Characterization of murine BATF: a negative regulator of activator protein-1 activity in the thymus.

BATF belongs to the AP-1/ATF superfamily of transcription factors and forms heterodimers with Jun proteins to bind AP-1 consensus DNA. Unlike Fos/Jun heterodimers which stimulate gene transcription, BATF/Jun heterodimers are transcriptionally inert and inhibit biological processes that are associated with the overstimulation of AP-1 activity. Here, we describe the murine BATF cDNA and genomic clones and map the BATF locus to chromosome 12 D2-3. Using in situ hybridization of BATF mRNA, we show that BATF gene expression is highly restricted, with the most prominent signals detected in the thymus. BATF mRNA levels are regulated differentially during discrete stages of T cell development and are up-regulated following activation of T cells in the periphery. To demonstrate the impact of BATF on AP-1 activity in vivo, AP-1 luciferase reporter mice were crossed to transgenic mice overexpressing BATF exclusively in thymic T cells. Results show that elevated levels of BATF protein correlate with reduced transactivation by AP-1. Since the differential regulation of AP-1 activity is linked to key transitions in the developing immune system, our observations support a critical role for BATF in determining the overall level of AP-1 activity, and thus AP-1 target gene expression, in specific T cell subtypes.

Down syndrome congenital heart disease: a narrowed region and a candidate gene.

PURPOSE: Down syndrome (DS) is a major cause of congenital heart disease (CHD) and the most frequent known cause of atrioventricular septal defects (AVSDs). Molecular studies of rare individuals with CHD and partial duplications of chromosome 21 established a candidate region that included D21S55 through the telomere. We now report human molecular and cardiac data that narrow the DS-CHD region, excluding two candidate regions, and propose DSCAM (Down syndrome cell adhesion molecule) as a candidate gene. METHODS: A panel of 19 individuals with partial trisomy 21 was evaluated using quantitative Southern blot dosage analysis and fluorescence in situ hybridization (FISH) with subsets of 32 BACs spanning the region defined by D21S16 (21q11.2) through the telomere. These BACs span the molecular markers D21S55, ERG, ETS2, MX1/2, collagen XVIII and collagen VI A1/A2. Fourteen individuals are duplicated for the candidate region, of whom eight (57%) have the characteristic spectrum of DS-CHD. RESULTS: Combining the results from these eight individuals suggests the candidate region for DS-CHD is demarcated by D21S3 (defined by ventricular septal defect), through PFKL (defined by tetralogy of Fallot). CONCLUSIONS: These data suggest that the presence of three copies of gene(s) from the region is sufficient for the production of subsets of DS-CHD. This region does not include genes located near D21S55, previously proposed as a "DS critical region," or the genes encoding collagens VI and XVIII. Of the potential gene candidates in the narrowed DS-CHD region, DSCAM is notable in that it encodes a cell adhesion molecule, spans more than 840 kb of the candidate region, and is expressed in the heart during cardiac development. Given these properties, we propose DSCAM as a candidate for DS-CHD.

In situ hybridization: use of 35S-labeled probes on paraffin tissue sections.

The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with (35)S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided.

Down syndrome cell adhesion molecule is conserved in mouse and highly expressed in the adult mouse brain.

Down Syndrome (DS) is a major cause of mental retardation and is associated with characteristic well-defined although subtle brain abnormalities, many of which arise after birth, with particular defects in the cortex, hippocampus and cerebellum. The neural cell adhesion molecule DSCAM (Down syndrome cell adhesion molecule) maps to 21q22.2-->q22.3, a region associated with DS mental retardation, and is expressed largely in the neurons of the central and peripheral nervous systems during development. In order to evaluate the contribution of DSCAM to postnatal morphogenetic and cognitive processes, we have analyzed the expression of the mouse DSCAM homolog, Dscam, in the adult mouse brain from 1 through 21 months of age. We have found that Dscam is widely expressed in the brain throughout adult life, with strongest levels in the cortex, the mitral and granular layers of the olfactory bulb, the granule cells of the dentate gyrus and the pyramidal cells of the CA1, CA2 and CA3 regions, the ventroposterior lateral nuclei of the thalamus, and in the Purkinje cells of the cerebellum. Dscam is also expressed ventrally in the adult spinal cord. Given the homology of DSCAM to cell adhesion molecules involved in development and synaptic plasticity, and its demonstrated role in axon guidance, we propose that DSCAM overexpression contributes not only to the structural defects seen in these regions of the DS brain, but also to the defects of learning and memory seen in adults with DS.

