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1.  Key pathways regulated by HoxA9,10,11/HoxD9,10,11 during limb development 
The 39 mammalian Hox genes show problematic patterns of functional overlap. In order to more fully define the developmental roles of Hox genes it is necessary to remove multiple combinations of paralogous and flanking genes. In addition, the downstream molecular pathways regulated by Hox genes during limb development remain incompletely delineated.
In this report we examine limb development in mice with frameshift mutations in six Hox genes, Hoxa9,10,11 and Hoxd9,10,11. The mice were made with a novel recombineering method that allows the simultaneous targeting of frameshift mutations into multiple flanking genes. The Hoxa9,10,11−/−/Hoxd9,10,11−/− mutant mice show a reduced ulna and radius that is more severe than seen in Hoxa11−/−/Hoxd11−/− mice, indicating a minor role for the flanking Hox9,10 genes in zeugopod development, as well as their primary function in stylopod development. The mutant mice also show severe reduction of Shh expression in the zone of polarizing activity, and decreased Fgf8 expression in the apical ectodermal ridge, thereby better defining the roles of these specific Hox genes in the regulation of critical signaling centers during limb development. Importantly, we also used laser capture microdissection coupled with RNA-Seq to characterize the gene expression programs in wild type and mutant limbs. Resting, proliferative and hypertrophic compartments of E15.5 forelimb zeugopods were examined. The results provide an RNA-Seq characterization of the progression of gene expression patterns during normal endochondral bone formation. In addition of the Hox mutants showed strongly altered expression of Pknox2, Zfp467, Gdf5, Bmpr1b, Dkk3, Igf1, Hand2, Shox2, Runx3, Bmp7 and Lef1, all of which have been previously shown to play important roles in bone formation.
The recombineering based frameshift mutation of the six flanking and paralogous Hoxa9,10,11 and Hoxd9,10,11 genes provides a resource for the analysis of their overlapping functions. Analysis of the Hoxa9,10,11−/−/Hoxd9,10,11−/− mutant limbs confirms and extends the results of previous studies using mice with Hox mutations in single paralogous groups or with entire Hox cluster deletions. The RNA-Seq analysis of specific compartments of the normal and mutant limbs defines the multiple key perturbed pathways downstream of these Hox genes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-015-0078-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4506574  PMID: 26186931
Hox genes; Limb development; RNA-Seq; Zone of polarizing activity; Apical ectodermal ridge; Zeugopod; Endochondral bone development; Sonic hedgehog; Fgf8; Lef1
2.  A Gene Expression Atlas of Early Craniofacial Development 
Developmental biology  2014;391(2):133-146.
We present a gene expression atlas of early mouse craniofacial development. Laser capture microdissection (LCM) was used to isolate cells from the principal critical micro-regions, whose development, differentiation and signaling interactions are responsible for the construction of the mammalian face. At E8.5, as migrating neural crest cells begin to exit the neural fold/epidermal ectoderm boundary, we examined the cranial mesenchyme, composed of mixed neural crest and paraxial mesoderm cells, as well as cells from adjacent neuroepithelium. At E9.5 cells from the cranial mesenchyme, overlying olfactory placode/epidermal ectoderm, and underlying neuroepithelium, as well as the emerging mandibular and maxillary arches were sampled. At E10.5, as the facial prominences form, cells from the medial and lateral prominences, the olfactory pit, multiple discrete regions of underlying neuroepithelium, the mandibular and maxillary arches, including both their mesenchymal and ectodermal components, as well as Rathke’s pouch, were similarly sampled and profiled using both microarray and RNA-seq technologies. Further, we performed single cell studies to better define the gene expression states of the early E8.5 pioneer neural crest cells and paraxial mesoderm. Taken together, and analyzable by a variety of biological network approaches, these data provide a complementing and cross-validating resource capable of fueling discovery of novel compartment specific markers and signatures whose combinatorial interactions of transcription factors and growth factors/receptors are responsible for providing the master genetic blueprint for craniofacial development.
PMCID: PMC4095820  PMID: 24780627
mammalian craniofacial development; gene expression atlas; RNA-seq; microarrays
3.  Molecular Anatomy of Palate Development 
PLoS ONE  2015;10(7):e0132662.
