Developmental Biology 266 (2004) 346–360 www.elsevier.com/locate/ydbio
DSCR1 gene expression is dependent on NFATc1 during cardiac valve formation and colocalizes with anomalous organ development in trisomy 16 mice
Alexander W. Lange, Jeffery D. Molkentin, and Katherine E. Yutzey*
Division of Molecular Cardiovascular Biology, Children’s Medical Center Cincinnati ML 7020, Cincinnati, OH 45229, USA
Received for publication 8 May 2003, revised 9 October 2003, accepted 30 October 2003
Abstract
The Down syndrome critical region 1 (DSCR1) gene is present in the region of human chromosome 21 and the syntenic region of mouse chromosome 16, trisomy of which is associated with congenital heart defects observed in Down syndrome. DSCR1 encodes a regulatory protein in the calcineurin/NFAT signal transduction pathway. During valvuloseptal development in the heart, DSCR1 is expressed in the endocardium of the developing atrioventricular and semilunar valves, the muscular interventricular septum, and the ventricular myocardium. Human DSCR1 contains an NFAT-rich calcineurin-responsive element adjacent to exon 4. Transgenic mice generated with a homologous regulatory region of the mouse DSCR1 gene linked to lacZ (DSCR1(e4)/lacZ) show gene activation in the endocardium of the developing valves and aorticopulmonary septum of the heart, recapitulating a specific subdomain of endogenous DSCR1 cardiac expression. DSCR1(e4)/ lacZ expression in the developing valve endocardium colocalizes with NFATc1 and, endocardial DSCR1(e4)/lacZ, is notably reduced or absent in NFATc1À/À embryos. Furthermore, expression of the endogenous DSCR1(e4) isoform is decreased in the outflow tract of NFATc1À/À hearts, and the DSCR1(e4) intragenic element is trans-activated by NFATc1 in cell culture. In trisomy 16 (Ts16) mice, expression of endogenous DSCR1 and DSCR1(e4)/lacZ colocalizes with anomalous valvuloseptal development, and transgenic Ts16 hearts have increased h- galactosidase activity. DSCR1 and DSCR1(e4)/lacZ also are expressed in other organ systems affected by trisomy 16 in mice or trisomy 21 in humans including the brain, eye, ear, face, and limbs. Together, these results show that DSCR1(e4) expression in the developing valve endocardium is dependent on NFATc1 and support a role for DSCR1 in normal cardiac valvuloseptal formation as well as the abnormal development of several organ systems affected in individuals with Down syndrome. D 2003 Elsevier Inc. All rights reserved.
Keywords: DSCR1; MCIP1; ADAPT78; NFATc1; Trisomy 16 mice; Congenital heart defects; Down syndrome
Introduction (MCIP1), ADAPT78, or calcipressin, encodes a regulatory protein in the calcineurin/NFAT signal transduction path- The Down Syndrome Critical Region 1 (DSCR1) gene, way that binds to calcineurin to regulate its phosphatase located at position 21q22.12 on human chromosome 21, is activity (Ermak et al., 2001; Fuentes et al., 2000; Roth- in the genetic interval associated with Down syndrome ermel et al., 2000). The human DSCR1 gene has four congenital heart defects (Fuentes et al., 1995; Korenberg et alternative first exons and multiple splice variants (Ermak al., 1994). The mouse DSCR1 gene is in a syntenic region et al., 2002). An intragenic promoter adjacent to exon 4, of murine chromosome 16 and is expressed in the devel- which is thought to regulate expression of the DSCR1(e4) oping mouse heart, brain, and central nervous system splice isoform, is responsive to increased calcineurin (Casas et al., 2001; Epstein et al., 1985; Fuentes et al., signaling, thereby creating a potential feedback regulatory 1997; Mural et al., 2002; Vega et al., 2003). DSCR1, also loop (Yang et al., 2000). Calcineurin signaling through the known as modulatory calcineurin-interacting protein 1 NFAT family of transcription factors regulates gene ex- pression in response to elevated Ca2+ levels in the heart, brain, and immune systems (Crabtree and Olson, 2002; * Corresponding author. Division of Molecular Cardiovascular Biol- ogy, Children’s Medical Center Cincinnati ML 7020, 3333 Burnet Avenue, Molkentin et al., 1998). Evidence for the importance of the Cincinnati, OH 45229. Fax: +1-513-636-5958. calcineurin/NFAT signaling pathway in embryonic heart E-mail address: [email protected] (K.E. Yutzey). development is provided by mice deficient in NFATc1
