.y. : 2000)  2013;51(11):763-776.
Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor-intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)-specific hypomethylated loci (EShypo-T-DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso-4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso-4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso-622-14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum-free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763–776. © 2013 Wiley Periodicals, Inc.
PMCID: PMC4237151  PMID: 23913699
epigenetics; induced pluripotent stem cells; translational research
14.  The ortholog of the human proto-oncogene ROS1 is required for epithelial development in C. elegans 
Genesis (New York, N.y. : 2000)  2013;51(8):545-561.
The orphan receptor ROS1 is a human proto-oncogene, mutations of which are found in an increasing number of cancers. Little is known about the role of ROS1, however in vertebrates it has been implicated in promoting differentiation programs in specialized epithelial tissues. In this study we show that the C. elegans ortholog of ROS1, the receptor tyrosine kinase ROL-3, has an essential role in orchestrating the morphogenesis and development of specialized epidermal tissues, highlighting a potentially conserved function in coordinating crosstalk between developing epithelial cells. We also provide evidence of a direct relationship between ROL-3, the mucin SRAP-1, and BCC-1, the homolog of mRNA regulating protein Bicaudal-C. This study answers a longstanding question as to the developmental function of ROL-3, identifies three new genes that are expressed and function in the developing epithelium of C. elegans, and introduces the nematode as a potentially powerful model system for investigating the increasingly important, yet poorly understood, human oncogene ROS1. genesis 51:545–561.
PMCID: PMC4232869  PMID: 23733356
ROS1 oncogene; Caenorhabditis elegans; ROL-3; cuticle; epithelial; seam cells
15.  Analysis of primitive erythroid cell proliferation and enucleation using a cyan fluorescent reporter in transgenic mice 
Genesis (New York, N.Y. : 2000)  2013;51(11):10.1002/dvg.22420.
Primitive erythropoiesis is a vital process for mammalian embryonic development. Here we report the generation and characterization of a new transgenic mouse line that expresses a histone H2B-CFP fusion protein in the nuclei of primitive erythroid cells. We demonstrate the potential of this ε-globin-histone H2B-CFP line for multicolor imaging and flow cytometry analysis. The ε-globin-H2B-CFP line was used to analyze the cell cycle distribution and proliferation of CFP-expressing primitive erythroblasts from E8.5–E13.5. We also evaluated phagocytosis of extruded CFP-positive nuclei by macrophages in fetal liver and placenta. The ε-globin-H2B-CFP transgenic mouse line adds to the available tools for studying the development of the primitive erythroid lineage.
PMCID: PMC3840118  PMID: 23913596
primitive erythropoiesis; transgenic mice; cyan fluorescent protein; histone fusion; fluorescent reporter; imaging
16.  Efficient, specific, developmentally appropriate cre-mediated recombination in anterior pituitary gonadotropes and thyrotropes 
Genesis (New York, N.Y. : 2000)  2013;51(11):785-792.
Tissue-specific expression of cre recombinase is a well-established genetic tool to analyze gene function, and it is limited only by the efficiency and specificity of available cre mouse strains. Here we report the generation of a transgenic line containing a cre cassette with codon usage optimized for mammalian cells (iCre) under the control of a mouse glycoprotein hormone α-subunit (αGSU) regulatory sequences in a bacterial artificial chromosome genomic clone. Initial analysis of this transgenic line, Tg(αGSU-iCre), with cre reporter strains reveals onset of cre activity in the differentiating cells of the developing anterior pituitary gland at embryonic day 12.5, with a pattern characteristic of endogenous αGSU. In adult mice, αGSU-iCre was active in the anterior lobe of the pituitary gland and in the cells that produce αGSU (gonadotropes and thyrotropes) with high penetrance. Little or no activity was observed in other tissues, including skeletal and cardiac muscle, brain, kidney, lungs, testis, ovary and liver. This αGSU-iCre line is suitable for efficient, specific and developmentally regulated deletion of floxed alleles in anterior pituitary gonadotropes and thyrotropes.
PMCID: PMC4007265  PMID: 23996951
transgenic mouse; chorionic gonadotropin alpha subunit; Cga; alpha-GSU; site specific recombination; BAC
17.  Molecular analysis of neurogenic placode development in a basal ray-finned fish 
Genesis (New York, N.Y. : 2000)  2011;49(4):278-294.
