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1.  WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature 
Nature  2005;437(7057):417-421.
Macrophages have a critical role in inflammatory and immune responses through their ability to recognize and engulf apoptotic cells1. Here we show that macrophages initiate a cell-death programme in target cells by activating the canonical WNT pathway. We show in mice that macrophage WNT7b is a short-range paracrine signal required for WNT-pathway responses and programmed cell death in the vascular endothelial cells of the temporary hyaloid vessels of the developing eye. These findings indicate that macrophages can use WNT ligands to influence cell-fate decisions—including cell death—in adjacent cells, and raise the possibility that they do so in many different cellular contexts.
PMCID: PMC4259146  PMID: 16163358
2.  Distinct functions for Wnt/β-catenin in hair follicle stem cell proliferation and survival and interfollicular epidermal homeostasis 
Cell stem cell  2013;13(6):720-733.
Wnt/β-catenin signaling is a central regulator of adult stem cells. Variable sensitivity of Wnt reporter transgenes, β-catenin’s dual roles in adhesion and signaling, and hair follicle degradation and inflammation resulting from broad deletion of epithelial β-catenin, have precluded clear understanding of Wnt/β-catenin’s functions in adult skin stem cells. By inducibly deleting β-catenin globally in skin epithelia, only in hair follicle stem cells, or only in interfollicular epidermis, and comparing the phenotypes with those caused by ectopic expression of the Wnt/β-catenin inhibitor Dkk1, we show that this pathway is necessary for hair follicle stem cell proliferation. However, β-catenin is not required within hair follicle stem cells for their maintenance, and follicles resume proliferating after removal of ectopic Dkk1, indicating persistence of functional progenitors. We further unexpectedly discovered a broader role for Wnt/β-catenin signaling in contributing to progenitor cell proliferation in non-hairy epithelia and interfollicular epidermis under homeostatic, but not inflammatory, conditions.
PMCID: PMC3900235  PMID: 24315444
3.  Myeloid WNT7b mediates the angiogenic switch and metastasis in breast cancer 
Cancer research  2014;74(11):2962-2973.
Oncogenic targets acting in both tumor cells and tumor stromal cells may offer special therapeutic appeal. Interrogation of the Oncomine database revealed that 52/53 human breast carcinomas showed substantial upregulation WNT family ligand WNT7B. Immunolabeling of human mammary carcinoma showed that WNT7B immunoreactivity was associated with both tumor cells and with tumor associated macrophages (TAMs). In the MMTV-PymT mouse model of mammary carcinoma, we found tumor progression relied upon Wnt7b produced by myeloid cells in the microenvironment. Wnt7b deletion in myeloid cells reduced the mass and volume of tumors due to a failure in the angiogenic switch. In the tumor overall, there was no change in expression of Wnt/β-catenin pathway target genes, but in vascular endothelial cells (VEC) expression of these genes was reduced suggesting that VEC respond to Wnt/β-catenin signaling. Mechanistic investigations revealed that failure of the angiogenic switch could be attributed to reduced Vegfa mRNA and protein expression in VECs, a source of Vegfa mRNA in the tumor which was limiting in the absence of myeloid Wnt7b. We also noted a dramatic reduction in lung metastasis associated with decreased macrophage-mediated tumor cell invasion. Together these results illustrated the critical role of myeloid Wnt7b in tumor progression, acting at the levels of angiogenesis, invasion and metastasis. We suggest that therapeutic suppression of Wnt7b signaling might be advantageous due to targeting multiple aspects of tumor progression.
PMCID: PMC4137408  PMID: 24638982
Wnt7b; macrophage; myeloid; tumor angiogenesis; tumor metastasis
4.  HIPPO Pathway Members Restrict SOX2 to the Inner Cell Mass Where It Promotes ICM Fates in the Mouse Blastocyst 
PLoS Genetics  2014;10(10):e1004618.
Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog.