0012-1606/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2003.10.036 A.W. Lange et al. / Developmental Biology 266 (2004) 346–360 347 which die in utero due to valvuloseptal defects (de la Materials and methods Pompa et al., 1998; Ranger et al., 1998). Although initial endocardial cushion formation occurs in NFATc1À/À em- Isolation of the mouse DSCR1 intragenic exon 4 (e4) bryos, mature semilunar valves are absent and develop- regulatory region and generation of DSCR1(e4)/lacZ ment of the AV valves and interventricular septum is transgenic mice affected (de la Pompa et al., 1998; Ranger et al., 1998). Still, the molecular mechanism(s) by which NFATc1 reg- DSCR1 exon 4 upstream sequence (À696 to À56) was ulates valvuloseptal development and its relationship to amplified from mouse genomic DNA by PCR using primers other calcineurin regulatory proteins including DSCR1 5V-GCCCTGCAACTACAGAAGG-3Vand 5V-GGCTT- remain undefined. GGCTTCCAGAGCGTCC-3V. The sequence was verified Mice trisomic for chromosome 16 (Ts16) are a murine and linked to the basal promoter hsp68/lacZ reporter gene model for Down syndrome and have a high incidence of (Kothary et al., 1989) to produce the DSCR1(e4)/lacZ trans- congenital heart defects as well as abnormalities in the brain, gene. The DSCR1(e4)/lacZ transgene was isolated away sensory organs, and skull (Hiltgen et al., 1996; Kola and from plasmid sequence and used for pronuclear microinjec- Hertzog, 1998; Ludwig et al., 1997; Miyabara et al., 1982; tions by the Cincinnati Children’s Transgenic Animal Core Webb et al., 1996). Ts16 mice exhibit high frequency of atrial Facility. Founder animals were identified by PCR amplifi- and ventricular septal defects at late gestation which arise cation of the lacZ transgene (Colbert et al., 1997). Three from malalignment of the heart and abnormal endocardial separate FVBN founder lines of DSCR1(e4)/lacZ transgenic cushion formation early in development (Hiltgen et al., 1996; mice with similar patterns of expression were used in the Webb et al., 1996, 1997, 1999). The high incidence of cardiac analyses. malformations in these mice supports the premise that valvu- loseptal development is affected by trisomy of genes on Genotyping and karyotyping chromosome 16 (Webb et al., 1997). Mouse orthologues have been identified for many of the approximately 200 DSCR1(e4)/lacZ transgenic embryos were identified by genes on human chromosome 21; however, the molecular PCR analysis of yolk sac DNA using primers that recognize mechanisms by which candidate genes contribute to the the lacZ transgene (Colbert et al., 1997). NFATc1 heterozy- cardiac malformations associated with mouse Ts16 or human gous mutant mice were obtained from Dr. Laurie Glimcher Ts21 have not been established (Gitton et al., 2002; Hattori et (Ranger et al., 1998). Genotyping for NFATc1 mutation was al., 2000; Reymond et al., 2002). performed by PCR using primers designed for the wild type The embryonic expression of mouse DSCR1 was ex- and targeted alleles as described in Ranger et al. (1998). amined during critical stages of valvuloseptal development. Ts16 embryos were generated by crossing Rb(6.16)24Lub/ Expression of the murine DSCR1(e4) NFAT-rich intragenic Rb(16.17)7BnrF1 males (obtained through the NIH/NICHD regulatory element was assessed during normal embryo- cytogenetic models