Neurogenic placodes are transient, thickened patches of embryonic vertebrate head ectoderm that give rise to the paired peripheral sense organs and most neurons in cranial sensory ganglia. We present the first analysis of gene expression during neurogenic placode development in a basal actinopterygian (ray-finned fish), the North American paddlefish (Polyodon spathula). Pax3 expression in the profundal placode confirms its homology with the ophthalmic trigeminal placode of amniotes. We report the conservation of expression of Pax2 and Pax8 in the otic and/or epibranchial placodes, Phox2b in epibranchial placode-derived neurons, Sox3 during epibranchial and lateral line placode development, and NeuroD in developing cranial sensory ganglia. We identify Sox3 as a novel marker for developing fields of electrosensory ampullary organs and for ampullary organs themselves. Sox3 is also the first molecular marker for actinopterygian ampullary organs. This is consistent with, though does not prove, a lateral line placode origin for actinopterygian ampullary organs.
PMCID: PMC4212515  PMID: 21381180
paddlefish; sensory ganglia; otic; epibranchial; electroreceptors; lateral line
18.  Generation and Characterization of a Tamoxifen-Inducible EomesCreER Mouse Line 
Genesis (New York, N.Y. : 2000)  2013;51(10):725-733.
Transgenic mouse lines expressing inducible forms of Cre-recombinase in a tissue-specific manner are powerful genetic tools for studying aspects of development and various processes in the adult. The T-box transcription factor eomesodermin (Eomes) plays critical roles for maintenance and differentiation of different pools of stem and progenitor cells from early embryonic stages to adulthood. These include trophoblast stem cells, epiblast cells during the generation of the primary germ layers, neurogenic intermediate progenitor cells in embryonic and adult cortical neurogenesis, and maturing natural killer and T cells. Here, we report on the generation and analysis of an EomesCreER-targeted allele by placing the tamoxifen-activatable Cre-recombinase (CreER) under the control of the Eomes genomic locus. We demonstrate that CreER expression recapitulates endogenous Eomes transcription within different progenitor cell populations. Tamoxifen administration specifically labels Eomes-expressing cells and their progeny as demonstrated by crossing EomesCreER animals to different Cre-inducible reporter strains. In summary, this novel EomesCreER allele can be used as elegant genetic tool that allows to follow the fate of Eomes-positive cells and to genetically manipulate them in a temporal specific manner.
PMCID: PMC4112203  PMID: 23897762
mouse; Eomesodermin; Tbr2; tamoxifen-activatable CreER; lineage tracing
19.  Conditional KIAA1549:BRAF mice reveal brain region- and cell type-specific effects 
Genesis (New York, N.Y. : 2000)  2013;51(10):10.1002/dvg.22415.
Low-grade brain tumors (pilocytic astrocytomas)that result from a genomic rearrangement in which the BRAF kinase domain is fused to the amino terminal of the KIAA1549 gene (KIAA1549:BRAF fusion; f-BRAF) commonly arise in the cerebellum of young children. To model this temporal and spatial specificity in mice, we generated conditional KIAA1549:BRAF strains that co-expresses green fluorescent protein (GFP). While both primary astrocytes and neural stem cells (NSCs) from these mice express f-BRAF and GFP as well as exhibit increased MEK activity, only f-BRAF-expressing NSCs exhibit increased proliferation in vitro. Using Cre driver lines in which KIAA1549:BRAF expression was directed to NSCs (f-BRAF; BLBP-Cre mice), astrocytes (f-BRAF; GFAP-Cre mice), and NG2 progenitor cells (f-BRAF; NG2-Cre mice), increased glial cell numbers were only observed in the cerebellum off-BRAF; BLBP-Cre mice in vivo. The availability of this unique KIAA1549:BRAF conditional transgenic mouse strain will enable future mechanistic studies aimed at defining the developmentally-regulated temporal and spatial determinants that underlie low-grade astrocytoma formation in children.
PMCID: PMC3808469  PMID: 23893969
genetically-engineered mice; brain tumor; glioma; pilocytic astrocytoma; astrocyte; neural stem cell
20.  Genetic inactivation of the allograft inflammatory factor-1 locus 
Genesis (New York, N.Y. : 2000)  2013;51(10):10.1002/dvg.22424.
