Towards large scale automated cage monitoring - Diurnal rhythm and impact of interventions on in-cage activity of C57BL/6J mice recorded 24/7 with a non-disrupting capacitive-based technique.

BACKGROUND AND AIMS: Automated recording of laboratory animal's home cage behavior is receiving increasing attention since such non-intruding surveillance will aid in the unbiased understanding of animal cage behavior potentially improving animal experimental reproducibility. MATERIAL AND METHODS: Here we investigate activity of group held female C57BL/6J mice (mus musculus) housed in standard Individually Ventilated Cages across three test-sites: Consiglio Nazionale delle Ricerche (CNR, Rome, Italy), The Jackson Laboratory (JAX, Bar Harbor, USA) and Karolinska Insititutet (KI, Stockholm, Sweden). Additionally, comparison of female and male C57BL/6J mice was done at KI. Activity was recorded using a capacitive-based sensor placed non-intrusively on the cage rack under the home cage collecting activity data every 250 msec, 24/7. The data collection was analyzed using non-parametric analysis of variance for longitudinal data comparing sites, weekdays and sex. RESULTS: The system detected an increase in activity preceding and peaking around lights-on followed by a decrease to a rest pattern. At lights off, activity increased substantially displaying a distinct temporal variation across this period. We also documented impact on mouse activity that standard animal handling procedures have, e.g. cage-changes, and show that such procedures are stressors impacting in-cage activity. These key observations replicated across the three test-sites, however, it is also clear that, apparently minor local environmental differences generate significant behavioral variances between the sites and within sites across weeks. Comparison of gender revealed differences in activity in the response to cage-change lasting for days in male but not female mice; and apparently also impacting the response to other events such as lights-on in males. Females but not males showed a larger tendency for week-to-week variance in activity possibly reflecting estrous cycling. CONCLUSIONS: These data demonstrate that home cage monitoring is scalable and run in real time, providing complementary information for animal welfare measures, experimental design and phenotype characterization.

A radiation hybrid transcript map of the mouse genome.

Expressed-sequence tag (EST) maps are an adjunct to sequence-based analytical methods of gene detection and localization for those species for which such data are available, and provide anchors for high-density homology and orthology mapping in species for which large-scale sequencing has yet to be done. Species for which radiation hybrid-based transcript maps have been established include human, rat, mouse, dog, cat and zebrafish. We have established a comprehensive first-generation-placement radiation hybrid map of the mouse consisting of 5,904 mapped markers (3,993 ESTs and 1,911 sequence-tagged sites (STSs)). The mapped ESTs, which often originate from small-EST clusters, are enriched for genes expressed during early mouse embryogenesis and are probably different from those localized in humans. We have confirmed by in situ hybridization that even singleton ESTs, which are usually not retained for mapping studies, may represent bona fide transcribed sequences. Our studies on mouse chromosomes 12 and 14 orthologous to human chromosome 14 show the power of our radiation hybrid map as a predictive tool for orthology mapping in humans.

Embryonic stem cell development in a chemically defined medium.

Vertebrate germ layer development is an intricately interwoven process with the organism operating as an integrated whole. To examine these processes we have used embryonic stem (ES) cell in vitro differentiation in a serum-free, chemically defined medium (CDM). In CDM, ES cells differentiate as embryoid bodies to neuroectoderm with upregulation of pax-6, without commensurate expression of Brachyury. In the presence of Activin A, pax-6 and Brachyury mRNAs are readily detectable, suggestive of both neuroectoderm and mesoderm formation, while in the presence of BMP-4 a process resembling primitive streak formation at the molecular level occurs. Neuroectoderm development in CDM alone is consistent with the view that this process can occur by default, as reported in Xenopus, due to the absence or sequestration of mesoderm-inducing factors. Additionally, these data show that BMP-4 alone is capable of instigating a process resembling primitive streak formation in ES cells and possibly in vivo.

Interleukin 6 influences germinal center development and antibody production via a contribution of C3 complement component.

