Generation of mice with a conditional null allele for Wntless

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. genesis 48:554–558, 2010. © 2010 Wiley‐Liss, Inc.     

Deficient Alk3-mediated BMP signaling causes prenatal omphalocele-like defect

BMP signaling plays important roles in many embryonic developmental processes. Alk3 is one of two BMP type I receptors that transduces BMP signal from the cell surface into cell. Conventional knockout of Alk3 resulted in early embryonic lethality around E7.5-E9.5. In this study, we have generated embryonic mesoderm-specific Alk3 conditional knockout by crossing Dermo1-Cre and floxed Alk3 mice. Abrogation of Alk3-mediated BMP signaling in this mouse resulted in severe defect of secondary ventral body wall formation, replicating the omphalocele phenotype in human. Our finding suggests that Alk3 plays an essential role in the formation of embryonic ventral abdominal wall, and abrogation of BMP signaling activity due to gene mutations in its signaling components could be one of the underlying causes of omphalocele at birth.     

Generation of a Magoh conditional allele in mice

Magoh encodes a core component of the exon junction complex (EJC), which binds mRNA and regulates mRNA metabolism. Magoh is highly expressed in proliferative tissues during development. EJC components have been implicated in several developmental disorders including TAR syndrome, Richieri–Costa–Pereira syndrome, and intellectual disability. Existing germline null Magoh mice are embryonic lethal as homozygotes and perinatal lethal as heterozygotes, precluding detailed analysis of embryonic and postnatal functions. Here, we report the generation of a new genetic tool to dissect temporal and tissue‐specific roles for Magoh in development and adult homeostasis. This Magoh conditional allele has two loxP sites flanking the second exon. Ubiquitous Cre‐mediated deletion of the floxed allele in a heterozygous mouse (Magoh^del/+) causes 50% reduction of both Magoh mRNA and protein. Magoh^del/+ mice exhibit both microcephaly and hypopigmentation, thus phenocopying germline haploinsufficient Magoh mice. Using Emx1‐Cre, we further show that conditional Magoh deletion in neural progenitors during embryonic development also causes microcephaly. We anticipate this novel conditional allele will be a valuable tool for assessing tissue‐specific roles for Magoh in mammalian development and postnatal processes. genesis 52:752–758, 2014. © 2014 Wiley Periodicals, Inc.     

A new Adamts9 conditional mouse allele identifies its non‐redundant role in interdigital web regression

ADAMTS9 is the most conserved member of a large family of secreted metalloproteases having diverse functions. Adamts9 null mice die before gastrulation, precluding investigations of its roles later in embryogenesis, in adult mice or disease models. We therefore generated a floxed Adamts9 allele to bypass embryonic lethality. In this mutant, unidirectional loxP sites flank exons 5–8, which encode the catalytic domain, including the protease active site. Mice homozygous for the floxed allele were viable, lacked an overt phenotype, and were fertile. Conversely, mice homozygous for a germ‐line deletion produced from the floxed allele by Cre‐lox recombination did not survive past gastrulation. Hemizygosity of the deleted Adamts9 in combination with mutant Adamts20 led to cleft palate and severe white spotting as previously described. Previously, Adamts9 haploinsufficiency combined with either Adamts20 or Adamts5 nullizygosity suggested a cooperative role in interdigital web regression, but the outcome of deletion of Adamts9 alone remained unknown. Here, Adamts9 was conditionally deleted in limb mesoderm using Prx1‐Cre mice. Unlike other ADAMTS single knockouts, limb‐specific Adamts9 deletion resulted in soft‐tissue syndactyly (STS) with 100% penetrance and concurrent deletion of Adamts5 increased the severity of STS. Thus, Adamts9 has both non‐redundant and cooperative roles in ensuring interdigital web regression. This new allele will be useful for investigating other biological functions of ADAMTS9. genesis 52:702–712, 2014. © 2014 Wiley Periodicals, Inc.     

Generation of novel conditional and hypomorphic alleles of the Smad2 gene

Smad2 is an intracellular mediator of the transforming growth factor beta signaling (TGFbeta) pathway. It has been previously shown that, in the mouse, ablation of functional Smad2 results in embryonic lethality due to gastrulation defects. To circumvent the early lethality and study the spatially and temporally specific functions of Smad2, we utilized the Cre-loxP system to generate a Smad2 conditional allele. Here we show that a conditional allele, Smad2(flox), was generated. In this allele, exons 9 and 10 are flanked by loxP sites and the gene is functionally wildtype. Cre-mediated recombination results in a deletion allele which phenocopies our previously reported Smad2(DeltaC) null mutation. To generate this conditional allele, we first made a targeted mutation which introduced a floxed neo cassette into intron 10. This allele (Smad2(3loxP)) functions hypomorphically when placed opposite a null allele, and unlike the other published Smad2 hypomorphic allele, can be maintained in the homozygous state.     