Telokin expression is restricted to smooth muscle tissues during mouse development.

Telokin is a 17-kDa protein with an amino acid sequence that is identical to the COOH terminus of the 130-kDa myosin light chain kinase (MLCK). Telokin mRNA is transcribed from a second promoter, located within an intron, in the 3' region of the MLCK gene. In the current study, we show by in situ mRNA hybridization that telokin mRNA is restricted to the smooth muscle cell layers within adult smooth muscle tissues. In situ mRNA analysis of mouse embryos also revealed that telokin expression is restricted to smooth muscle tissues during embryonic development. Telokin mRNA expression was first detected in mouse gut at embryonic day 11.5; no telokin expression was detected in embryonic cardiac or skeletal muscle. Expression of telokin was also found to be regulated during postnatal development of the male and female reproductive tracts. In both uterus and vas deferens, telokin protein expression greatly increased between days 7 and 14 of postnatal development. The increase in telokin expression correlated with an increase in the expression of several other smooth muscle-restricted proteins, including smooth muscle myosin and alpha-actin.

Transcriptional regulation of S100A1 and expression during mouse heart development.

S100A1, a member of the large EF-hand family of Ca(2+)-binding proteins, is mainly expressed in the mammalian heart. To assess the underlying mechanisms for cell- and tissue-specific expression we isolated and characterized the mouse S100A1 gene. The gene displays a high degree of homology to the human and rat genes, especially in the exonic sequences. In its promoter region and the first intron, we identified regulatory elements characteristic for cardiac and slow skeletal muscle restricted genes. Transfection assays with luciferase constructs containing different parts of the S100A1 gene demonstrated the active expression in primary mouse cardiomyocytes and that its 5'-upstream region containing a putative cardiac enhancer showed a greatly increased activity. Furthermore, we investigated the expression of the S100A1 mRNA during embryonic mouse development, using in situ hybridization. S100A1 transcripts were first detected in the primitive heart at embryonic day (E) 8, with equal levels in the atrium and ventricle. During development up to E17.5 we detected a shift in the S100A1 expression pattern with lower levels in atrial and high levels in ventricular myocardium. The regulatory elements identified in the mouse S100A1 promoter correspond well with the observed expression pattern and suggest that S100A1 has an important function during heart muscle development.

Jumonji, a nuclear protein that is necessary for normal heart development.

Jumonji (jmj) was cloned in a gene trap screen to identify and mutagenize genes important for heart development. To investigate the role of jmj in heart development, we generated mice homozygous for the jmj mutation. The jmj homozygous mouse embryos showed heart malformations, including ventricular septal defect, noncompaction of the ventricular wall, double-outlet right ventricle, and dilated atria. The jmj mutants died soon after birth, apparently as a result of respiratory insufficiency caused by rib and sternum defects in addition to the heart defects. In situ hybridization analyses suggested that cardiomyocytes were differentiated but developmental regulation of chamber-specific genes was defective in fetal hearts. Expression of jmj was detected in the myocardium, especially in the interventricular septum, ventricular wall, and outflow tract, which correlated well with the locations of defects observed in the hearts of mutant mice. Homozygous embryos failed to express the jmj transcript in all tissues except in the nervous system. Confocal microscopic examination using anti-JMJ antibodies indicated that the JMJ protein was localized in the nuclei of cells transfected with jmj. These data demonstrate that JMJ is a nuclear protein, which is essential for normal heart development and function.

Filamin isogene expression during mouse myogenesis.