Allograft inflammatory factor-1 (Aif-1) is a 17 kDa EF hand motif-bearing protein expressed primarily in developing spermatids and cells of monocyte/macrophage lineage. Increased Aif-1 expression has been identified in clinically important conditions, including rheumatoid arthritis, systemic sclerosis, endometriosis, and transplant-associated arteriosclerosis. Largely similar gene products arising from the same locus are known as ionized Ca2+ binding adapter-1 (Iba1), microglial response factor-1 (MRF1), and daintain; Iba1 in particular has emerged as a histologic marker of microglia and their activation in pathologic CNS conditions, including the response to facial nerve axotomy and stroke, uveitis, and experimental autoimmune neuritis and encephalomyelitis. Nevertheless, how aif-1 gene products affect cellular function is only partly understood, and the physiologic significance of these products for male fertility, immune system development, and inflammation has not been described. To permit such investigations, we generated a mouse line with targeted deletion of the coding regions of the aif-1 gene. Here we report that mice lacking Aif-1 breed well and show normal post-natal growth, but show resistance to disease in a model of collagen-induced arthritis. We anticipate that these mice will be useful for studies of Aif-1 function in a variety of immune and inflammatory disease models.
PMCID: PMC3808495  PMID: 23929822
Aif-1; Iba1; Daintain; MRF-1; spermatid; macrophage
21.  The Ptch1DL mouse: a new model to study lambdoid craniosynostosis and basal cell nevus syndrome associated skeletal defects 
Genesis (New York, N.Y. : 2000)  2013;51(10):677-689.
Mouse models provide valuable opportunities for probing the underlying pathology of human birth defects. Employing an ENU-based screen for recessive mutations affecting craniofacial anatomy we isolated a mouse strain, Dogface-like (DL), with abnormal skull and snout morphology. Examination of the skull indicated that these mice developed craniosynostosis of the lambdoid suture. Further analysis revealed skeletal defects related to the pathology of basal cell nevus syndrome (BCNS) including defects in development of the limbs, scapula, ribcage, secondary palate, cranial base, and cranial vault. In humans, BCNS is often associated with mutations in the Hedgehog receptor PTCH1 and genetic mapping in DL identified a point mutation at a splice donor site in Ptch1. Using genetic complementation analysis we determined that DL is a hypomorphic allele of Ptch1, leading to increased Hedgehog signaling. Two aberrant transcripts are generated by the mutated Ptch1DL gene, which would be predicted to reduce significantly the levels of functional Patched1 protein. This new Ptch1 allele broadens the mouse genetic reagents available to study the Hedgehog pathway and provides a valuable means to study the underlying skeletal abnormalities in BCNS. In addition, these results strengthen the connection between elevated Hedgehog signaling and craniosynostosis.
PMCID: PMC3918964  PMID: 23897749
Hedgehog; Craniosynostosis; Polydactyly; Craniofacial Defects; Omphalocele
22.  Troponin T3 expression in skeletal and smooth muscle is required for growth and postnatal survival: Characterization of Tnnt3tm2a(KOMP)Wtsi mice 
Genesis (New York, N.Y. : 2000)  2013;51(9):667-675.
The troponin complex, which consists of three regulatory proteins (troponin C, troponin I and troponin T), is known to regulate muscle contraction in skeletal and cardiac muscle, but its role in smooth muscle remains controversial. Troponin T3 (TnnT3) is a fast skeletal muscle troponin believed to be expressed only in skeletal muscle cells. To determine the in vivo function and tissue specific expression of Tnnt3, we obtained the heterozygous Tnnt3+/flox/lacZ mice from Knockout Mouse Project (KOMP) Repository. Tnnt3lacZ/+ mice are smaller than their WT littermates throughout development, but do not display any gross phenotypes. Tnnt3lacZ/lacZ embryos are smaller than heterozygotes, and die shortly after birth. Histology revealed hemorrhagic tissue in Tnnt3lacZ/lacZ liver and kidney, which was not present in Tnnt3lacZ/+ or WT, but no other gross tissue abnormalities. X-gal staining for Tnnt3 promoter-driven lacZ transgene expression revealed positive staining in skeletal muscle and diapharam, and smooth muscle cells located in the aorta, bladder, and bronchus. Collectively, these findings suggest that troponins are expressed in smooth muscle, and are required for normal growth and breathing for postnatal survival. Moreover, future studies with this mouse model can explore TnnT3 function in adult muscle function using the conditional-inducible gene deletion approach.
PMCID: PMC3787964  PMID: 23775847
Troponin; Knockout Mice; Muscle; Development
23.  Proliferation of embryonic cardiomyocytes in zebrafish requires the sodium channel scn5Lab 
Genesis (New York, N.Y. : 2000)  2013;51(8):562-574.