resource maintained by Jackson Labs) genesis as well as in mice with cardiac valvuloseptal with nontransgenic FVBN or DSCR1(e4)/lacZ transgenic defects as a result of Ts16 or loss of NFATc1. Transgenic females. Ts16 embryos were identified by standard karyo- mice generated with the NFAT-rich DSCR1(e4) promoter type analysis of chromosomes isolated from embryonic yolk linked to a lacZ reporter gene show expression in the sacs taken before E13 or from liver cells of later stage developing valvuloseptal endocardium in addition to other embryos (Waller et al., 2000; Webb et al., 1996). Giemsa- structures affected in human Down syndrome. Expression stained metaphase spreads were examined for the presence of the DSCR1(e4)/lacZ transgene in the developing valves of metacentric chromosomes indicative of Robertsonian colocalizes with a subset of endocardial cells expressing translocations, and trisomic embryos were identified by NFATc1, and the mouse DSCR1(e4) intragenic promoter the presence of two metacentric chromosomes (Epstein element is activated by NFATc1 in cell culture. Loss of and Vekemans, 1990). NFATc1 in homozygous null gene targeted mice results in a marked reduction or complete absence of endocardial Analysis of transgene expression DSCR1(e4)/lacZ expression, as well as decreased expres- sion of the endogenous calcineurin-responsive DSCR1(e4) LacZ expression in E9.5–E14.5 embryos was analyzed transcript. Endogenous DSCR1 and DSCR1(e4)/lacZ trans- by X-gal detection of h-galactosidase (h-gal) activity as gene expression in Ts16 mice colocalizes with anomalous previously described (Searcy et al., 1998). h-gal activity valvuloseptal development in the heart and craniofacial was quantified in cell lysates of whole hearts from E13.5 and limb structures affected with trisomy 16 in mice or transgenic embryos. Dissected hearts were placed in 60 trisomy 21 in humans. Together, these studies support a Al of Tropix lysis buffer containing 0.5 mM DTT for regulatory role for NFATc1 and DSCR1 in normal valvu- sonication, and h-gal activity in lysates was determined as loseptal development and reinforce the association of previously described (Liberatore et al., 2002). For histol- DSCR1 with Down syndrome-related developmental ogy of X-gal stained embryos, individual hearts and heads anomalies. were infiltrated in sucrose and embedded in OCT (Stern, 348 A.W. Lange et al. / Developmental Biology 266 (2004) 346–360
1993). Cryostat sections (10–25 Am) were placed on Immunohistochemistry Superfrost/Plus slides and cover slips were mounted with 50% glycerol/PBS. Images were obtained using an Olym- Localized protein expression of NFATc1 and h-gal in pus BX60 microscope with a Spot 3.0.4 digital camera. developing hearts of DSCR1(e4)/lacZ transgenic embryos was examined by immunohistochemistry and confocal In situ hybridization microscopy. DSCR1(e4)/lacZ transgenic embryos were isolated at E10.5–E13.5, fixed overnight in 4% parafor- Embryos were isolated at E8.5–E13.5 and fixed over- maldehyde/PBS at 4jC, and processed for cryosectioning night at 4jC in 4% paraformaldehyde/PBS. For whole mount in situ hybridization, fixed embryos were dehy- drated in a graded methanol/PTW (PBS, 0.01% Tween-20) series and stored at À20jC in 100% methanol. Whole mount in situ hybridizations were performed as described by Wilkinson (1993) with reported modifications (Ehrman and Yutzey, 1999). E12.5–E14.5 hearts were bisected with a razor blade before hybridization to visualize the devel- oping valves and septa. ProteinaseK digestions were per- formed for 10–15 min and color reactions were incubated 2 h to overnight using NBT/BCIP (Roche). Embryos used for in situ hybridization of histological sections were stored in PBS at 4jC following fixation and subsequently infiltrated in sucrose and embedded in OCT (Stern, 1993). Cryostat