The developmental pattern of filamin gene expression has been studied in mouse embryos by using in situ hybridization. The probes used were isoform specific, (35)S-labeled antisense complementary ribonucleic acids (cRNAs) to the 3; untranslated region (3; UTR) of muscle-specific and nonmuscle-specific filamin genes. Northern blot and in situ hybridization results showed that nonmuscle-specific filamin transcripts had a size of 9.5 kb and were expressed in all nonmuscle tissues. Labeling was most intense in tissues containing a substantial proportion of epithelial and smooth muscle cells. Muscle-specific filamin transcripts had a size of 10 kb and were expressed primarily in cardiac and skeletal muscle. The expression of muscle-specific filamin messenger ribonucleicacids (mRNAs) was detected in heart at 8.0 days after coitum, whereas that in the myotomes of somites was not detected until 10.5 days after coitum. The expression of muscle-specific filamin mRNAs in heart and in skeletal muscle continued through the subsequent days of myogenesis. The results showed that muscle-specific filamin gene transcripts are detected before the formation of myotubes in vivo. This is the first study of filamin gene expression at the early stages of skeletal muscle development. Dev Dyn 2000;217:99-108.

Spatial and temporal changes in myosin heavy chain gene expression in skeletal muscle development.

Seven myosin heavy chains (MyHC) are expressed in mammalian skeletal muscle in spatially and temporally regulated patterns. The timing, distribution, and quantitation of MyHC expression during development and early postnatal life of the mouse are reported here. The three adult fast MyHC RNAs (IIa, IIb, and IId/x) are expressed in the mouse embryo and each mRNA has a distinct temporal and spatial distribution. In situ hybridization analysis demonstrates expression of IIb mRNA by 14.5 dpc, which proceeds developmentally in a rostral to caudal pattern. IId/x and IIa mRNAs are detectable 2 days later. Ribonuclease protection assays demonstrate that the three adult fast genes are expressed at approximately equal levels relative to each other in the embryo but at quite low levels relative to the two developmental isoforms, embryonic and perinatal. Just after birth major changes in the relative proportions of different MyHC RNAs and protein occur. In all cases, RNA expression and protein expression appear coincident. The changes in MyHC RNA and protein expression are distinct in different muscles and are restricted in some cases to particular regions of the muscle and do not always reflect their distribution in the adult.

Identification of NFI-binding sites and cloning of NFI-cDNAs suggest a regulatory role for NFI transcription factors in olfactory neuron gene expression.

Olfactory receptor neurons are responsible for the detection and signal transduction of odor ligands. Several genes associated with this activity are preferentially or exclusively expressed in these neurons. Among these genes are those coding for olfactory receptors, adenylyl cyclase type III, the cyclic nucleotide gated olfactory channel 1 (OcNC-1), Galpha(olf) and the olfactory marker protein (OMP). Promoter analyses of these genes identified a binding site for the new transcription factor family O/E whose initial member, Olf-1, is abundantly expressed in olfactory neurons. We report here that the proximal promoters of three of these genes, that are selectively expressed in olfactory neurons, each contains a functional NFI binding site and that the sites have different affinities for NFI proteins indicating a regulatory role for NFI proteins in olfactory gene expression. We further demonstrate, by cloning, that all four NFI genes are expressed in the olfactory nasal mucosa. Analysis by in situ hybridization illustrates that at least three of these gene products are expressed in the neuroepithelium in which the olfactory neurons reside. NFI proteins are capable of functioning as positive or negative regulators of transcription depending on the tissue, cell-type, age, and gene in question. These multivalent functions of NFI could be achieved by temporally and spatially regulated expression of distinct subsets of NFI isoforms. It now remains to characterize the tissue and cell specific patterns of expression of distinct NFI transcription factors during ontogeny and their roles in regulating gene expression.

Expression of the Nkx3.1 homobox gene during pre and postnatal development.

We report here the expression pattern of Nkx3.1, a murine homolog of Drosophila bagpipe, in different stages of embryos and in neonates. Nkx3.1 was expressed in paraxial mesoderm at day 7.5 p.c., in the somite by day 8.0 p.c. and in a subset of vascular smooth muscle cells by day 9.5 p.c. In later stage embryos and neonates, Nkx3.1 was expressed in subpopulations of epithelial cells, particularly in epithelial outgrowths. Nkx3.1 was also expressed in restricted regions of the central nervous system.