A conditional allele of Rspo3 reveals redundant function of R‐spondins during mouse limb development

Summary: R‐spondins are secreted ligands that bind cell surface receptors and activate Wnt/β‐catenin signaling. Human mutations and gene inactivation studies in mice have revealed a role for these four proteins (RSPO1‐4) in diverse developmental processes ranging from sex determination to limb development. Among the genes coding for R‐spondins, only inactivation of Rspo3 shows early embryonic lethality (E10.5 in mice). Therefore, a conditional allele of this gene is necessary to understand the function of R‐spondins throughout murine development. To address this need, we have produced an allele in which loxP sites flank exons 2–4 of Rspo3, allowing tissue‐specific deletion of these exons in the presence of Cre recombinase. We used these mice to investigate the role of Rspo3 during limb development and found that limbs ultimately developed normally in the absence of Rspo3 function. However, severe hindlimb truncations resulted when Rspo3 and Rspo2 mutations were combined, demonstrating redundant function of these genes. genesis 50:741–749, 2012. © 2012 Wiley Periodicals, Inc.     

Generation of an Frs2alpha conditional null allele

The fibroblast growth factor (FGF) signaling family controls a broad spectrum of cellular processes in development and adult tissue homeostasis and function, which is expressed in almost all tissues at all stages. FGF receptor substrate 2 alpha (FRS2alpha) is an adaptor protein that recruits downstream substrates to the FGF receptor (FGFR) tyrosine kinase. Disruption of Frs2alpha gene in mice abrogates activation of the mitogen-activated protein kinase pathway by the FGFR and leads to embryonic lethality at day E7.5 post copulation. To circumvent the embryonic lethality resulting from disruption of the Frs2alpha gene, which hinders further characterization of the role of FRS2alpha in adult tissue function and homeostasis, we generated an Frs2alpha conditional null allele for temporally- and tissue-specific disruption of the Frs2alpha gene. Using gene targeting in mouse embryonic stem cells, we introduced two loxP sites flanking the largest coding exon, exon 5, in the Frs2alpha allele. Our results indicate that the floxed Frs2alpha (Frs2alpha(flox)) allele is a true conditional null allele that encodes wildtype activity and is converted to a null allele after Cre recombinase mediated recombination.     

Generation of mice with a conditional allele for <Emphasis Type="Italic">Ift172</Emphasis>

Ift172 encodes a gene product that is part of a complex that mediates intraflagellar transport (IFT), a process necessary for the genesis and maintenance of cilia. Genetic studies in mice have offered evidence that Ift172 also plays a role in hedgehog signaling. Disruption of Ift172 in mice is associated with lethality at about embryonic day 11, limiting studies to understand the role for Ift172 in later development and the adult. To further our understanding of the later roles of Ift172, we have generated mice with a conditional allele for Ift172. We have confirmed the phenotype of the disrupted allele by using CRE expression directed by the prx1 enhancer to disrupt the conditional Ift172 allele in the developing limb.     

Generation of mice with a conditional null allele of the Jagged2 gene

The Notch signaling pathway is an evolutionarily‐conserved intercellular signaling mechanism, and mutations in its components disrupt embryonic development in many organisms and cause inherited diseases in humans. The Jagged2 (Jag2) gene, which encodes a ligand for Notch pathway receptors, is required for craniofacial, limb, and T cell development. Mice homozygous for a Jag2 null allele die at birth from cleft palate, precluding study of Jag2 function in postnatal and adult mice. We have generated a Jag2 conditional null allele by flanking the first two exons of the Jag2 gene with loxP sites. Cre‐mediated deletion of the Jag2^flox allele generates the Jag2^del2 allele, which behaves genetically as a Jag2 null allele. This Jag2 conditional null allele will enable investigation of Jag2 function in a variety of tissue‐specific contexts. genesis 48:390–393, 2010. © 2010 Wiley‐Liss, Inc.     

A key role for Pak4 in proliferation and differentiation of neural progenitor cells

The Pak4 serine/threonine kinase regulates cytoskeletal organization, and controls cell growth, proliferation, and survival. Deletion of Pak4 in mice results in embryonic lethality prior to embryonic day 11.5. Pak4 knockout embryos exhibit abnormalities in the nervous system, the heart, and other tissues. In this study a conditional deletion of Pak4 was generated in order to study the function of Pak4 in the development of the brain. Nervous system-specific conditional deletion of Pak4 was accomplished by crossing mice with a floxed allele of Pak4 with transgenic mice expressing Cre recombinase under the control of the nestin promoter. The conditional Pak4 knockout mice were born normally, but displayed growth retardation and died prematurely. The brains showed a dramatic decrease in proliferation of cortical and striatal neuronal progenitor cells. In vitro analyses revealed a reduced proliferation and self-renewing capacity of neural progenitor cells isolated from Pak4 knockout brains. The mice also exhibited cortical thinning, impaired neurogenesis and loss of neuroepithelial adherens junctions. By the time the mice died, by 4 weeks after birth, severe hydrocephalus could also be seen. These results suggest that Pak4 plays a critical role in the regulation of neural progenitor cell proliferation and in establishing the foundation for development of the adult brain.     