In mice, homozygous deletion of the cardiac sodium channel Scn5a results in defects in cardiac morphology and embryonic death before robust sodium current can be detected. In zebrafish, morpholino knockdown of cardiac sodium channel orthologs scn5Laa and scn5Lab perturbs specification of pre-cardiac mesoderm and inhibits growth of the embryonic heart. It is not known which developmental processes are perturbed by sodium channel knockdown and whether reduced cell number is from impaired migration of cardiac progenitors into the heart, impaired myocyte proliferation, or both. We found that embryos deficient in scn5Lab displayed defects in primary cardiogenesis specific to loss of nkx2.5, but not nkx2.7. We generated kaede reporter fish and demonstrated that embryos treated with anti-scn5Lab morpholino showed normal secondary differentiation of cardiomyocytes at the arterial pole between 30 and 48 hours post-fertilization. However, while proliferating myocytes were readily detected at 48 hpf in wild type embryos, there were no BrdU-positive cardiomyocytes in embryos subjected to anti-scn5Lab treatment. Proliferating myocytes were present in embryos injected with anti-tnnt2 morpholino to phenocopy the silent heart mutation, and absent in embryos injected with anti-tnnt2 and anti-scn5Lab morpholinos, indicating cardiac contraction is not required for the loss of proliferation. These data demonstrate that the role of scn5Lab in later heart growth does not involve contribution of the secondary heart field, but rather proliferation of cardiomyocytes, and appears unrelated to the role of the channel in cardiac electrogenesis.
PMCID: PMC3750094  PMID: 23650201
heart; development; sodium channel; myocardium
24.  Functional defect of peripheral neutrophils in mice with induced deletion of CXCR2 
Genesis (New York, N.Y. : 2000)  2013;51(8):587-595.
Type 2 CXC chemokine receptor CXCR2 plays roles in development, tumorigenesis and inflammation. CXCR2 also promotes demyelination and decreases remyelination by actions toward hematopoietic cells and non-hematopoietic cells. Germline CXCR2 deficient (Cxcr2−/−) mice reported in 1994 revealed the complexity of CXCR2 function and its differential expression in varied cell-types. Here, we describe Cxcr2fl/fl mice for which the targeting construct was generated by recombineering based on homologous recombination in E. coli. Without recombination Cxcr2fl/fl mice have CXCR2 expression on neutrophils in peripheral blood, bone marrow and spleen. Cxcr2fl/fl mice were crossed to Mx-Cre mice in which Cre recombinase is induced by type I interferons, elicited by injection with polyinosinic-polycytidylic acid (poly(I:C)). CXCR2-deficient neutrophils were observed in poly(I:C) treated Cxcr2fl/fl::Mx-Cre+ (Cxcr2-CKO) mice, but not in poly(I:C) treated Cxcr2f//+::Mx-Cre+ mice. CXCR2 deletion was mainly observed peripherally but not in the CNS. Cxcr2-CKO mice showed impaired neutrophil migration in sterile peritonitis. Cxcr2-CKO mice reported here will provide a genetic reagent to dissect roles of CXCR2 in the neutrophil granulocyte lineage. Furthermore Cxcr2fl/fl mice will provide useful genetic models to evaluate CXCR2 function in varied cell populations.
PMCID: PMC3753089  PMID: 23650205
CXCR2; chemokine; chemokine receptor; conditional KO mice; neutrophil
25.  Morphological defects in a Novel Rdh10 Mutant that has Reduced Retinoic Acid Biosynthesis and Signaling 
Genesis (New York, N.Y. : 2000)  2012;50(5):415-423.
Retinoic acid (RA) signaling is necessary for proper patterning and morphogenesis during embryonic development. Tissue specific RA signaling requires precise spatial and temporal synthesis of RA from retinal by retinaldehyde dehydrogenases (Raldh) and the conversion of retinol to retinal by retinol dehydrogenases (Rdh) of the short-chain dehydrogenase/reducatase gene family (SDR). The SDR, retinol dehydrogenase 10 (RDH10), is a major contributor to retinal biosynthesis during mid-gestation. We have identified a missense mutation in the Rdh10 gene (Rdh10m366Asp) using an N-ethyl-N-nitrosourea (ENU)-induced forward genetic screen that result in reduced RA levels and signaling during embryonic development. Rdh10m366Asp mutant embryos have unique phenotypes, such as edema, a massive midline facial cleft, and neurogenesis defects in the forebrain, that will allow the identification of novel RA functions.
PMCID: PMC4118640  PMID: 22162152
Retinol dehydrogenase; ENU-induced mutagenesis; embryo development; organogenesis

Results 1-25 (270)