The NIH FACEBASE consortium was established in part to create a central resource for craniofacial researchers. One purpose is to provide a molecular anatomy of craniofacial development. To this end we have used a combination of laser capture microdissection and RNA-Seq to define the gene expression programs driving development of the murine palate. We focused on the E14.5 palate, soon after medial fusion of the two palatal shelves. The palate was divided into multiple compartments, including both medial and lateral, as well as oral and nasal, for both the anterior and posterior domains. A total of 25 RNA-Seq datasets were generated. The results provide a comprehensive view of the region specific expression of all transcription factors, growth factors and receptors. Paracrine interactions can be inferred from flanking compartment growth factor/receptor expression patterns. The results are validated primarily through very high concordance with extensive previously published gene expression data for the developing palate. In addition selected immunostain validations were carried out. In conclusion, this report provides an RNA-Seq based atlas of gene expression patterns driving palate development at microanatomic resolution. This FACEBASE resource is designed to promote discovery by the craniofacial research community.
PMCID: PMC4500583  PMID: 26168040
4.  Pathogenic pathways are activated in each major cell type of the glomerulus in the Cd2ap mutant mouse model of focal segmental glomerulosclerosis 
BMC Nephrology  2015;16:71.
Mutations in several genes expressed in podocytes, including Cd2ap, have been associated with focal segmental glomerulosclerosis in humans. Mutant mouse models provide an opportunity to better understand the molecular pathology that drives these diseases.
In this report we use a battery of transgenic-GFP mice to facilitate the purification of all three major cell types of the glomerulus from Cd2ap mutant mice. Both microarrays and RNA-seq were used to characterize the gene expression profiles of the podocytes, mesangial cells and endothelial cells, providing a global dual platform cross-validating dataset.
The mesangial cells showed increased expression of profibrotic factors, including thrombospondin, Tgfb2 and Tgfb3, as well as the angiogenesis factor Vegf. They also showed upregulation of protective genes, including Aldh1a2, involved in retinoic acid synthesis and Decorin, a Tgfb antagonist. Of interest, the mesangial cells also showed significant expression of Wt1, which has generally been considered podocyte specific. The Cd2ap mutant podocytes showed upregulation of proteases as well as genes involved in muscle and vasculature development and showed a very strong gene expression signature indicating programmed cell death. Endothelial cells showed increased expression of the leukocyte adhesion associated factors Vcam1 and Sele, as well as Midkine (promoting angiogenesis), endothelin and many genes responsive to cytokines and interferons.
This study provides a comprehensive analysis of the changing properties of the three cell types of the glomerulus in Cd2ap mutants, identifying activated and repressed pathways and responsible genes, thereby delivering a deeper molecular understanding of this genetic disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s12882-015-0063-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4430919  PMID: 25968128
Cd2ap; Focal segmental glomerulosclerosis; Podocytes; Mesangial cells; Endothelial cells; RNA-seq; Pathogenic pathways
5.  SP8 regulates signaling centers during craniofacial development 
Developmental biology  2013;381(2):312-323.
Much of the bone, cartilage and smooth muscle of the vertebrate face is derived from neural crest (NC) cells. During craniofacial development, the anterior neural ridge (ANR) and olfactory pit (OP) signaling centers are responsible for driving the outgrowth, survival, and differentiation of NC populated facial prominences, primarily via FGF. While much is known about the functional importance of signaling centers, relatively little is understood of how these signaling centers are made and maintained. In this report we describe a dramatic craniofacial malformation in mice mutant for the zinc finger transcription factor gene Sp8. At E14.5 they show facial prominences that are reduced in size and underdeveloped, giving an almost faceless phenotype. At later times they show severe midline defects, excencephaly, hyperterlorism, cleft palate, and a striking loss of many NC and paraxial mesoderm derived cranial bones. Sp8 expression was primarily restricted to the ANR and OP regions during craniofacial development. Analysis of an extensive series of conditional Sp8 mutants confirmed the critical role of Sp8 in signaling centers, and not directly in the NC and paraxial mesoderm cells. The NC cells of the Sp8 mutants showed increased levels of apoptosis and decreased cell proliferation, thereby explaining the reduced sizes of the facial prominences. Perturbed gene expression in the Sp8 mutants was examined by laser capture microdissection coupled with microarrays, as well as in situ hybridization and immunostaining. The most dramatic differences included striking reductions in Fgf8 and Fgf17 expression in the ANR and OP signaling centers. We were also able to achieve genetic and pharmaceutical partial rescue of the Sp8 mutant phenotype by reducing Sonic Hedgehog (SHH) signaling. These results show that Sp8 primarily functions to promote Fgf expression in the ANR and OP signaling centers that drive the survival, proliferation, and differentiation of the NC and paraxial mesoderm that make the face.
