GPI-specific phospholipase D (GPI-PLD) is expressed during mouse development and is localized to the extracellular matrix of the developing mouse skeleton

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and has a well-characterized biochemistry; however, its physiological role is completely unknown. Previous investigations into GPI-PLD have focused on the adult animal or on in vitro systems and a putative role in development has been neither proposed nor investigated. We describe the first evidence of GPI-PLD expression during mouse embryonic ossification. GPI-PLD expression was detected predominantly at sites of skeletal development, increasing during the course of gestation. GPI-PLD was observed during both intramembraneous and endochondral ossification and localized predominantly to the extracellular matrix of chondrocytes and to primary trabeculae of the skeleton. In addition, the mouse chondrocyte cell line ATDC5 expressed GPI-PLD after experimental induction of differentiation. These results implicate GPI-PLD in the process of bone formation during mouse embryogenesis.

CA125/MUC16 is dispensable for mouse development and reproduction.

Cancer antigen 125 (CA125) is a blood biomarker that is routinely used to monitor the progression of human epithelial ovarian cancer (EOC) and is encoded by MUC16, a member of the mucin gene family. The biological function of CA125/MUC16 and its potential role in EOC are poorly understood. Here we report the targeted disruption of the of the Muc16 gene in the mouse. To generate Muc16 knockout mice, 6.0 kb was deleted that included the majority of exon 3 and a portion of intron 3 and replaced with a lacZ reporter cassette. Loss of Muc16 protein expression suggests that Muc16 homozygous mutant mice are null mutants. Muc16 homozygous mutant mice are viable, fertile, and develop normally. Histological analysis shows that Muc16 homozygous mutant tissues are normal. By the age of 1 year, Muc16 homozygous mutant mice appear normal. Downregulation of transcripts from another mucin gene (Muc1) was detected in the Muc16 homozygous mutant uterus. Lack of any prominent abnormal phenotype in these Muc16 knockout mice suggests that CA125/MUC16 is not required for normal development or reproduction. These knockout mice provide a unique platform for future studies to identify the role of CA125/MUC16 in organ homeostasis and ovarian cancer.

Functional studies of homeobox gene {\it Cart1\/} during mouse development

Cart1 is a paired-class homeobox-containing gene that is expressed in head mesenchyme, branchial arches, limb buds, and various cartilages during embryogenesis. To understand the role of Cart1 during mammalian development, I generated Cart1-mutant mice by gene targeting in mouse embryonic stem cells. Cart1-homozygous mutants were born alive but all died soon after birth. Most had acrania (absence of the cranial vault) and meroanencephaly (absence of part of the brain). In situ hybridization studies showed that Cart1 is expressed specifically in forebrain mesenchyme but not in midbrain or hindbrain mesenchyme nor in the neural tube. Developmental studies revealed a transient deficiency of forebrain mesenchyme cells due to apoptosis associated with a delay in neural tube closure in that region. Subsequently, the forebrain region became filled with mesenchyme and closed, however, the midbrain neural tube region never initiated closure and remained open. These results suggest that Cart1 is required for the survival of forebrain mesenchyme and that its absence disrupts cranial neural tube morphogenesis by blocking the initiation of closure in the midbrain region, and this ultimately leads to the generation of lethal craniofacial defects. Prenatal treatment of Cart1 homozygous mutants with folic acid suppressed the development of the acrania/meroanencephaly phenotype. Thus, Cart1 mutant mice provide a novel animal model for understanding the cellular, molecular, and genetic etiology of neural tube defects and for the development of prenatal therapeutic protocols using folic acid. ^

Use of a hypomorphic allele of myogenin to analyze Myogenin-dependent processes in mouse development