Mrj encodes a DnaJ-related co-chaperone that is essential for murine placental development.

We have identified a novel gene in a gene trap screen that encodes a protein related to the DnaJ co-chaperone in E. coli. The gene, named Mrj (mammalian relative of DnaJ) was expressed throughout development in both the embryo and placenta. Within the placenta, expression was particularly high in trophoblast giant cells but moderate levels were also observed in trophoblast cells of the chorion at embryonic day 8.5, and later in the labyrinth which arises from the attachment of the chorion to the allantois (a process called chorioallantoic fusion). Insertion of the ROSAbetageo gene trap vector into the Mrj gene created a null allele. Homozygous Mrj mutants died at mid-gestation due to a failure of chorioallantoic fusion at embryonic day 8.5, which precluded formation of the mature placenta. At embryonic day 8.5, the chorion in mutants was morphologically normal and expressed the cell adhesion molecule beta4 integrin that is known to be required for chorioallantoic fusion. However, expression of the chorionic trophoblast-specific transcription factor genes Err2 and Gcm1 was significantly reduced. The mutants showed no abnormal phenotypes in other trophoblast cell types or in the embryo proper. This study indicates a previously unsuspected role for chaperone proteins in placental development and represents the first genetic analysis of DnaJ-related protein function in higher eukaryotes. Based on a survey of EST databases representing different mouse tissues and embryonic stages, there are 40 or more DnaJ-related genes in mammals. In addition to Mrj, at least two of these genes are also expressed in the developing mouse placenta. The specificity of the developmental defect in Mrj mutants suggests that each of these genes may have unique tissue and cellular activities.

Characterization of myocyte enhancer factor 2 (MEF2) expression in B and T cells: MEF2C is a B cell-restricted transcription factor in lymphocytes.

Our studies examined the expression and DNA binding activity of myocyte enhancer factor 2 (MEF2A-D) transcription factors in lymphopoietic tissues, cell lines, and primary lymphocytes. Our analyses demonstrate that mef2C expression is restricted to B cells within the lymphocyte lineage. Using in situ hybridization, mef2C is detected in foci in fetal liver and postnatal thymic medulla, and both mef2B and mef2C are expressed in areas of the postnatal spleen and lymph node that also express kappa light chain (Ckappa), a B cell-specific marker. Reverse transcriptase-PCR (RT-PCR) analyses demonstrate that all mef2 family members are expressed in B cell lines, and all except mef2C are expressed in T cell lines. Immunoblot analyses of cell lines and primary thymic and splenic lymphocytes show that MEF2C and MEF2D proteins are expressed in B cells and that MEF2D is expressed in T cells; however, MEF2A protein is not detected in lymphocytes. Electrophoretic mobility shift assays (EMSA) demonstrate that B cell lines have MEF2C-containing, MEF2-specific DNA binding complexes whereas T cells do not. Our data is the first to describe mef2C expression in the lymphocyte lineage, and this finding suggests possible roles for MEF2C activity in B cell development and function.

Requirement of the MADS-box transcription factor MEF2C for vascular development.

The embryonic vasculature develops from endothelial cells that form a primitive vascular plexus which recruits smooth muscle cells to form the arterial and venous systems. The MADS-box transcription factor MEF2C is expressed in developing endothelial cells and smooth muscle cells (SMCs), as well as in surrounding mesenchyme, during embryogenesis. Targeted deletion of the mouse MEF2C gene resulted in severe vascular abnormalities and lethality in homozygous mutants by embryonic day 9.5. Endothelial cells were present and were able to differentiate, but failed to organize normally into a vascular plexus, and smooth muscle cells did not differentiate in MEF2C mutant embryos. These vascular defects resemble those in mice lacking the vascular-specific endothelial cell growth factor VEGF or its receptor Flt-1, both of which are expressed in MEF2C mutant embryos. These results reveal multiple roles for MEF2C in vascular development and suggest that MEF2-dependent target genes mediate endothelial cell organization and SMC differentiation.