Author Summary
Pluripotent stem cells can give rise to any cell type in the body, making them an attractive tool for regenerative medicine. Pluripotent stem cells can be derived from the mammalian embryo at the blastocyst stage or they can be created from mature adult cells by reprogramming. During reprogramming, SOX2 helps establish pluripotency, but it is not clear how SOX2 establishes pluripotency in the blastocyst. We evaluated where SOX2 is present, how SOX2 is regulated, and where SOX2 is active during blastocyst formation. Our data show that the roles and the regulation of SOX2 are unique compared to other pluripotency/reprogramming factors, such as OCT4 and NANOG. SOX2 marks pluripotent cells earlier than do other factors, but does not regulate pluripotency until several days later. Rather, the earlier role of SOX2 is to help establish the yolk sac lineage, which is essential for gestation.
PMCID: PMC4207610  PMID: 25340657
5.  Length of Day during Early Gestation as a Predictor of Risk for Severe Retinopathy of Prematurity 
Ophthalmology  2013;120(12):2706-2713.
Fetal mice require light exposure in utero during early gestation for normal vascular development in the eye. Because angiogenic abnormalities in retinopathy of prematurity (ROP) are manifested in preterm infants, we investigated whether day length during early gestation was associated with severe ROP (SROP).
Single-center, retrospective cohort study.
We included a total of 343 premature infants (401–1250 g birth weight [BW], from 1998–2002): 684 eyes (1 eye each of 2 patients excluded) with 76 eyes developing SROP, defined as (1) classic threshold ROP in zone I or II, (2) type 1 ROP in zone I, or (3) in a few eyes, type 1 ROP in posterior zone II that was treated.
For each infant, average day length (ADL) was calculated during different cumulative time periods and time windows after the estimated date of conception (EDC). Multiple logistic regression analysis (with generalized estimating equations to account for inter-eye correlation) was performed.
Main Outcome Measures
Association of ADL during early gestation with SROP.
In a model evaluating all 684 eyes with 76 eyes developing SROP, BW, gestational age, multiple births, race, per capita income in the mother's residence ZIP code, and ADL during the first 90 days after the EDC were factors associated with the development of SROP. Each additional hour of ADL (90 days) decreased the likelihood of SROP by 28% (P = 0.015; odds ratio [OR], 0.72; 95% confidence interval [CI], 0.55–0.94). In a model evaluating the subset of 146 prethreshold ROP eyes with 76 eyes developing SROP, each additional hour of ADL during the first 105 days after the EDC decreased the likelihood of SROP by 46% (P = 0.001; OR, 0.54; 95% CI, 0.37–0.78). Time windows when ADL was most closely associated with SROP were 31 to 60 days and 61 to 90 days after the EDC for the all eyes and the prethreshold ROP eyes models, respectively.
Higher ADL during early gestation was associated with a lower risk for SROP and may imply a role for prophylactic light treatment during early gestation to decrease the risk of SROP.
Financial Disclosure(s)
Proprietary or commercial disclosure may be found after the references.
PMCID: PMC4164223  PMID: 24139125
Developmental biology  2013;379(1):38-52.
Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease.
PMCID: PMC3699333  PMID: 23523683
Wntless; Wnt; lung development; endothelium; epithelium
7.  RhoA GTPase controls cytokinesis and programmed necrosis of hematopoietic progenitors 
The Journal of Experimental Medicine  2013;210(11):2371-2385.
The GTPase RhoA is required for the appropriate division and survival of hematopoietic progenitor cells.
Hematopoietic progenitor cells (HPCs) are central to hematopoiesis as they provide large numbers of lineage-defined blood cells necessary to sustain blood homeostasis. They are one of the most actively cycling somatic cells, and their precise control is critical for hematopoietic homeostasis. The small GTPase RhoA is an intracellular molecular switch that integrates cytokine, chemokine, and adhesion signals to coordinate multiple context-dependent cellular processes. By using a RhoA conditional knockout mouse model, we show that RhoA deficiency causes a multilineage hematopoietic failure that is associated with defective multipotent HPCs. Interestingly, RhoA−/− hematopoietic stem cells retained long-term engraftment potential but failed to produce multipotent HPCs and lineage-defined blood cells. This multilineage hematopoietic failure was rescued by reconstituting wild-type RhoA into the RhoA−/− Lin−Sca-1+c-Kit+ compartment. Mechanistically, RhoA regulates actomyosin signaling, cytokinesis, and programmed necrosis of the HPCs, and loss of RhoA results in a cytokinesis failure of HPCs manifested by an accumulation of multinucleated cells caused by failed abscission of the cleavage furrow after telophase. Concomitantly, the HPCs show a drastically increased death associated with increased TNF–RIP-mediated necrosis. These results show that RhoA is a critical and specific regulator of multipotent HPCs during cytokinesis and thus essential for multilineage hematopoiesis.