Mice rendered deficient for interleukin (IL) 6 by gene targeting were evaluated for their response to T cell-dependent antigens. Antigen-specific immunoglobulin (Ig)M levels were unaffected whereas all IgG isotypes showed varying degrees of alteration. Germinal center reactions occurred but remained physically smaller in comparison to those in the wild-type mice. This concurred with the observations that molecules involved in initial signaling events leading to germinal center formation were not altered (e.g., B7.2, CD40 and tumor necrosis factor R1). T cell priming was not impaired nor was a gross imbalance of T helper cell (Th) 1 versus Th2 cytokines observed. However, B7.1 molecules, absent from wild-type counterparts, were detected on germinal center B cells isolated from the deficient mice suggesting a modification of costimulatory signaling. A second alteration involved impaired de novo synthesis of C3 both in serum and germinal center cells from IL-6-deficient mice. Indeed, C3 provided an essential stimulatory signal for wild-type germinal center cells as both monoclonal antibodies that interrupted C3-CD21 interactions and sheep anti-mouse C3 antibodies caused a significant decrease in antigen-specific antibody production. In addition, germinal center cells isolated from C3-deficient mice produced a similar defect in isotype production. Low density cells with dendritic morphology were the local source of IL-6 and not the germinal center lymphocytes. Adding IL-6 in vitro to IL-6-deficient germinal center cells stimulated cell cycle progression and increased levels of antibody production. These findings reveal that the germinal center produces and uses molecules of the innate immune system, evolutionarily pirating them in order to optimally generate high affinity antibody responses.

Analysis of factors controlling primary germ layer formation and early hematopoiesis using embryonic stem cell in vitro differentiation.

Differentiation and subsequent development are intricately interwoven processes operating as an integrated whole to form the organism. As an approach to examine these interactions in early mammalian development, we used embryonic stem (ES) cell in vitro differentiation. ES cells can, depending upon the environment differentiated to neuroectoderm, mesoderm and hematopoietic cells. We developed a serum-free, chemically defined medium (CDM) in which ES cells survive and differentiate. In CDM, in the absence of exogenous factors, ES cells form neuroectoderm, upregulating the early neural marker Pax-6. This is consistent with the view that neuroectoderm development can represent a default state, where the absence or sequestration of mesoderm inducing factors permits neuroectoderm formation. In contrast, if CDM is supplemented with bone morphogenetic protein (BMP) 2 or 4 a process resembling primitive streak formation, least at the molecular level occurs, with the formation of mesoderm and subsequently endothelial and hematopoietic cells. If used with care, ES cell in vitro differentiation can act as a guide in understanding the environment which controls early differentiation events in mammals.

Transforming growth factor-beta 3 is required for secondary palate fusion.

Mice lacking TGF-beta 3 exhibit an incompletely penetrant failure of the palatal shelves to fuse leading to cleft palate. The defect appears to result from impaired adhesion of the apposing medial edge epithelia of the palatal shelves and subsequent elimination of the mid-line epithelial seam. No craniofacial abnormalities were observed. This result demonstrates that TGF-beta 3 affects palatal shelf fusion by an intrinsic, primary mechanism rather than by effects secondary to craniofacial defects.

A new approach to the study of haematopoietic development in the yolk sac and embryoid bodies.

To understand the mechanisms that control the differentiation of uncommitted mesoderm precursors into haematopoietic stem cells (HSCs) and the activation of haematopoiesis, we conducted a study to identify genes expressed at the earliest stages of both in vivo and in vitro haematopoietic development. Our strategy was to utilize Differential Display by means of the Polymerase Chain Reaction (DD-PCR) to compare patterns of gene expression between mRNA populations representing different levels of haematopoietic activity obtained from the mouse embryo, embryoid bodies (EBs) and mouse cell lines. We report the molecular cloning of two groups of genes expressed in the yolk sac: a group of genes expressed in the day-8.5 yolk sac at higher levels than in the day-8.5 embryo proper and up-regulated during EB development, and another group of day-8.5 yolk sac genes not expressed in the day-8.5 embryo proper or in EBs. Specifically, we describe the molecular cloning of the first nucleobase permease gene to be found in vertebrates, yolk sac permease-like molecule 1 (Ysp11). The Ysp11 gene has the unique property of encoding both intracellular, transmembrane and extracellular protein forms, revealing novel aspects of nucleotide metabolism that may be relevant during mammalian development.