Myogenin is a muscle-specific transcription factor essential for skeletal muscle differentiation. A severe reduction in the number of fused myotubes is seen in myogenin-null mice, and the expression of genes characteristic of differentiated skeletal muscle is reduced. Additionally, sternebrae defects are seen in myogenin-null mice, a secondary defect in the sternal cartilage precursors. Very little is known about the quantitative requirement for myogenin in muscle differentiation and thoracic skeletal development in vivo. In this thesis I describe experiments utilizing a mouse line harboring a hypomorphic allele of myogenin, generated by gene targeting techniques in embryonic stem cells. The nature of the hypomorphism was due to lowered levels of myogenin from this allele. In embryos homozygous for the hypomorphic allele, normal sternum formation and extensive muscle differentiation was observed. However, muscle hypoplasia and reduced muscle-specific gene expression were apparent in these embryos, and the mice were not viable after birth. These results suggest skeletal muscle differentiation is highly sensitive to the absolute amounts of myogenin, and reveal distinct threshold requirements for myogenin in skeletal muscle differentiation, sternum formation, and viability in vivo. The hypomorphic allele was utilized as a genetically sensitized background to identify other components of myogenin-mediated processes. Using a candidate gene approach I crossed null mutations in MEF2C and MRF4 into the hypomorphic background and examined whether these mutations affected muscle differentiation and skeleton formation in the myogenin hypomorph. Although MEF2C mutation did not affect any phenotypes seen in the hypomorphic background, MRF4 was observed to be an essential component of myogenin-mediated processes of thoracic skeletal development. Additionally, the hypomorphic allele was very sensitive to genetic effects, suggesting the existence of mappable genetic modifiers of the hypomorphic allele of myogenin. ^

Analysis of Lim1 LIM domains in mouse development

Transcriptional regulation is fundamental for the precise development of all organisms. Through tight regulation, necessary genes are activated at proper spatial and temporal patterns, while unnecessary genes are repressed. A large family of regulator proteins that have been demonstrated to be involved in various developmental processes by activation and repression of target genes is the homeodomain family of proteins. To date, the function of many of these homeoproteins has been elucidated in diverse species. However, the molecular mechanism underlying the function of these proteins has not been fully understood. In this study, the molecular mechanism of the function of a LIM-homeoprotein, Lim1, was examined. In addition to the homeodomain, Lim1 contains two LIM domains that are highly conserved among species. This high conservation along with data from in vitro studies on Xenopus Lim1 suggests that the LIM domains might be important for the function of Lim1 as a transcriptional regulator. Here, the functional importance of the LIM domains of Lim1 was determined by using a novel gene-targeting strategy in mouse embryonic stem (ES) cells. A cre-loxP system was used in conjunction with the unique genomic organization of Lim1 to obtain four types of mutant ES cell lines that would allow for the in vivo analysis of the function of both the LIM domains of Lim1 together and also singularly. These four mutant Lim1 alleles either contained base-pair changes at the LIM encoding exons that alters zinc-binding amino acids of the LIM domains or contained only exogenous loxP sequences in the first intron of Lim1, which serves as the control allele. These mutations in the LIM domains would presumably abolish the zinc-finger tertiary structure of the domain and thus render the domain non-functional. Mice carrying mutations at both the LIM domains of Lim1, L1L2, die around E10 without anterior head structures anterior to rhombomere 3, identical in phenotype to the Lim1 null mutants in spite of the presence of mutant Lim1 RNA. This result demonstrates that the integrity of both the LIM domains are essential for the function of Lim1. This is further supported by the phenotype of mice carrying mutation at only the second LIM domain of Lim1, L2. The L2 mice although still carrying one intact Lim1 LIM domain, also die in utero. The L2 mice die at varying times, from around E8 to E10 with anterior defects in addition to other axial defects which have yet to be fully characterized. The results of this study so far demonstrates that the integrity of both LIM domains are required for the function of Lim1. ^

Glutathione synthesis is essential for mouse development but not for cell growth in culture

Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of γ-glutamylcysteine synthetase (γGCS-HStm1), an essential enzyme in GSH synthesis. Embryos homozygous for γGCS-HStm1 fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.

Profilin I is essential for cell survival and cell division in early mouse development

Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1ko) allele in mice. Homozygous pfn1ko/ko mice are not viable. Pfn1ko/ko embryos died as early as the two-cell stage, and no pfn1ko/ko blastocysts were detectable. Adult pfn1ko/wt mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1ko/wt embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.