PMCID: PMC3804933  PMID: 24101377
8.  Methane Emission by Camelids 
PLoS ONE  2014;9(4):e94363.
Methane emissions from ruminant livestock have been intensively studied in order to reduce contribution to the greenhouse effect. Ruminants were found to produce more enteric methane than other mammalian herbivores. As camelids share some features of their digestive anatomy and physiology with ruminants, it has been proposed that they produce similar amounts of methane per unit of body mass. This is of special relevance for countrywide greenhouse gas budgets of countries that harbor large populations of camelids like Australia. However, hardly any quantitative methane emission measurements have been performed in camelids. In order to fill this gap, we carried out respiration chamber measurements with three camelid species (Vicugna pacos, Lama glama, Camelus bactrianus; n = 16 in total), all kept on a diet consisting of food produced from alfalfa only. The camelids produced less methane expressed on the basis of body mass (0.32±0.11 L kg−1 d−1) when compared to literature data on domestic ruminants fed on roughage diets (0.58±0.16 L kg−1 d−1). However, there was no significant difference between the two suborders when methane emission was expressed on the basis of digestible neutral detergent fiber intake (92.7±33.9 L kg−1 in camelids vs. 86.2±12.1 L kg−1 in ruminants). This implies that the pathways of methanogenesis forming part of the microbial digestion of fiber in the foregut are similar between the groups, and that the lower methane emission of camelids can be explained by their generally lower relative food intake. Our results suggest that the methane emission of Australia's feral camels corresponds only to 1 to 2% of the methane amount produced by the countries' domestic ruminants and that calculations of greenhouse gas budgets of countries with large camelid populations based on equations developed for ruminants are generally overestimating the actual levels.
PMCID: PMC3981797  PMID: 24718604
9.  Distinct Requirements for Cranial Ectoderm and Mesenchyme-Derived Wnts in Specification and Differentiation of Osteoblast and Dermal Progenitors 
PLoS Genetics  2014;10(2):e1004152.
The cranial bones and dermis differentiate from mesenchyme beneath the surface ectoderm. Fate selection in cranial mesenchyme requires the canonical Wnt effector molecule β-catenin, but the relative contribution of Wnt ligand sources in this process remains unknown. Here we show Wnt ligands are expressed in cranial surface ectoderm and underlying supraorbital mesenchyme during dermal and osteoblast fate selection. Using conditional genetics, we eliminate secretion of all Wnt ligands from cranial surface ectoderm or undifferentiated mesenchyme, to uncover distinct roles for ectoderm- and mesenchyme-derived Wnts. Ectoderm Wnt ligands induce osteoblast and dermal fibroblast progenitor specification while initiating expression of a subset of mesenchymal Wnts. Mesenchyme Wnt ligands are subsequently essential during differentiation of dermal and osteoblast progenitors. Finally, ectoderm-derived Wnt ligands provide an inductive cue to the cranial mesenchyme for the fate selection of dermal fibroblast and osteoblast lineages. Thus two sources of Wnt ligands perform distinct functions during osteoblast and dermal fibroblast formation.
Author Summary
Craniofacial abnormalities are relatively common congenital birth defects, and the Wnt signaling pathway and its effectors have key roles in craniofacial development. Wntless/Gpr177 is required for the efficient secretion of all Wnt ligands and maps to a region that contains SNPs strongly associated with reduced bone mass, and heterozygous deletion is associated with facial dysmorphology. Here we test the role of specific sources of secreted Wnt proteins during early stages of craniofacial development and obtained dramatic craniofacial anomalies. We found that the overlying cranial surface ectoderm Wnts generate an instructive cue of Wnt signaling for skull bone and skin cell fate selection and transcription of additional Wnts in the underlying mesenchyme. Once initiated, mesenchymal Wnts may maintain Wnt signal transduction and function in an autocrine manner during differentiation of skull bones and skin. These results highlight how Wnt ligands from two specific tissue sources are integrated for normal craniofacial patterning and can contribute to complex craniofacial abnormalities.