Alpha 6 integrins participate in pro-T cell homing to the thymus.

During embryogenesis, colonization of the thymic rudiment by hemopoietic progenitor cells depends on the adhesion of these cells to the jugular endothelium. Previously, we showed that progenitor T cells (pro-T cells) interact with alpha 6 integrins present on vascular endothelium. Here, we demonstrate that anti-alpha 6 integrin antibodies reduced the number of thymocytes up to 80% in a congenic mouse model for thymus colonization by pro-T cells. In organotypic thymus cultures, the anti-alpha 6 integrin antibodies did not influence T cell development and proliferation. From this, we conclude that alpha 6 integrin participates in thymus homing. During mouse thymus ontogeny, alpha 6 integrin mRNA and protein expression was found as early as day 10 of development; at day 11, perithymic endothelial cells were alpha 6 integrin positive. Two alpha 6 integrin mRNA exist which are produced by alternative exon usage. The longer form, alpha 6 integrin, predominates during early embryonic stages, while the shorter alpha 6A form was present later during development. Although alpha 6 integrins can be displayed by immature thymocytes, strongest expression was found on intra- and perithymic vascular endothelium. These data suggest that alpha 6 integrins are involved in the homing of pro-T cells to the developing thymus by mediating adhesion of pro-T cells to the vascular endothelium.

Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development.

Xenopus in vitro studies have implicated both transforming growth factor beta (TGF-beta) and fibroblast growth factor (FGF) families in mesoderm induction. Although members of both families are present during mouse mesoderm formation, there is little evidence for their functional role in mesoderm induction. We show that mouse embryonic stem cells, which resemble primitive ectoderm, can differentiate to mesoderm in vitro in a chemically defined medium (CDM) in the absence of fetal bovine serum. In CDM, this differentiation is responsive to TGF-beta family members in a concentration-dependent manner, with activin A mediating the formation of dorsoanterior-like mesoderm and bone morphogenetic protein 4 mediating the formation of ventral mesoderm, including hematopoietic precursors. These effects are not observed in CDM alone or when TGF-beta 1, -beta 2, or -beta 3, acid FGF, or basic FGF is added individually to CDM. In vivo, at day 6.5 of mouse development, activin beta A RNA is detectable in the decidua and bone morphogenetic protein 4 RNA is detectable in the egg cylinder. Together, our data strongly implicate the TGF-beta family in mammalian mesoderm development and hematopoietic cell formation.

An in vitro differentiation system for the examination of transgene activation in mouse macrophages.

In an effort to study basic principles of marker gene activation during myeloid lineage development, we established an in vitro differentiation system for macrophages based on mouse embryonic stem (ES) cells. Under the influence of defined cytokines, ES cells gave rise to a cell population consisting predominantly of macrophages. We could show, that expression of the mouse lysozyme M gene is a faithful internal standard for indicating the proportion of macrophage cells in the differentiation culture. This controlled in vitro differentiation system can be used for quantitative studies on transgene activation. Undifferentiated ES cells were stably transfected with a construct carrying the chicken lysozyme gene locus, which had been shown previously to express lysozyme RNA cell type specifically and position independently in macrophages of transgenic mice. In undifferentiated transfected ES cell clones, the transgene was consistently inactive. Upon in vitro differentiation, the transgene was expressed exclusively in macrophages and its level of activity was independent of the chromosomal site of integration. The in vitro cell differentiation system presented here will be useful to study the cis- and trans-regulatory requirements of myeloid-specific gene activation and the influence of hematopoietic regulators on myelopoiesis through their effect on transfected marker gene expression.

Far upstream regions of class II MHC Ea are necessary for position-independent, copy-dependent expression of Ea transgene.