sections (14 Am) were place on Superfrost/Plus slides, and non-radioactive in situ hybridization was per- formed as described by Toresson et al. (1999).Color reactions were carried out using BM Purple AP substrate (Roche) for 16–72 h and cover slips were mounted with 50% glycerol/PBS. Digoxigenin UTP-labeled antisense RNA probes were generated for mouse DSCR1/MCIP1 and NFATc1.A mouse DSCR1 cDNA probe (nt 404–2055) was isolated by RT-PCR of E10.5 head RNA based on published sequence (GenBank #AF260717) (Casas et al., 2001). Primers for DSCR1 amplification were 5V-AGACC- CAAGCCCAAAATCATCC-3V and 5V-TAGCCTCTC- CGGTTCAAAGTGC-3V. The PCR-generated DNA fragment was subcloned into pGEMT-vector (Promega), verified by sequencing and used to generate DSCR1 antisense probe with T7 polymerase after linearization with Not1. Alternatively embryos were hybridized with antisense probe for mouse MCIP1 (GenBank #AF237790) coding sequence (597 bp) initiated from exon 4 (Roth- ermel et al., 2000). MCIP1 coding sequence (provided by Fig. 1. DSCR1 is expressed during cardiac valvuloseptal development. DSCR1 expression in the developing heart was assessed in whole embryos Dr. R.S. Williams, Duke University) was inserted into (A), bisected hearts (B), or 14-Am sections (C–F). (A) At E10.5, DSCR1 is pBluescript and used for probe synthesis by T7 polymerase expressed in the outflow tract and atrioventricular canal of the developing after linearization of plasmid with Xba1. The separate heart (arrows). (B) At E12.5, DSCR1 is present in the developing mitral and DSCR1 and MCIP1 probes contain overlapping sequences, tricuspid valves (arrows) as well as the surrounding atrioventricular canal had similar expression patterns, and were used inter- (black arrowhead) and the muscular interventricular septum (white arrowhead). (C–F) Transverse sections of E13.5 embryos. (C) DSCR1 is changeably. Mouse NFATc1 (GenBank #AF239169) se- strongly expressed in the muscular interventricular septum and ventricular quence (nt. 1313–2459) was isolated from E14.5 myocardium of the heart as well as the dorsal root ganglion (arrowhead) ventricular cDNA using primers 5V-GCTGTTCATTGG- and ventral neural tube (arrow). (D) Increased magnification of boxed area GACGGCTGACGA-3V and 5V-CACGTCACCAGGGGA- in C reveals DSCR1 expression in the endocardium of the developing mitral CAGCATTAT-3V and subcloned into pBluescript. The and tricuspid valves (arrows). (E–F) DSCR1 is also expressed in the endocardium of the developing aortic valves (F, arrowheads). (F) Increased resulting plasmid was verified by sequencing and linear- magnification of boxed area in E. ot, outflow tract; avc, atrioventricular ized with Not1 for generation of NFATc1 antisense RNA canal; tv, tricuspid valve; mv, mitral valve; ivs, interventricular septum, probe with T7 polymerase. AoV, aortic valve. A.W. Lange et al. / Developmental Biology 266 (2004) 346–360 349
(7 Am) as described above. Sections were washed in PBS, labeling, sections were washed several times in PBS and then blocked for 30 min in 10% normal goat serum cover slips were mounted with Vectashield Hard Set (Sigma) in PBS and incubated with a rabbit polyclonal mounting medium (Vector Labs). Fluorescent microscopic anti-h-galactosidase primary antibody (1:250, Eppendorf 5 analysis was performed using a Nikon PCM 2000 confocal Prime, Inc.) for 30 min in blocking serum. After three microscope and images were obtained using SimplePCI washes in PBS, sections were incubated with Alexa Fluor software. 