PMCID: PMC3930509  PMID: 24586192
10.  Crim1 maintains retinal vascular stability during development by regulating endothelial cell Vegfa autocrine signaling 
Development (Cambridge, England)  2014;141(2):448-459.
Angiogenesis defines the process in which new vessels grow from existing vessels. Using the mouse retina as a model system, we show that cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is highly expressed in angiogenic endothelial cells. Conditional deletion of the Crim1 gene in vascular endothelial cells (VECs) causes delayed vessel expansion and reduced vessel density. Based on known Vegfa binding by Crim1 and Crim1 expression in retinal vasculature, where angiogenesis is known to be Vegfa dependent, we tested the hypothesis that Crim1 is involved in the regulation of Vegfa signaling. Consistent with this hypothesis, we showed that VEC-specific conditional compound heterozygotes for Crim1 and Vegfa exhibit a phenotype that is more severe than each single heterozygote and indistinguishable from that of the conditional homozygotes. We further showed that human CRIM1 knockdown in cultured VECs results in diminished phosphorylation of VEGFR2, but only when VECs are required to rely on an autocrine source of VEGFA. The effect of CRIM1 knockdown on reducing VEGFR2 phosphorylation was enhanced when VEGFA was also knocked down. Finally, an anti-VEGFA antibody did not enhance the effect of CRIM1 knockdown in reducing VEGFR2 phosphorylation caused by autocrine signaling, but VEGFR2 phosphorylation was completely suppressed by SU5416, a small-molecule VEGFR2 kinase inhibitor. These data are consistent with a model in which Crim1 enhances the autocrine signaling activity of Vegfa in VECs at least in part via Vegfr2.
PMCID: PMC3879820  PMID: 24353059
Crim1; Vegfa; Endothelial cell; Angiogenesis
11.  Stem cell factor Sox2 and its close relative Sox3 have differentiation functions in oligodendrocytes 
Development (Cambridge, England)  2014;141(1):39-50.
Neural precursor cells of the ventricular zone give rise to all neurons and glia of the central nervous system and rely for maintenance of their precursor characteristics on the closely related SoxB1 transcription factors Sox1, Sox2 and Sox3. We show in mouse spinal cord that, whereas SoxB1 proteins are usually downregulated upon neuronal specification, they continue to be expressed in glial precursors. In the oligodendrocyte lineage, Sox2 and Sox3 remain present into the early phases of terminal differentiation. Surprisingly, their deletion does not alter precursor characteristics but interferes with proper differentiation. Although a direct influence on myelin gene expression may be part of their function, we provide evidence for another mode of action. SoxB1 proteins promote oligodendrocyte differentiation in part by negatively controlling miR145 and thereby preventing this microRNA from inhibiting several pro-differentiation factors. This study presents one of the few cases in which SoxB1 proteins, including the stem cell factor Sox2, are associated with differentiation rather than precursor functions.
PMCID: PMC3865748  PMID: 24257626
Glia; Myelin; Transcriptional control; High mobility group; MicroRNA
12.  A direct and melanopsin-dependent fetal light response regulates mouse eye development 
Nature  2013;494(7436):243-246.
Vascular patterning is critical for organ function. In the eye, there is simultaneous regression of embryonic hyaloid vasculature1 (important to clear the optical path) and formation of the retinal vasculature2 (important for the high metabolic demands of retinal neurons). These events occur postnatally in the mouse. Here we have identified a light-response pathway that regulates both processes. We show that when mice are mutated in the gene (Opn4) for the atypical opsin melanopsin3–5, or are dark-reared from late gestation, the hyaloid vessels are persistent at 8 days post-partum and the retinal vasculature overgrows. We provide evidence that these vascular anomalies are explained by a light-response pathway that suppresses retinal neuron number, limits hypoxia and, as a consequence, holds local expression of vascular endothelial growth factor (VEGFA) in check. We also show that the light response for this pathway occurs in late gestation at about embryonic day 16 and requires the photopigment in the fetus and not the mother. Measurements show that visceral cavity photon flux is probably sufficient to activate melanopsin-expressing retinal ganglion cells in the mouse fetus. These data thus show that light—the stimulus for function of the mature eye—is also critical in preparing the eye for vision by regulating retinal neuron number and initiating a series of events that ultimately pattern the ocular blood vessels.