PMCID: PMC4078980  PMID: 23872235
Craniofacial development; neural crest; anterior neural ridge; SP8; FGF8; FGF17; cyclopamine; olfactory pits; signaling center
6.  RNA-Seq defines novel genes, RNA processing patterns and enhancer maps for the early stages of nephrogenesis: Hox supergenes 
Developmental Biology  2012;368(1):4-17.
During kidney development the cap mesenchyme progenitor cells both self renew and differentiate into nephrons. The balance between renewal and differentiation determines the final nephron count, which is of considerable medical importance. An important goal is to create a precise genetic definition of the early differentiation of cap mesenchyme progenitors. We used RNA-Seq to transcriptional profile the cap mesenchyme progenitors and their first epithelial derivative, the renal vesicles. The results provide a global view of the changing gene expression program during this key period, defining expression levels for all transcription factors, growth factors, and receptors. The RNA-Seq was performed using two different biochemistries, with one examining only polyadenylated RNA and the other total RNA. This allowed the analysis of noncanonical transcripts, which for many genes were more abundant than standard exonic RNAs. Since a large fraction of enhancers are now known to be transcribed the results also provide global maps of potential enhancers. Further, the RNA-Seq data defined hundreds of novel splice patterns and large numbers of new genes. Particularly striking was the extensive sense/antisense transcription and changing RNA processing complexities of the Hox clusters.
PMCID: PMC3400938  PMID: 22664176
kidney development; RNA-Seq; Hox genes; renal vesicle; cap mesenchyme; induction
7.  The FaceBase Consortium: A comprehensive program to facilitate craniofacial research 
Developmental biology  2011;355(2):175-182.
The FaceBase Consortium consists of ten interlinked research and technology projects whose goal is to generate craniofacial research data and technology for use by the research community through a central data management and integrated bioinformatics hub. Funded by the National Institute of Dental and Craniofacial Research (NIDCR) and currently focused on studying the development of the middle region of the face, the Consortium will produce comprehensive datasets of global gene expression patterns, regulatory elements and sequencing; will generate anatomical and molecular atlases; will provide human normative facial data and other phenotypes; conduct follow up studies of a completed genome-wide association study; generate independent data on the genetics of craniofacial development, build repositories of animal models and of human samples and data for community access and analysis; and will develop software tools and animal models for analyzing and functionally testing and integrating these data. The FaceBase website ( will serve as a web home for these efforts, providing interactive tools for exploring these datasets, together with discussion forums and other services to support and foster collaboration within the craniofacial research community.
PMCID: PMC3440302  PMID: 21458441
Craniofacial development; Cleft lip and palate; Human genetics; Animal models; Database; Morphometrics
8.  Changes in the gene expression programs of renal mesangial cells during diabetic nephropathy 
BMC Nephrology  2012;13:70.
Diabetic nephropathy is the leading cause of end stage renal disease. All three cell types of the glomerulus, podocytes, endothelial cells and mesangial cells, play important roles in diabetic nephropathy. In this report we used Meis1-GFP transgenic mice to purify mesangial cells from normal mice and from db/db mice, which suffer diabetic nephropathy. The purpose of the study is to better define the unique character of normal mesangial cells, and to characterize their pathogenic and protective responses during diabetic nephropathy.