Expression of the mitotic motor protein CHO1/MKLP1 in postmitotic neurons.

The kinesin-related motor protein CHO1/MKLP1 was initially thought to be expressed only in mitotic cells, where it presumably transports oppositely oriented microtubules relative to one another in the spindle mid-zone. We have recently shown that CHO1/MKLP1 is also expressed in cultured neuronal cells, where it is enriched in developing dendrites [Sharp et al. (1997a) J. Cell Biol., 138, 833-843]. The putative function of CHO1/MKLP1 in these postmitotic cells is to intercalate minus-end-distal microtubules among oppositely oriented microtubules within developing dendrites, thereby establishing their non-uniform microtubule polarity pattern. Here we used in situ hybridization to determine whether CHO1/MKLP1 is expressed in a variety of rodent neurons both in vivo and in vitro. These analyses revealed that CHO1/MKLP1 is expressed within various neuronal populations of the brain including those in the cerebral cortex, hippocampus, olfactory bulb and cerebellum. The messenger ribonucleic acid (mRNA) levels are high within these neurons well after the completion of their terminal mitotic division and throughout the development of their dendrites. After this, the levels decrease and are relatively low within the adult brain. Parallel analyses on developing hippocampal neurons in culture indicate that the levels of expression increase dramatically just prior to dendritic development, and then decrease somewhat after the dendrites have differentiated. Dorsal root ganglion neurons, which generate axons but not dendrites, express significantly lower levels of mRNA for CHO1/MKLP1 than hippocampal or sympathetic neurons. These results are consistent with the proposed role of CHO1/MKLP1 in establishing the dendritic microtubule array.

Expression of the mitotic motor protein Eg5 in postmitotic neurons: implications for neuronal development.

It is well established that the microtubules of the mitotic spindle are organized by a variety of motor proteins, and it appears that the same motors or closely related variants organize microtubules in the postmitotic neuron. Specifically, cytoplasmic dynein and the kinesin-related motor known as CHO1/MKLP1 are used within the mitotic spindle, and recent studies suggest that they are also essential for the establishment of the axonal and dendritic microtubule arrays of the neuron. Other motors are required to tightly regulate microtubule behaviors in the mitotic spindle, and it is attractive to speculate that these motors might also help to regulate microtubule behaviors in the neuron. Here we show that a homolog of the mitotic kinesin-related motor known as Eg5 continues to be expressed in rodent neurons well after their terminal mitotic division. In neurons, Eg5 is directly associated with the microtubule array and is enriched within the distal regions of developing processes. This distal enrichment is transient, and typically lost after a process has been clearly defined as an axon or a dendrite. Strong expression can resume later in development, and if so, the protein concentrates within newly forming sprouts at the distal tips of dendrites. We suggest that Eg5 generates forces that help to regulate microtubule behaviors within the distal tips of developing axons and dendrites.

Vertebrate homologs of tinman and bagpipe: roles of the homeobox genes in cardiovascular development.