488 goat anti-rabbit secondary antibody (1:100 in PBS, Molecular Probes) for 15 min and washed three times in Cell culture and transient transfections PBS. Subsequently, immunolabeling for NFATc1 was per- formed using the Vector M.O.M. Basic immunodetection C2C12 myoblasts were cultured in DMEM (Cellgro) kit for detecting mouse primary antibodies on mouse tissue supplemented with 15% FBS, pen/strep (Gibco, 100 units/ according to the manufacturer’s recommendations (Vector ml), and L-glutamine (Gibco, 100 Ag/ml). Expression Labs). Briefly, sections were blocked for 1 h, then incu- vectors for wild-type and constitutively active Nfatc1 bated with mouse monoclonal anti-NFATc1 (7A6) (1:100, (wt-Nfatc1 and ca-Nfatc1, respectively) and pBJ5 have Santa Cruz Biotechnology, Inc.) for 1 h in M.O.M. diluent been previously described (Beals et al., 1997; Porter et solution, washed in PBS, and incubated with Alexa Fluor al., 2000). The approximately 600 bp DSCR1(e4) intra- 568 goat anti-mouse IgG (1:100 in PBS, Molecular genic element was ligated into the Kpn1 site of the pGL3- Probes) for 15 min. After immunofluorescent double Promoter vector (Promega) to generate the DSCR1(e4)-
Fig. 2. The mouse DSCR1 exon 4 regulatory sequence contains multiple conserved NFAT consensus binding sequences. (A) The sequence of the mouse DSCR1 exon 4 intragenic regulatory sequence (À696 to À56) homologous to the human DSCR1/MCIP1 calcineurin response element (Yang et al., 2000). The mouse DSCR1(e4) regulatory region contains 10 NFAT consensus binding sequences (blue) and a conserved AP-1 consensus binding sequence (red) (Rao et al., 1997). Nucleotides conserved with human DSCR1 NFAT and AP-1 consensus binding sequences in the same positions are underscored with stars (Yang et al., 2000). (B) Schematic representation of the mouse DSCR1 gene comprised of 7 exons. The mouse DSCR1 sequence À696 to À56 relative to exon 4 was linked to the hsp68-lacZ reporter gene (Kothary et al., 1989) to generate the DSCR1(e4)/lacZ transgene. 350 A.W. Lange et al. / Developmental Biology 266 (2004) 346–360 luciferase reporter construct. C2C12 cells were transfected RNA was isolated using TRIZOL (Life Technologies) with 0.3 Ag of reporter plasmid and 0.1 Ag of expression according to the manufacturer’s instructions. cDNA generat- vector using FuGENE 6 Transfection Reagent (Roche). All ed with oligo(dT) primers and the SuperScript First-Strand transfections included 0.01 Ag of pRL-TK (Promega) as an Synthesis kit (Invitrogen) was used for quantitative real-time internal control for transfection efficiency. Cells were RT-PCR using the MJ Research Opticon Monitor II system harvested 36–48 h after transfection and reporter activity (95jC, 30 s; 52jC, 30 s; 72jC, 30 s; 35 cycles). Reactions was measured according to protocols for the Dual Lucif- included 0.1Â Sybr Green (Molecular Probes) and fluores- erase Assay system (Promega) using a Monolight 3010 cence was monitored at 80jC. Primers for specific DSCR1 luminometer. Experiments were repeated three times in splice isoforms were: DSCR1(e1) 5V-ATGGAGGAGGTG- triplicate and statistical significance was determined by GATCTGC-3Vand 5V-TTTATCCGGACACGTTTGAAG-3V; Student’s t test. DSCR1(e4) 5V-GTGTGGCAAACGATGATGTC-3V and 5V- AGGAACTCGGTCTTGTGCAG-3V. Gene expression lev- Quantitative real time PCR els were quantified based on the threshold cycle [C(t)] calibrated to a standard curve generated using E14.5 whole The AVendocardial cushions and OT were dissected from heart cDNA and normalized to h-actin (Broberg et al., 2003). E12.5 hearts of embryos from Nfatc1+/À matings and total Reactions were repeated twice using technical duplicates