PMCID: PMC3746810  PMID: 23334418
13.  In Vivo Depletion of CD11c+ Dendritic Cells Abrogates Priming of CD8+ T Cells by Exogenous Cell-Associated Antigens 
Immunity  2002;17(2):211-220.
Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules. On most cells, these peptides are exclusively of endogenous, cytosolic origin. Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens. This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections. Here, we report a novel diphtheria toxin-based system that allows the inducible, short-term ablation of dendritic cells (DC) in vivo. We show that in vivo DC are required to cross-prime CTL precursors. Our results thus define a unique in vivo role of DC, i.e., the sensitization of the immune system for cell-associated antigens. DC-depleted mice fail to mount CTL responses to infection with the intracellular bacterium Listeria monocytogenes and the rodent malaria parasite Plasmodium yoelii.
PMCID: PMC3689299  PMID: 12196292
14.  Generation of Mice with a Conditional Null Allele for Wntless 
Genesis (New York, N.Y. : 2000)  2010;48(9):554-558.
The Wnt-signaling pathway is necessary in a variety of developmental processes and has been implicated in numerous pathologies. Wntless (Wls) binds to Wnt proteins and facilitates Wnt sorting and secretion. Conventional deletion of Wls results in early fetal lethality due to defects in body axis establishment. To gain insight into the function of Wls in later stages of development, we have generated a conditional null allele. Homozygous germline deletion of Wls confirmed prenatal lethality and failure of embryonic axis formation. Deletion of Wls using Wnt1-cre phenocopied Wnt1 null abnormalities in the midbrain and hindbrain. In addition, conditional deletion of Wls in pancreatic precursor cells resulted in pancreatic hypoplasia similar to that previously observed after conditional β-catenin deletion. This Wls conditional null allele will be valuable in detecting novel Wnt functions in development and disease.
PMCID: PMC3689319  PMID: 20614471
Wnt; Evi; Gpr177; Sprinter; Wnt transporter
15.  Obligatory participation of macrophages in an angiopoietin 2-mediated cell death switch 
Development (Cambridge, England)  2007;134(24):4449-4458.
Macrophages have a critical function in the recognition and engulfment of dead cells. In some settings, macrophages also actively signal programmed cell death. Here we show that during developmentally scheduled vascular regression, resident macrophages are an obligatory participant in a signaling switch that favors death over survival. This switch occurs when the signaling ligand angiopoietin 2 has the dual effect of suppressing survival signaling in vascular endothelial cells (VECs) and stimulating Wnt ligand production by macrophages. In response to the Wnt ligand, VECs enter the cell cycle and in the absence of survival signals, die from G1 phase of the cell cycle. We propose that this mechanism represents an adaptation to ensure that the macrophage and its disposal capability are on hand when cell death occurs.
PMCID: PMC3675770  PMID: 18039971
Macrophage; Angiopoietin; Wnt; Programmed cell death; Vascular regression; Cell cycle
16.  Shroom3 and a Pitx2-N-cadherin pathway function cooperatively to generate asymmetric cell shape changes during gut morphogenesis 
Developmental biology  2011;357(1):227-234.