Comprehensive gene expression states of the normal and diseased mesangial cells were defined with microarrays. By comparing the gene expression profiles of mesangial cells with those of multiple other renal cell types, including podocytes, endothelial cells and renal vesicles, it was possible to better define their exceptional nature, which includes smooth muscle, phagocytic and neuronal traits.
The complete set of mesangial cell expressed transcription factors, growth factors and receptors were identified. In addition, the analysis of the mesangial cells from diabetic nephropathy mice characterized their changes in gene expression. Molecular functions and biological processes specific to diseased mesangial cells were characterized, identifying genes involved in extracellular matrix, cell division, vasculogenesis, and growth factor modulation. Selected gene changes considered of particular importance to the disease process were validated and localized within the glomuerulus by immunostaining. For example, thrombospondin, a key mediator of TGFβ signaling, was upregulated in the diabetic nephropathy mesangial cells, likely contributing to fibrosis. On the other hand the decorin gene was also upregulated, and expression of this gene has been strongly implicated in the reduction of TGFβ induced fibrosis.
The results provide an important complement to previous studies examining mesangial cells grown in culture. The remarkable qualities of the mesangial cell are more fully defined in both the normal and diabetic nephropathy diseased state. New gene expression changes and biological pathways are discovered, yielding a deeper understanding of the diabetic nephropathy pathogenic process, and identifying candidate targets for the development of novel therapies.
PMCID: PMC3416581  PMID: 22839765
Mesangial cells; Diabetic nephropathy; Fibrosis
9.  The GUDMAP database – an online resource for genitourinary research 
Development (Cambridge, England)  2011;138(13):2845-2853.
The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium working to generate gene expression data and transgenic mice. GUDMAP includes data from large-scale in situ hybridisation screens (wholemount and section) and microarray gene expression data of microdissected, laser-captured and FACS-sorted components of the developing mouse genitourinary (GU) system. These expression data are annotated using a high-resolution anatomy ontology specific to the developing murine GU system. GUDMAP data are freely accessible at via easy-to-use interfaces. This curated, high-resolution dataset serves as a powerful resource for biologists, clinicians and bioinformaticians interested in the developing urogenital system. This paper gives examples of how the data have been used to address problems in developmental biology and provides a primer for those wishing to use the database in their own research.
PMCID: PMC3188593  PMID: 21652655
Database; Gene expression; Genitourinary; In situ hybridisation; Microarray
10.  Defining the Molecular Character of the Developing and Adult Kidney Podocyte 
PLoS ONE  2011;6(9):e24640.
The podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Although mesodermal in origin it sends out axonal like projections that wrap around the capillaries. These extend yet finer projections, the foot processes, which interdigitate, leaving between them the slit diaphragms, through which the glomerular filtrate must pass. The podocytes are a subject of keen interest because of their key roles in kidney development and disease.
Methodology/Principal Findings
In this report we identified and characterized a novel transgenic mouse line, MafB-GFP, which specifically marked the kidney podocytes from a very early stage of development. These mice were then used to facilitate the fluorescent activated cell sorting based purification of podocytes from embryos at E13.5 and E15.5, as well as adults. Microarrays were then used to globally define the gene expression states of podocytes at these different developmental stages. A remarkable picture emerged, identifying the multiple sets of genes that establish the neuronal, muscle, and phagocytic properties of podocytes. The complete combinatorial code of transcription factors that create the podocyte was characterized, and the global lists of growth factors and receptors they express were defined.
The complete molecular character of the in vivo podocyte is established for the first time. The active molecular functions and biological processes further define their unique combination of features. The results provide a resource atlas of gene expression patterns of developing and adult podocytes that will help to guide further research of these incredible cells.
PMCID: PMC3169617  PMID: 21931791
11.  Microdissection of the gene expression codes driving nephrogenesis 
Organogenesis  2010;6(4):263-269.