In Drosophila, dorsal mesodermal specification is regulated by the homeobox genes tinman and bagpipe. Vertebrate homologs of tinman and bagpipe have been isolated in various species. Moreover, there are at least four different genes related to tinman in the vertebrate, which indicates that this gene has been duplicated during evolution. One of the murine homologs of tinman is the cardiac homeobox gene Csx or Nkx2.5. Gene targeting of Csx/Nkx2.5 showed that this gene is required for completion of the looping morphogenesis of the heart. However, it is not essential for the specification of the heart cell lineage. Early cardiac development might therefore be regulated by other genes, which may act either independently or in concert with Csx/Nkx2.5. Possible candidates might be other members of the NK2 class of homeobox proteins like Tix/Nkx2.6, Nkx2.3, nkx2.7, or cNkx2.8. Murine Tix/Nkx2.6 mRNA has been detected in the heart and pharyngeal endoderm (this study). Xenopus XNkx2.3 and chicken cNkx2.3 are expressed in the heart as well as in pharyngeal and gut endoderm. In contrast, murine Nkx2.3 is expressed in the gut and pharyngeal arches but not the heart. In zebrafish and chicken, two new NK-2 class homeoproteins, nkx2.7 and cNkx2.8, have been identified. Zebrafish nkx2.7 is expressed in both, the heart and pharyngeal endoderm. In the chicken, cNkx2.8 is expressed in the heart primordia and the primitive heart tube and becomes undetectable after looping. No murine homologs of nkx2.7 or cNkx2.8 have been found so far. The overlapping expression pattern of NK2 class homeobox genes in the heart and the pharynx may suggest a common origin of these two organs. In the Drosophila genome, the tinman gene is linked to another NK family gene named bagpipe. A murine homolog of bagpipe, Bax/Nkx3.1, is expressed in somites, blood vessels, and the male reproductive system during embryogenesis (this study), suggesting that this gene's function may be relevant for the development of these organs. A bagpipe homolog in Xenopus, Xbap, is expressed in the gut masculature and a region of the facial cartilage during development. In this paper, we discuss molecular mechanisms of cardiovascular development with particular emphasis on roles of transcription factors.

Akt2 mRNA is highly expressed in embryonic brown fat and the AKT2 kinase is activated by insulin.

Akt2 encodes a protein-serine/threonine kinase containing a pleckstrin homology domain characteristic of many signaling molecules. Although there has been extensive interest in the mechanism by which the closely-related Akt kinase participates in phosphatidylinositol 3-kinase-mediated signaling, comparatively little is known regarding the expression and function of Akt2. This manuscript is the first to describe Akt2 mRNA expression in the developing mouse and the activation of AKT2 by insulin. These studies demonstrate that Akt2 is especially abundant in brown fat and, to a lesser extent, skeletal muscle and liver, tissues which are highly insulin-responsive and play a role in glucose metabolism. Endogenous Akt2 expression also is upregulated in fully-differentiated C2C12 myotubes and 3T3-L1 adipocytes, suggesting that these murine cell lines represent useful in vitro models for studies of Akt2 function. We show that HA-tagged AKT2 is activated in response to insulin stimulation in vitro and that activation of AKT2 is not induced in cells pretreated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase. These data suggest that Akt2 expression is fundamental to the differentiated state of fat and muscle cells and that activation of AKT2 kinase by insulin is mediated through the phosphatidylinositol 3-kinase signaling pathway.

DSCAM: a novel member of the immunoglobulin superfamily maps in a Down syndrome region and is involved in the development of the nervous system.

Down syndrome (DS), a major cause of mental retardation, is characterized by subtle abnormalities of cortical neuroanatomy, neurochemistry and function. Recent work has shown that chromosome band 21q22 is critical for many of the neurological phenotypes of DS. A gene, DSCAM (Down syndrome cell adhesion molecule), has now been isolated from chromosome band 21q22.2-22.3. Homology searches indicate that the putative DSCAM protein is a novel member of the immunoglobulin (Ig) superfamily that represents a new class of neural cell adhesion molecules. The sequence of cDNAs indicates alternative splicing and predicts two protein isoforms, both containing 10 Ig-C2 domains, with nine at the N-terminus and the tenth located between domains 4 and 5 of the following array of six fibronectin III domains, with or without the following transmembrane and intracellular domains. Northern analyses reveals the transcripts of 9.7, 8.5 and 7.6 kb primarily in brain. These transcripts are differentially expressed in substructures of the adult brain. Tissue in situ hybridization analyses of a mouse homolog of the DSCAM gene revealed broad expression within the nervous system at the time of neuronal differentiation in the neural tube, cortex, hippocampus, medulla, spinal cord and most neural crest-derived tissues. Given its location on chromosome 21, its specific expression in the central nervous system and neural crest, and the homologies to molecules involved in neural migration, differentiation, and synaptic function, we propose that DSCAM is involved in neural differentiation and contributes to the central and peripheral nervous system defects in DS.