Fig. 3. Cardiac expression of DSCR1(e4)/lacZ is dependent on NFATc1. (A–D) DSCR1(e4)/lacZ is expressed in a subset of NFATc1-expressing cells in the developing heart. (A, C) Cryosections (10 Am) of DSCR1(e4)/lacZ transgenic mouse hearts stained with X-gal. (A) h-galactosidase is detected in the endocardium of the developing mitral and tricuspid valves of an E13.5 transgenic heart (arrowheads). (C) DSCR1(e4)/lacZ is also expressed in the endocardial endothelium of the developing aortic valve of an E12.5 transgenic heart (arrowheads). (B, D) In situ hybridization of histological sections (14 Am) for NFATc1. NFATc1 is expressed in the endocardium of the developing mitral and tricuspid valves (B) as well as the developing pulmonary valve (D). (E–F) DSCR1(e4)/ lacZ expression in the developing mitral valve is lost in NFATc1À/À embryonic hearts. (E) Histological section (10 Am) of an E12.5 DSCR1(e4)/lacZ;NFATc1+/+ heart stained with X-gal shows normal transgene expression in the developing mitral valve (arrow). (F) h-gal activity is not detected in the mitral valve of an E12.5 DSCR1(e4)/lacZ;NFATc1À/À heart stained with X-gal (arrow). A.W. Lange et al. / Developmental Biology 266 (2004) 346–360 351 from three independent samples of each genotype and statis- to the developing AV valves (Fig. 1B). In sectioned tical significance was determined using Student’s t test. E13.5 embryos, DSCR1 expression in the heart is detected throughout the ventricular myocardium but is enriched in the muscular IVS (Figs. 1C–E). DSCR1 Results expression is also apparent in the dorsal root ganglion and ventral portion of the neural tube (Fig. 1C). In the DSCR1 is expressed in the developing valves and septa of developing valves, DSCR1 gene expression is restricted the heart to the endocardial cells (Figs. 1C–F).Thus,DSCR1 expression in the embryonic heart, notably in the ven- The expression of the Down syndrome candidate gene tricular septum, AV canal, and developing valve endo- DSCR1 was examined during embryonic heart formation cardium, is consistent with a role in valvuloseptal in the mouse. Previous studies demonstrated that DSCR1 formation. is expressed in the developing mouse heart at E9.5 to E10.5 but expression during valvuloseptal development Expression of DSCR1(e4) in the developing valve was not reported (Casas et al., 2001; Vega et al., 2003). endocardium is dependent on NFATc1 A more complete analysis of DSCR1 expression was obtained by in situ hybridization of whole mount and Human DSCR1 contains an intragenic promoter adjacent sectioned embryos. DSCR1 is expressed in the developing to exon 4 that is responsive to calcineurin signaling via mouse heart as early as E8.5 (data not shown). At E10.5, clustered NFAT consensus-binding sequences (Yang et al., DSCR1 expression is detected in the outflow tract (OT) 2000). A homologous sequence in mouse DSCR1 was and atrioventricular canal (AVC) where the endocardial identified after isolation of the gene. Sequence analysis of cushions are induced (Fig. 1A). These structures later the region 5V to exon 4 in mouse and human DSCR1 shows contribute to the valves and septa of the heart as it a high degree of homology between the murine sequence divides into four chambers. In bisected E12.5 hearts, and the human DSCR1/MCIP1 intragenic calcineurin-re- DSCR1 expression is apparent in the muscular interven- sponsive promoter. The approximately 700 bp adjacent to tricular septum (IVS) and AVC myocardium in addition mouse DSCR1 exon 4 contains 10 NFAT-binding sites based
Fig. 4. DSCR1(e4)/lacZ transgene expression colocalizes with NFATc1 in the developing valve endocardium. NFATc1 (red) and h-galactosidase (green) localization was examined by fluorescent confocal microscopy on E11.5 DSCR1(e4)/lacZ transgenic hearts. (A–C) Valvuloseptal endocardium of the embryonic outflow tract. (D–F) Atrioventricular endocardial cushions. Arrowheads denote endothelial endocardial cells with colocalized expression of NFATc1 and h-gal. Note that only a subset of endocardial valve cells that express NFATc1 (arrows, D and F) have colocalized h-gal expression. 