Neural crest-directed gene transfer demonstrates Wnt1 role in melanocyte expansion and differentiation during mouse development

Wnt1 signaling has been implicated as one factor involved in neural crest-derived melanocyte (NC-M) development. Mice deficient for both Wnt1 and Wnt3a have a marked deficiency in trunk neural crest derivatives including NC-Ms. We have used cell lineage-directed gene targeting of Wnt signaling genes to examine the effects of Wnt signaling in mouse neural crest development. Gene expression was directed to cell lineages by infection with subgroup A avian leukosis virus vectors in lines of transgenic mice that express the retrovirus receptor tv-a. Transgenic mice with tva in either nestin-expressing neural precursor cells (line Ntva) or dopachrome tautomerase (DCT)-expressing melanoblasts (line DCTtva) were analyzed. We overstimulated Wnt signaling in two ways: directed gene transfer of Wnt1 to Ntva+ cells and transfer of β-catenin to DCTtva+ NC-M precursor cells. In both methods, NC-M expansion and differentiation were effected. Significant increases were observed in the number of NC-Ms [melanin+ and tyrosinase-related protein 1 (TYRP1)+ cells], the differentiation of melanin− TYRP1+ cells to melanin+ TYRP1+ NC-Ms, and the intensity of pigmentation per NC-M. These data are consistent with Wnt1 signaling being involved in both expansion and differentiation of migrating NC-Ms in the developing mouse embryo. The use of lineage-directed gene targeting will allow the dissection of signaling molecules involved in NC development and is adaptable to other mammalian developmental systems.

Capturing genes encoding membrane and secreted proteins important for mouse development.

A strategy based on the gene trap was developed to prescreen mouse embryonic stem cells for insertional mutations in genes encoding secreted and membrane-spanning proteins. The "secretory trap" relies on capturing the N-terminal signal sequence of an endogenous gene to generate an active beta-galactosidase fusion protein. Insertions were found in a cadherin gene, an unc6-related laminin (netrin) gene, the sek receptor tyrosine kinase gene, and genes encoding two receptor-linked protein-tyrosine phosphatases, LAR and PTP kappa. Analysis of homozygous mice carrying insertions in LAR and PTP kappa showed that both genes were effectively disrupted, but neither was essential for normal embryonic development.

Expression analysis of the Epha1 receptor tyrosine kinase and its high-affinity ligands Efna1 and Efna3 during early mouse development

Interaction of Eph receptor tyrosine kinases with their membrane bound ephrin ligands initiates bidirectional signaling events that regulate cell migratory and adhesive behavior. Whole-mount in situ hybridization revealed overlapping expression of the Epha1 receptor and its high-affinity ligands ephrin A1 (Efna1) and ephrin A3 (Efna3) in the primitive streak and the posterior paraxial mesoderm during early mouse development. These results show complex and dynamic expression for all three genes with expression domains that are successively complementary, overlapping, and divergent. (c) 2006 Elsevier B.V. All rights reserved.