The chromatin upstream of the class II MHC Ea gene contains specific, DNase I hypersensitive (DH) sites (groups I-V), overlapping and extending the promoter proximal and distal control regions. To determine whether the Ea DH groups I-V define a functionally important chromatin domain or locus control region (LCR), we have used wild type Ead gene constructs to generate transgenic mouse lines from strains that do not express an endogenous Ea gene product. Constructs contained either DH groups I-V 'Longs' or DH groups I-II 'Shorts', of the hypersensitive sites defined within 20 kb 5' of Ea. We show that position-independent, copy number-dependent expression of the Ead gene occurs only with the Long construct (8/8 transgenic mouse lines, over a range of copy numbers, 1-30 copies); in contrast, the Short constructs are subject to position-dependent effects. This suggests that the region delineated by Ea DH groups I-II is necessary but not sufficient as an LCR, which requires the presence of the upstream regions containing DH III-V for complete position-independent, copy number-dependent expression. These results introduce an immunologically-important, putative LCR which can be used to target genes to cells of the B cell lineage, as well as to other class II MHC expressing cells, and highlight the importance of chromatin structure analysis as a means to locate DNA regions of regulatory interest which are dispersed over a large distance.

Phylogeny of immunoglobulin heavy chain isotypes: structure of the constant region of Ambystoma mexicanum upsilon chain deduced from cDNA sequence.

An RNA polymerase chain reaction strategy was used to amplify and clone a cDNA segment encoding for the complete constant part of the axolotl IgY heavy (C upsilon) chain. C upsilon is 433 amino acids long and organized into four domains (C upsilon 1-C upsilon 4); each has the typical internal disulfide bond and invariant tryptophane residues. Axolotl C upsilon is most closely related to Xenopus C upsilon (40% identical amino acid residues) and C upsilon 1 shares 46.4% amino acid residues among these species. The presence of additional cysteines in C upsilon 1 and C upsilon 2 domains is consistent with an additional intradomain S-S bond similar to that suggested for Xenopus C upsilon and C chi, and for the avian C upsilon and the human C epsilon. C upsilon 4 ends with the Gly-Lys dipeptide characteristic of secreted mammalian C gamma 3, human C epsilon 4, and avian and anuran C upsilon 4, and contains the consensus [G/GT(AA)] nucleotide splice signal sequence for joining C upsilon 4 to the transmembrane region. These results are consistent with the hypothesis of an ancestral structural relationship between amphibian, avian upsilon chains, and mammalian epsilon chains. However, these molecules have different biological properties: axolotl IgY is secretory Ig, anuran and avian IgY behave like mammalian IgG, and mammalian IgE is implicated in anaphylactic reactions.

Hematopoietic commitment during embryonic stem cell differentiation in culture.

We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation, and by day 6, more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction profile of marker genes for primitive endoderm (collagen alpha IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1, GATA-3, and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis, indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors, including interleukin-3 (IL-3), IL-1, IL-6, IL-11, erythropoietin, and Kit ligand. At days 10 and 14 of differentiation, EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and IL-11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first, approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next, and precursors of the mast cell lineage develop last. The kinetics of precursor development, as well as the growth factor responsiveness of these early cells, is similar to that found in the yolk sac and early fetal liver, indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo.

Evolution of vertebrate IgM: complete amino acid sequence of the constant region of Ambystoma mexicanum mu chain deduced from cDNA sequence.