The cytoskeletal protein Shroom3 is a potent inducer of epithelial cell shape change and is required for lens and neural plate morphogenesis. Analysis of gut morphogenesis in Shroom3 deficient mouse embryos revealed that the direction of gut rotation is also disrupted. It was recently established that Pitx2-dependent, asymmetrical cellular behaviors in the dorsal mesentery (DM) of the early mid-gut, a structure connecting the gut-tube to the rest of the embryo, contribute to the direction of gut rotation in chicken embryos by influencing the direction of the dorsal mesenteric tilt. Asymmetric cell shapes in the DM epithelium are hypothesized to contribute to the tilt, however, it is unclear what lies downstream of Pitx2 to alter epithelial cell shape. The cells of the left DM epithelium in either Pitx2 or Shroom3 deficient embryos are shorter and wider than those in control embryos and resemble the shape of those on the right, demonstrating that like Pitx2, Shroom3 is required for cell shape asymmetry and the leftward DM tilt. Because N-cadherin expression is specific to the left side and is Pitx2 dependent, we determined whether Shroom3 and N-cadherin function together to regulate cell shape in the left DM epithelium. Analysis of mouse embryos lacking one allele of both Shroom3 and N-cadherin revealed that they possess shorter and wider left epithelial DM cells when compared with Shroom3 or N-cadherin heterozygous embryos. This indicates a genetic interaction. Together these data provide evidence that Shroom3 and N-cadherin function cooperatively downstream of Pitx2 to directly regulate cell shape changes necessary for early gut tube morphogenesis.
PMCID: PMC3619216  PMID: 21726547
17.  Left-right locomotor circuitry depends upon RhoA-driven organization of the neuroepithelium in the developing spinal cord 
RhoA is a key regulator of cytoskeletal dynamics with a variety of effects on cellular processes. Loss of RhoA in neural progenitor cells disrupts adherens junctions and causes disorganization of the neuroepithelium in the developing nervous system. However, it remains largely unknown how the loss of RhoA physiologically affects neural circuit formation. Here we show that proper neuroepithelial organization maintained by RhoA GTPase in both the ventral and dorsal spinal cord is critical for left-right locomotor behavior. We examined the roles of RhoA in the ventral and dorsal spinal cord by deleting the gene in neural progenitors using Olig2-Cre and Wnt1-Cre mice, respectively. RhoA-deleted neural progenitors in both mutants exhibit defects in the formation of apical adherens junctions and disorganization of the neuroepithelium. Consequently, the ventricular zone and lumen of the dysplastic region are lost, causing the left and right sides of the gray matter to be directly connected. Furthermore, the dysplastic region lacks ephrinB3 expression at the midline that is required for preventing EphA4-expressing corticospinal neurons and spinal interneurons from crossing the midline. As a result, aberrant neuronal projections are observed in that region. Finally, both RhoA mutants develop a rabbit-like hopping gait. These results demonstrate that RhoA functions to maintain neuroepithelial structures in the developing spinal cord and that proper organization of the neuroepithelium is required for appropriate left-right motor behavior.
PMCID: PMC3424512  PMID: 22836272
18.  Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival 
Developmental biology  2011;360(1):30-43.
Morphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/β-catenin/Rap1/Nectin-based cell-cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover.
PMCID: PMC3215831  PMID: 21945075
Rac1 GTPase; lens fibers; conditional knockout; migration; cell adhesion
19.  Metchnikoff’s Policemen—Macrophages in Development, Homeostasis and Regeneration 
Trends in molecular medicine  2011;17(12):743-752.
Over the past decade, modern genetic tools have permitted scientists to study the function of myeloid lineage cells, including macrophages, as never before. Macrophages were first detected more than a century ago as cells that ingested bacteria and other microbes, but it is now known that their functional roles are far more numerous. In this review, we focus on the prevailing functions of macrophages beyond their role in innate immunity. We highlight examples of macrophages acting as regulators of development, tissue homoeostasis, remodeling (the reorganization or renovation of existing tissues), and repair. We also detail how modern genetic tools have facilitated new insights into these mysterious cells.
PMCID: PMC3225647  PMID: 21890411
20.  Generation of an Rx-tTA: TetOp-Cre Knock-In Mouse Line for Doxycycline Regulated Cre Activity in the Rx Expression Domain 
PLoS ONE  2012;7(11):e50426.
Genetic deletion of mouse genes has played a crucial role in our understanding of embryonic eye development. Transgenic, tissue specific Cre recombinase expression in various eye structures has facilitated these experiments. However, an early expressing, temporally-regulated, optic vesicle-specific Cre line has not been available. In this report, we detail the generation and analysis of a knock-in, inducible Cre line designed to drive recombination specifically within the Rx expression domain. Crossing this line with a reporter line demonstrates that recombination can be mediated within the early optic vesicle and throughout retinal development. Recombination can also be mediated in the Rx-expressing, ventral diencephalon and future posterior pituitary gland. Furthermore, it was demonstrated that dietary doxycycline could effectively modulate Cre activity. This line has the potential to facilitate conditional knock-out experimentation to study early retina and/or posterior pituitary development.