Plexin B2 in mouse and zebrafish development

Plexins (plxn) are receptors of semaphorins (sema), which were originally characterized as axon guidance cues. Semaphorin-plexin signalling has now been implicated in many other developmental and pathological processes. In this thesis, my first aim was to study the expression of plexins during mouse development. My second aim was to study the function of Plexin B2 in the development of the kidney. Thirdly, my objective was to elucidate the evolutionary conservation of Plexin B2 by investigating its sequence, expression and function in developing zebrafish. I show by in situ hybridisation that plexins are widely expressed also in the non-neuronal tissues during mouse development. Plxnb1 and Plxnb2, for example, are expressed also in the ureteric epithelium, developing glomeruli and undifferentiated metanephric mesenchyme of the developing kidney. Plexin B2-deficient (Plxnb2-/-) mice die before birth and have severe defects in the nervous system. I demonstrate that they develop morphologically normal but hypoplastic kidneys. The ureteric epithelium of Plxnb2-/- kidneys has fewer branches and a lower rate of proliferating cells. 10% of the embryos show unilateral double ureters and kidneys. The defect in the branching is intrinsic to the epithelium as the isolated ureteric epithelium grown in vitro fails to respond to Glial-cell-line-derived neurotrophic factor (Gdnf). We prove by co-immunoprecipitation that Plexin B2 interacts with the Gdnf-receptor Ret. Sema4C, the Plexin B2 ligand, increases branching of the ureteric epithelium in controls but not in Plxnb2-/- kidney explants. These results suggest that Sema4C-Plexin B2 signalling modulates ureteric branching in a positive manner, possibly through directly regulating the activation of Ret. I cloned the zebrafish orthologs of Plexin B2, Plexin B2a and B2b. The corresponding proteins contain the conserved domains the B-subfamily plexins. Especially the expression pattern of plxnb2b recapitulates many aspects of the expression pattern of Plxnb2 in mouse. Plxnb2a and plxnb2b are expressed, for example, in the pectoral fins and at the midbrain-hindbrain region during zebrafish development. The nearly complete knockdown of Plexin B2a alone or together with the 45% knockdown of Plexin B2b did not interfere with the normal development of the zebrafish. In conclusion, my thesis reveals that plexins are broadly expressed during mouse embryogenesis. It also shows that Sema4C-Plexin B2 signalling modulates the branching of the ureteric epithelium during kidney development, perhaps through a direct interaction with Ret. Finally, I show that the sequence and expression of Plexin B2a and B2b are conserved in zebrafish. Their knockdown does not, however, result in the exencephaly phenotype of Plxnb2-/- mice.

Targeted Deletion Of Functionally Validated Enhancers Defines Their Role in Mouse Limb Development

Transcriptional enhancers are genomic DNA sequences that contain clustered transcription factor (TF) binding sites. When combinations of TFs bind to enhancer sequences they act together with basal transcriptional machinery to regulate the timing, location and quantity of gene transcription. Elucidating the genetic mechanisms responsible for differential gene expression, including the role of enhancers, during embryological and postnatal development is essential to an understanding of evolutionary processes and disease etiology. Numerous methods are in use to identify and characterize enhancers. Several high-throughput methods generate large datasets of enhancer sequences with putative roles in embryonic development. However, few enhancers have been deleted from the genome to determine their roles in the development of specific structures, such as the limb. Manipulation of enhancers at their endogenous loci, such as the deletion of such elements, leads to a better understanding of the regulatory interactions, rules and complexities that contribute to faithful and variant gene transcription – the molecular genetic substrate of evolution and disease. To understand the endogenous roles of two distinct enhancers known to be active in the mouse embryo limb bud we deleted them from the mouse genome. I hypothesized that deletion of these enhancers would lead to aberrant limb development. The enhancers were selected because of their association with p300, a protein associated with active transcription, and because the human enhancer sequences drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. To confirm that the orthologous mouse enhancers, mouse 280 and 1442 (M280 and M1442, respectively), regulate expression in the developing limb we generated stable transgenic lines, and examined lacZ expression. In M280-lacZ mice, expression was detected in E11.5 fore- and hindlimbs in a region that corresponds to digits II-IV. M1442-lacZ mice exhibited lacZ expression in posterior and anterior margins of the fore- and hindlimbs that overlapped with digits I and V and several wrist bones. We generated mice lacking the M280 and M1442 enhancers by gene targeting. Intercrosses between M280 -/+ and M1442 -/+, respectively, generated M280 and M1442 null mice, which are born at expected Mendelian ratios and manifest no gross limb malformations. Quantitative real-time PCR of mutant E11.5 limb buds indicated that significant changes in transcriptional output of enhancer-proximal genes accompanied the deletion of both M280 and M1442. In neonatal null mice we observed that all limb bones are present in their expected positions, an observation also confirmed by histology of E18.5 distal limbs. Fine-scale measurement of E18.5 digit bone lengths found no differences between mutant and control embryos. Furthermore, when the developmental progression of cartilaginous elements was analyzed in M280 and M1442 embryos from E13.5-E15.5, transient development defects were not detected. These results demonstrate that M280 and M1442 are not required for mouse limb development. Though M280 is not required for embryonic limb development it is required for the development and/or maintenance of body size – adult M280 mice are significantly smaller than control littermates. These studies highlight the importance of experiments that manipulate enhancers in situ to understand their contribution to development.