cDNA clones coding for the constant region of the Mexican axolotl (Ambystoma mexicanum) mu heavy immunoglobulin chain were selected from total spleen RNA, using a cDNA polymerase chain reaction technique. The specific 5'-end primer was an oligonucleotide homologous to the JH segment of Xenopus laevis mu chain. One of the clones, JHA/3, corresponded to the complete constant region of the axolotl mu chain, consisting of a 1362-nucleotide sequence coding for a polypeptide of 454 amino acids followed in 3' direction by a 179-nucleotide untranslated region and a polyA+ tail. The axolotl C mu is divided into four typical domains (C mu 1-C mu 4) and can be aligned with the Xenopus C mu with an overall identity of 56% at the nucleotide level. Percent identities were particularly high between C mu 1 (59%) and C mu 4 (71%). The C-terminal 20-amino acid segment which constitutes the secretory part of the mu chain is strongly homologous to the equivalent sequences of chondrichthyans and of other tetrapods, including a conserved N-linked oligosaccharide, the penultimate cysteine and the C-terminal lysine. The four C mu domains of 13 vertebrate species ranging from chondrichthyans to mammals were aligned and compared at the amino acid level. The significant number of mu-specific residues which are conserved into each of the four C mu domains argues for a continuous line of evolution of the vertebrate mu chain. This notion was confirmed by the ability to reconstitute a consistent vertebrate evolution tree based on the phylogenic parsimony analysis of the C mu 4 sequences.

Rescue of erythroid development in gene targeted GATA-1- mouse embryonic stem cells.

Development of definitive (fetal liver-derived) red cells is blocked by a targeted mutation in the gene encoding the transcription factor GATA-1. We used in vitro differentiation of GATA-1- mouse embryonic stem (ES) cells to reveal a requirement for GATA-1 during primitive (yolk sac-derived) erythropoiesis and to establish a rescue assay. We show that the block to development includes primitive, as well as definitive, erythroid cells and is complete at the level of globin RNA expression; that the introduction of a normal GATA-1 gene restores developmental potential both in vivo and in vitro; and that efficient rescue is dependent on a putative autoregulatory GATA-motif in the distal promoter. Use of in vitro differentiated ES cells bridges a gap between conventional approaches to gene function in cell lines and analysis of loss of function mutations in the whole animal.

Interleukin-7 expression during mouse thymus development.

We have monitored the expression of interleukin-7 (IL-7) in the developing embryonic mouse thymus by a combination of quantitative polymerase chain reaction (PCR) and immunofluorescence microscopy. A strong specific signal for IL-7 mRNA was detected by day 12 in the developing fetal thymus. IL-7 mRNA was found to be maximally expressed on day 15, and then decreased over the next 5 days. Immunofluorescence staining of fetal thymus sections using an anti-IL-7 antibody confirmed these PCR data. IL-7 protein expression was first detected at day 13 of development. At 14 days the intensity of the staining increased by a factor of three and stayed at this level over the next 4 days. The same anti-IL-7 antibody used for immunofluorescence, blocked the proliferation of fetal thymocytes in organotypic cultures. In addition, we detected mRNA coding for IL-2 and SCF (also known as the steel factor or KL) in embryonic thymocytes. The implications of these findings for early thymocyte growth are discussed.

Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture.

When embryonic stem cells are cultured directly in semisolid media (methyl cellulose), they proliferate and differentiate to generate colonies known as embryoid bodies (EBs). These EBs consist of differentiated cells from a number of lineages including those of the hematopoietic system. Following 10 days of culture in the presence of 10% fetal calf serum, more than 40% of all EBs from three different ES cell lines, CCEG2, D3 and SQ1.2S8 contained visible erythropoietic cells (i.e. red with hemoglobin). Beta H1 (z globin) mRNA is detectable in EBs within 5 days of differentiation, whilst beta(maj)-globin RNA appears by day 6. In the presence of erythropoietin (Epo), the frequency of EBs with erythropoietic activity increases to greater than 60%; Epo also prolongs this erythropoietic activity. Interleukin-3 (IL-3) does not significantly increase the frequency of EBs that contain erythroid cells, but increases slightly the number of erythropoietic cells associated with them. In the presence of IL-3, in addition to cells of the erythroid lineage, macrophages, mast cells and in some instances neutrophils are found within differentiating EBs. The development of macrophages is significantly enhanced by the addition of IL-3 alone or in combination with IL-1 and M-CSF or GM-CSF. When well-differentiated EBs are allowed to attach onto tissue-culture plates and grown in the presence of IL-3, a long-term output of cells from the mast cell lineage is observed.(ABSTRACT TRUNCATED AT 250 WORDS)