PMCID: PMC3507682  PMID: 23209739
21.  High calorie diet triggers hypothalamic angiopathy 
Molecular Metabolism  2012;1(1-2):95-100.
Obesity, type 2 diabetes, and related diseases represent major health threats to modern society. Related pathophysiology of impaired neuronal function in hypothalamic control centers regulating metabolism and body weight has been dissected extensively and recent studies have started focusing on potential roles of astrocytes and microglia. The hypothalamic vascular system, however, which maintains the microenvironment necessary for appropriate neuronal function, has been largely understudied. We recently discovered that high fat/high sucrose diet exposure leads to increased hypothalamic presence of immunoglobulin G (IgG1). Investigating this phenomenon further, we have discovered a significant increase in blood vessel length and density in the arcuate nucleus (ARC) of the hypothalamus in mice fed a high fat/high sucrose diet, compared to matched controls fed standard chow diet. We also found a clearly increased presence of α-smooth muscle actin immunoreactive vessels, which are rarely present in the ARC and indicate an increase in the formation of new arterial vessels. Along the blood brain barrier, an increase of degenerated endothelial cells are observed. Moreover, such hypothalamic angiogenesis was not limited to rodent models. We also found an increase in the number of arterioles of the infundibular nucleus (the human equivalent of the mouse ARC) in patients with type 2 diabetes, suggesting angiogenesis occurs in the human hypothalamus of diabetics. Our discovery reveals novel hypothalamic pathophysiology, which is reminiscent of diabetic retinopathy and suggests a potential functional involvement of the hypothalamic vasculature in the later stage pathogenesis of metabolic syndrome.
PMCID: PMC3757646  PMID: 24024123
Obesity; Diabetes; Blood brain barrier; Capillary; Fluorescent angiography; Endothelial cell
22.  Co-operative roles for E-cadherin and N-cadherin during lens vesicle separation and lens epithelial cell survival 
Developmental biology  2008;326(2):403-417.
The classical cadherins are known to have both adhesive and signaling functions. It has also been proposed that localized regulation of cadherin activity may be important in cell assortment during development. In the context of eye development, it has been suggested that cadherins are important for separation of the invaginated lens vesicle from the surface ectoderm. To test this hypothesis, we conditionally deleted N-cadherin or E-cadherin from the presumptive lens ectoderm of the mouse. Conditional deletion of either cadherin alone did not produce a lens vesicle separation defect. However, these conditional mutants did exhibit common structural deficits, including microphthalmia, severe iris hyperplasia, persistent vacuolization within the fibre cell region, and eventual lens epithelial cell deterioration. To assess the co-operative roles of E-cadherin and N-cadherin within the developing lens, double conditional knockout embryos were generated. These mice displayed distinct defects in lens vesicle separation and persistent expression of another classical cadherin, P-cadherin, within the cells of the persistent lens stalk. Double mutant lenses also exhibited severe defects in lens epithelial cell adhesion and survival. Finally, the severity of the lens phenotype was shown to be sensitive to the number of wild-type E- and N-cadherin alleles. These data suggest that the co-operative expression of both E- and N-cadherin during lens development is essential for normal cell sorting and subsequent lens vesicle separation.
PMCID: PMC3408230  PMID: 18996109
23.  The EYA Tyrosine Phosphatase Activity Is Pro-Angiogenic and Is Inhibited by Benzbromarone 
PLoS ONE  2012;7(4):e34806.