Bone morphogenetic protein receptor 1A signaling is dispensable for hematopoietic development but essential for vessel and atrioventricular endocardial cushion formation

Bone morphogenetic protein 4 (BMP4) is crucial for the formation of FLK1-expressing (FLK1(+)) mesodermal cells. To further define the requirement for BMP signaling in the differentiation of blood, endothelial and smooth muscle cells from FLK1(+) mesoderm, we inactivated Alk3 (Bmpr1a) in FLK1(+) cells by crossing Alk3(floxed/floxed) and Flk1(+/Cre)Alk3(+/floxed) mice. Alk3 conditional knockout (CKO) mice died between E10.5 and E11.5. Unexpectedly, Alk3 CKO embryos did not show any hematopoietic defects. However, Alk3 CKO embryos displayed multiple abnormalities in vascular development, including vessel remodeling and maturation, which contributed to severe abdominal hemorrhage. Alk3 CKO embryos also displayed defects in atrioventricular canal (AVC) endocardial cushion formation in the heart. Collectively, our studies indicate a crucial role for ALK3 in vessel remodeling, vessel integrity and endocardial cushion formation during the development of the circulation system.     

Jelly belly trans‐synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture

In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans‐synaptic signaling. Here, we show that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb–Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild‐type postsynaptic Alk expression in Alk partial loss‐of‐function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb–Alk signaling triggers the Ras‐MAP kinase cascade in both pre‐ and postsynaptic compartments. These novel roles for Jeb–Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Postnatal male germ‐cell expression of cre recombinase in Tex101‐iCre transgenic mice

We have generated a transgenic mouse line that expresses improved Cre recombinase (iCre) under the control of the testis‐expressed gene 101 (Tex101) promoter. This transgenic mouse line was named Tex101‐iCre. Using the floxed ROSA reporter mice, we found that robust Cre recombinase activity was detected in postnatal testes with weak or no activity in other tissues. Within the testis, Cre recombinase was active in spermatogenic cells as early as the prospermatogonia stage at day 1 after birth. In 30‐ and 60‐day‐old mice, positive Cre recombinase activity was detected not only in prospermatogonia but also in spermatogenic cells at later stages of spermatogenesis. There was little or no Cre activity in interstitial cells. Breeding wild‐type females with homozygous floxed fibroblast growth factor receptor 2 (Fgfr2) males carrying the Tex101‐iCre transgene did not produce any progeny with the floxed Fgfr2 allele. All the progeny inherited a recombined Fgfr2 allele, indicating that complete deletion of the floxed Fgfr2 allele by Tex101‐iCre can be achieved in the male germline. Furthermore, FGFR2 protein was not detected in spermatocytes and spermatids of adult Fgfr2^fl/fl;Tex101‐iCre mice. Taken together, our results suggest that the Tex101‐iCre mouse line allows the inactivation of a floxed gene in spermatogenic cells in adult mice, which will facilitate the functional characterization of genes in normal spermatogenesis and male fertility. genesis 48:717–722, 2010. © 2010 Wiley‐Liss, Inc.     

Generation of a conditional allele of the mouse prostaglandin EP4 receptor

Genetic disruption of the mouse EP4 receptor results in perinatal lethality associated with persistent patent ductus areteriosus (PDA). To circumvent this, an EP4 allele amenable to conditional deletion using the Cre/loxP system was generated. The targeting construct was comprised of a floxed exon2 in tandem with the neomycin-resistance gene in intron 2, flanked by third 3' LoxP site. Mice homozygous for the targeted allele (EP4(lox+neo/lox+neo)), or following its Cre-mediated deletion (EP4(del/del)), also die within hours of birth with PDA. In contrast, mice homozygous for a partially recombined allele, retaining exon2 but lacking neo (EP4(flox/flox)), are viable and show no overt phenotype. Postnatal deletion of the floxed EP4 gene is efficiently achieved in the liver and kidney in a transgenic mouse expressing the inducible Mx1Cre recombinase. The EP4(flox) mouse should provide a useful reagent with which to examine the physiologic roles of the EP4 receptor.     

Establishment of conditional knockout alleles for the gene encoding the regulator of telomere length (RTEL)

Regulator of telomere length (RTEL) is a DNA helicase-like protein that has recently been demonstrated to be required for the maintenance of telomere length and genomic stability. Rtel null mice are embryonic lethal with the defects in the nervous system, the heart, the vasculature, and extra-embryonic tissues. Rtel could also be important for the postnatal development as its expression is strongly induced in the proliferating adult cells. To further characterize the role of RTEL in adult tissue function and homeostasis, we have generated the floxed (loxP-flanked) alleles allowing to inactivate RTEL through Cre-mediated recombination in a cell- or tissue-specific manner and also to circumvent the embryonic lethality of the Rtel null allele. Mice heterozygous or homozygous for these alleles are viable and fertile. Crossing the floxed Rtel allele with a ubiquitous Cre transgenic line resulted in embryonic defects identical to those previously described for the Rtel null embryos. These conditional alleles will therefore be the important genetic tools for dissecting the spatial and temporal roles of RTEL in the regulation of telomere length and genomic stability during postnatal development and tumorigenesis.