The kidney represents an excellent model system for learning the principles of organogenesis. It is intermediate in complexity, and employs many commonly used developmental processes. As such, kidney development has been the subject of intensive study, using a variety of techniques, including in situ hybridization, organ culture and gene targeting, revealing many critical genes and pathways. Nevertheless, proper organogenesis requires precise patterns of cell type specific differential gene expression, involving very large numbers of genes. This review is focused on the use of global profiling technologies to create an atlas of gene expression codes driving development of different mammalian kidney compartments. Such an atlas allows one to select a gene of interest, and to determine its expression level in each element of the developing kidney, or to select a structure of interest, such as the renal vesicle, and to examine its complete gene expression state. Novel component specific molecular markers are identified, and the changing waves of gene expression that drive nephrogenesis are defined. As the tools continue to improve for the purification of specific cell types and expression profiling of even individual cells it is possible to predict an atlas of gene expression during kidney development that extends to single cell resolution.
PMCID: PMC3055652  PMID: 21220959
kidney development; microarrays; organogenesis; nephrogenesis; RNA-Seq; gene expression atlas
12.  Microarrays and RNA-Seq identify molecular mechanisms driving the end of nephron production 
The production of nephrons suddenly ends in mice shortly after birth when the remaining cells of the multi-potent progenitor mesenchyme begin to differentiate into nephrons. We exploited this terminal wave of nephron production using both microarrays and RNA-Seq to serially evaluate gene transcript levels in the progenitors. This strategy allowed us to define the changing gene expression states following induction and the onset of differentiation after birth.
Microarray and RNA-Seq studies of the progenitors detected a change in the expression profiles of several classes of genes early after birth. One functional class, a class of genes associated with cellular proliferation, was activated. Analysis of proliferation with a nucleotide analog demonstrated in vivo that entry into the S-phase of the cell cycle preceded increases in transcript levels of genetic markers of differentiation. Microarrays and RNA-Seq also detected the onset of expression of markers of differentiation within the population of progenitors prior to detectable Six2 repression. Validation by in situ hybridization demonstrated that the markers were expressed in a subset of Six2 expressing progenitors. Finally, the studies identified a third set of genes that provide indirect evidence of an altered cellular microenvironment of the multi-potential progenitors after birth.
These results demonstrate that Six2 expression is not sufficient to suppress activation of genes associated with growth and differentiation of nephrons. They also better define the sequence of events after induction and suggest mechanisms contributing to the rapid end of nephron production after birth in mice.
PMCID: PMC3065427  PMID: 21396121
13.  Gene Expression Programs of Mouse Endothelial Cells in Kidney Development and Disease 
PLoS ONE  2010;5(8):e12034.
Endothelial cells are remarkably heterogeneous in both morphology and function, and they play critical roles in the formation of multiple organ systems. In addition endothelial cell dysfunction can contribute to disease processes, including diabetic nephropathy, which is a leading cause of end stage renal disease. In this report we define the comprehensive gene expression programs of multiple types of kidney endothelial cells, and analyze the differences that distinguish them. Endothelial cells were purified from Tie2-GFP mice by cell dissociation and fluorescent activated cell sorting. Microarrays were then used to provide a global, quantitative and sensitive measure of gene expression levels. We examined renal endothelial cells from the embryo and from the adult glomerulus, cortex and medulla compartments, as well as the glomerular endothelial cells of the db/db mutant mouse, which represents a model for human diabetic nephropathy. The results identified the growth factors, receptors and transcription factors expressed by these multiple endothelial cell types. Biological processes and molecular pathways were characterized in exquisite detail. Cell type specific gene expression patterns were defined, finding novel molecular markers and providing a better understanding of compartmental distinctions. Further, analysis of enriched, evolutionarily conserved transcription factor binding sites in the promoters of co-activated genes begins to define the genetic regulatory network of renal endothelial cell formation. Finally, the gene expression differences associated with diabetic nephropathy were defined, providing a global view of both the pathogenic and protective pathways activated. These studies provide a rich resource to facilitate further investigations of endothelial cell functions in kidney development, adult compartments, and disease.
PMCID: PMC2919381  PMID: 20706631
14.  Atlas of Gene Expression in the Developing Kidney at Microanatomic Resolution 
Developmental cell  2008;15(5):781-791.