Eyes Absents (EYA) are multifunctional proteins best known for their role in organogenesis. There is accumulating evidence that overexpression of EYAs in breast and ovarian cancers, and in malignant peripheral nerve sheath tumors, correlates with tumor growth and increased metastasis. The EYA protein is both a transcriptional activator and a tyrosine phosphatase, and the tyrosine phosphatase activity promotes single cell motility of mammary epithelial cells. Since EYAs are expressed in vascular endothelial cells and cell motility is a critical feature of angiogenesis we investigated the role of EYAs in this process. Using RNA interference techniques we show that EYA3 depletion in human umbilical vein endothelial cells inhibits transwell migration as well as Matrigel-induced tube formation. To specifically query the role of the EYA tyrosine phosphatase activity we employed a chemical biology approach. Through an experimental screen the uricosuric agents Benzbromarone and Benzarone were found to be potent EYA inhibitors, and Benzarone in particular exhibited selectivity towards EYA versus a representative classical protein tyrosine phosphatase, PTP1B. These compounds inhibit the motility of mammary epithelial cells over-expressing EYA2 as well as the motility of endothelial cells. Furthermore, they attenuate tubulogenesis in matrigel and sprouting angiogenesis in the ex vivo aortic ring assay in a dose-dependent fashion. The anti-angiogenic effect of the inhibitors was also demonstrated in vivo, as treatment of zebrafish embryos led to significant and dose-dependent defects in the developing vasculature. Taken together our results demonstrate that the EYA tyrosine phosphatase activity is pro-angiogenic and that Benzbromarone and Benzarone are attractive candidates for repurposing as drugs for the treatment of cancer metastasis, tumor angiogenesis, and vasculopathies.
PMCID: PMC3335822  PMID: 22545090
24.  RhoA of the Rho Family Small GTPases Is Essential for B Lymphocyte Development 
PLoS ONE  2012;7(3):e33773.
RhoA is a member of the Rho family small GTPases that are implicated in various cell functions including proliferation and survival. However, the physiological role of RhoA in vivo remains largely unknown. Here, we deleted RhoA in the B cell and hematopoietic stem cell (HSC) populations in RhoAflox/flox mice with CD19 and Mx promoter-driven Cre expression, respectively. Deletion of RhoA by CD19Cre/+ significantly blocked B cell development in spleen, leading to a marked reduction in the number of transitional, marginal zone, and follicular B cells. Surprisingly, neither B cell proliferation in response to either LPS or B cell receptor (BCR) engagement nor B cell survival rate in vivo was affected by RhoA deletion. Furthermore, RhoA−/− B cells, like control cells, were rescued from apoptosis by BCR crosslinking in vitro. In contrast, RhoA deficiency led to a defect in B cell activating factor (BAFF)-mediated B cell survival that was associated with a dampened expression of BAFF receptor and a loss of BAFF-mediated Akt activation. Finally, HSC deletion of RhoA by Mx-Cre severely reduced proB/preB and immature B cell populations in bone marrow while common lymphoid progenitors were increased, indicating that RhoA is also required for B cell progenitor/precursor differentiation. Taken together, our results uncover an important role for RhoA at multiple stages of B cell development.
PMCID: PMC3306291  PMID: 22438996
25.  CRIM1 Complexes with ß-catenin and Cadherins, Stabilizes Cell-Cell Junctions and Is Critical for Neural Morphogenesis 
PLoS ONE  2012;7(3):e32635.
In multicellular organisms, morphogenesis is a highly coordinated process that requires dynamically regulated adhesion between cells. An excellent example of cellular morphogenesis is the formation of the neural tube from the flattened epithelium of the neural plate. Cysteine-rich motor neuron protein 1 (CRIM1) is a single-pass (type 1) transmembrane protein that is expressed in neural structures beginning at the neural plate stage. In the frog Xenopus laevis, loss of function studies using CRIM1 antisense morpholino oligonucleotides resulted in a failure of neural development. The CRIM1 knockdown phenotype was, in some cases, mild and resulted in perturbed neural fold morphogenesis. In severely affected embryos there was a dramatic failure of cell adhesion in the neural plate and complete absence of neural structures subsequently. Investigation of the mechanism of CRIM1 function revealed that it can form complexes with ß-catenin and cadherins, albeit indirectly, via the cytosolic domain. Consistent with this, CRIM1 knockdown resulted in diminished levels of cadherins and ß-catenin in junctional complexes in the neural plate. We conclude that CRIM1 is critical for cell-cell adhesion during neural development because it is required for the function of cadherin-dependent junctions.
PMCID: PMC3299674  PMID: 22427856

Results 1-25 (41)