Kidney development is based on differential cell type specific expression of a vast number of genes. While multiple critical genes and pathways have been elucidated, a genomewide analysis of gene expression within individual cellular and anatomic structures is lacking. Accomplishing this could provide significant new insights into fundamental developmental mechanisms such as mesenchymal-epithelial transition, inductive signaling, branching morphogenesis and segmentation. We describe here a comprehensive gene expression atlas of the developing mouse kidney based on the isolation of each major compartment by either laser capture microdissection or fluorescent activated cell sorting, followed by microarray profiling. The resulting data agrees with known expression patterns and additional in situ hybridizations. This kidney atlas allows a comprehensive analysis of the progression of gene expression states during nephrogenesis, as well as discovery of novel growth factor-receptor interactions. In addition, the results provide deeper insight into the genetic regulatory mechanisms of kidney development.
PMCID: PMC2653061  PMID: 19000842
15.  Epigenetic inheritance based evolution of antibiotic resistance in bacteria 
The evolution of antibiotic resistance in bacteria is a topic of major medical importance. Evolution is the result of natural selection acting on variant phenotypes. Both the rigid base sequence of DNA and the more plastic expression patterns of the genes present define phenotype.
We investigated the evolution of resistant E. coli when exposed to low concentrations of antibiotic. We show that within an isogenic population there are heritable variations in gene expression patterns, providing phenotypic diversity for antibiotic selection to act on. We studied resistance to three different antibiotics, ampicillin, tetracycline and nalidixic acid, which act by inhibiting cell wall synthesis, protein synthesis and DNA synthesis, respectively. In each case survival rates were too high to be accounted for by spontaneous DNA mutation. In addition, resistance levels could be ramped higher by successive exposures to increasing antibiotic concentrations. Furthermore, reversion rates to antibiotic sensitivity were extremely high, generally over 50%, consistent with an epigenetic inheritance mode of resistance. The gene expression patterns of the antibiotic resistant E. coli were characterized with microarrays. Candidate genes, whose altered expression might confer survival, were tested by driving constitutive overexpression and determining antibiotic resistance. Three categories of resistance genes were identified. The endogenous β-lactamase gene represented a cryptic gene, normally inactive, but when by chance expressed capable of providing potent ampicillin resistance. The glutamate decarboxylase gene, in contrast, is normally expressed, but when overexpressed has the incidental capacity to give an increase in ampicillin resistance. And the DAM methylase gene is capable of regulating the expression of other genes, including multidrug efflux pumps.
In this report we describe the evolution of antibiotic resistance in bacteria mediated by the epigenetic inheritance of variant gene expression patterns. This provides proof in principle that epigenetic inheritance, as well as DNA mutation, can drive evolution.
PMCID: PMC2262874  PMID: 18282299
16.  Mutation of an axonemal dynein affects left–right asymmetry in inversus viscerum mice 
Nature  1997;389(6654):963-966.
The development of characteristic visceral asymmetries along the left–right (LR) axis in an initially bilaterally symmetrical embryo is an essential feature of vertebrate patterning. The allelic mouse mutations inversus viscerum (iv)1,2 and legless (lgl)3,4 produce LR inversion, or situs inversus, in half of live-born homozygotes. This suggests that the iv gene product drives correct LR determination, and in its absence this process is randomized2. These mutations provide tools for studying the development of LR-handed asymmetry and provide mouse models of human lateralization defects. At the molecular level, the normally LR asymmetric expression patterns of nodal5 and lefty6 are randomized in iv/iv embryos, suggesting that iv functions early in the genetic hierarchy of LR specification. Here we report the positional cloning of an axonemal dynein heavy-chain gene, left/right-dynein (lrd), that is mutated in both lgl and iv. lrd is expressed in the node of the embryo at embryonic day 7.5, consistent with its having a role in LR development7. Our findings indicate that dynein, a micro-tubule-based motor, is involved in the determination of LR-handed asymmetry and provide insight into the early molecular mechanisms of this process.
PMCID: PMC1800588  PMID: 9353118
17.  Targeted deletion of the ATP binding domain of left-right dynein confirms its role in specifying development of left-right asymmetries 
Development (Cambridge, England)  1999;126(23):5495-5504.
Vertebrates develop distinct asymmetries along the left-right axis, which are consistently aligned with the anteroposterior and dorsoventral axes. The mechanisms that direct this handed development of left-right asymmetries have been elusive, but recent studies of mutations that affect left-right development have shed light on the molecules involved. One molecule implicated in left-right specification is left-right dynein (LRD), a microtubule-based motor protein. In the LRD protein of the inversus viscerum (iv) mouse, there is a single amino acid difference at a conserved position, and the lrd gene is one of many genes deleted in the legless (lgl) mutation. Both iv and lgl mice display randomized left-right development. Here we extend the analysis of the lrd gene at the levels of sequence, expression and function. The complete coding sequence of the lrd gene confirms its classification as an axonemal, or ciliary, dynein. Expression of lrd in the node at embryonic day 7.5 is shown to be symmetric. At embryonic day 8.0, however, a striking asymmetric expression pattern is observed in all three germ layers of the developing headfold, suggesting roles in both the establishment and maintenance of left-right asymmetries. At later times, expression of lrd is also observed in the developing floorplate, gut and limbs. These results suggest function for LRD protein in both cilitated and non-ciliated cells, despite its sequence classification as axonemal. In addition, a targeted mutation of lrd was generated that deletes the part of the protein required for ATP binding, and hence motor function. The resulting left-right phenotype, randomization of laterality, is identical to that of iv and lgl mutants. Gross defects in ciliary structure were not observed in lrd/lrd mutants. Strikingly, however, the monocilia on mutant embryonic node cells were immotile. These results prove the identity of the iv and lrd genes. Further, they argue that LRD motor function, and resulting nodal monocilia movement, are required for normal left-right development.
PMCID: PMC1797880  PMID: 10556073
Left-right asymmetry; Situs inversus; Dynein; ATP; Mouse
18.  Pygo1 and Pygo2 roles in Wnt signaling in mammalian kidney development 
BMC Biology  2007;5:15.
The pygopus gene of Drosophila encodes an essential component of the Armadillo (β-catenin) transcription factor complex of canonical Wnt signaling. To better understand the functions of Pygopus-mediated canonical Wnt signaling in kidney development, targeted mutations were made in the two mammalian orthologs, Pygo1 and Pygo2.
Each mutation deleted >80% of the coding sequence, including the critical PHD domain, and almost certainly resulted in null function. Pygo2 homozygous mutants, with rare exception, died shortly after birth, with a phenotype including lens agenesis, growth retardation, altered kidney development, and in some cases exencephaly and cleft palate. Pygo1 homozygous mutants, however, were viable and fertile, with no detectable developmental defects. Double Pygo1/Pygo2 homozygous mutants showed no apparent synergy in phenotype severity. The BAT-gal transgene reporter of canonical Wnt signaling showed reduced levels of expression in Pygo1-/-/Pygo2-/- mutants, with tissue-specific variation in degree of diminution. The Pygo1 and Pygo2 genes both showed widespread expression in the developing kidney, with raised levels in the stromal cell compartment. Confocal analysis of the double mutant kidneys showed disturbance of both the ureteric bud and metanephric mesenchyme-derived compartments. Branching morphogenesis of the ureteric bud was altered, with expanded tips and reduced tip density, probably contributing to the smaller size of the mutant kidney. In addition, there was an expansion of the zone of condensed mesenchyme capping the ureteric bud. Nephron formation, however, proceeded normally. Microarray analysis showed changed expression of several genes, including Cxcl13, Slc5a2, Klk5, Ren2 and Timeless, which represent candidate Wnt targets in kidney development.
The mammalian Pygopus genes are required for normal branching morphogenesis of the ureteric bud during kidney development. Nevertheless, the relatively mild phenotype observed in the kidney, as well as other organ systems, indicates a striking evolutionary divergence of Pygopus function between mammals and Drosophila. In mammals, the Pygo1/Pygo2 genes are not absolutely required for canonical Wnt signaling in most developing systems, but rather function as quantitative transducers, or modulators, of Wnt signal intensity.
PMCID: PMC1858683  PMID: 17425782

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