352 A.W. Lange et al. / Developmental Biology 266 (2004) 346–360 on the A/TGGAAAN(A/T/C) consensus sequence (Fig. 2A) cell layer as NFATc1 in corresponding cardiac structures. (Rao et al., 1997; Yang et al., 2000). Eight of these sites are Interestingly, NFATc1 expression is not apparent in the 100% conserved with the human sequence and an additional muscular ventricular septum or ventricular myocardium. pair of NFAT sites at À572 to À556 is unique to the mouse Thus, DSCR1 and NFATc1 expression overlap in the endo- DSCR1(e4) element. In addition to the NFAT sites, a thelium of the developing AV and semilunar valves, but conserved consensus binding sequence for AP-1, a known DSCR1 alone is expressed in the developing muscular NFAT cofactor, is present adjacent to an NFAT site (À104 to ventricular septum and ventricular myocardium. Therefore, À91) in the mouse DSCR1(e4) regulatory element and both the DSCR1 expression subdomain revealed by DSCR1(e4)/ of these sites are 100% conserved in the human sequence (Macian et al., 2001; Yang et al., 2000). NFATs also can function with GATA or MEF-2 transcription factors but no GATA/NFAT or MEF-2/NFAT consensus binding sequences are present in the DSCR1(e4) NFAT-rich regulatory se- quence (Molkentin et al., 1998; Nemer and Nemer, 2002; Wu et al., 2000). The high degree of homology and conservation of NFAT consensus binding sequences be- tween the mouse and human DSCR1(e4) sequence is evi- dence for conserved calcineurin-sensitive regulation of DSCR1 gene expression through activated NFATs (Yang et al., 2000). The embryonic expression of mouse DSCR1 via the NFAT-rich regulatory sequence adjacent to exon 4 was examined in transgenic mice in which the DSCR1(e4) regulatory sequence (À696 to À56 relative to exon 4) was linked to the hsp68/lacZ reporter gene (Fig. 2B). Whole mount X-gal staining of transgenic embryonic hearts for h- galactosidase activity revealed DSCR1(e4)/lacZ transgene expression in the developing OT and AV valves of the embryonic heart (Fig. 3). Similar cardiac expression of DSCR1(e4)/lacZ was observed for three independent trans- genic lines. Transgene expression in the OT is detected as early as E9.5, and DSCR1(e4)/lacZ is expressed in the endothelial cell layer of the OT endocardial cushions and aorticopulmonary septum at E11.5–E12.5 (data not shown). During endocardial cushion remodeling and valve leaflet maturation (E13.5–E15.5), DSCR1(e4)/lacZ is expressed in a subset of endocardial cells in the developing AV and semilunar valves (Figs. 3A, C). Although DSCR1(e4)/lacZ Fig. 5. DSCR1(e4) expression is responsive to NFATc1 through the is expressed in both nascent AV valves between E12.5 and intragenic promoter. (A) Endogenous DSCR1 transcript expression in the E14.5, predominant expression is observed in the develop- OT of Nfatc1 mutant hearts. Expression levels of DSCR1(e1) and ing mitral valve (Figs. 3A, E). DSCR1(e4)/lacZ expression DSCR1(e4) in isolated OT were measured by quantitative real-time RT- was down-regulated after E14.5 and no expression was PCR using primers for specific alternative transcripts. Expression levels of observed in the adult heart (data not shown). Unlike calcineurin-insensitive DSCR1(e1) are maintained despite loss of NFATc1, but NFATc1 deficiency results in decreased expression of the calcineurin- endogenous DSCR1, expression of the DSCR1(e4)/lacZ responsive DSCR1(e4) isoform. DSCR1(e4) levels quantified in the transgene is not apparent in the ventricular myocardium experiments were consistently less abundant (approximately .19X) than and was only rarely observed in the IVS in a punctate DSCR1(e1) expression levels. Reactions were repeated twice using three manner (data not shown). Thus, the cardiac expression of independent samples from each genotype, and expression levels were h the DSCR1(e4)/lacZ transgene recapitulates a specific sub- quantified in relation to a standard curve and normalization to -actin. Graphs represent the average fold change in expression relative to domain of embryonic expression of endogenous DSCR1. Nfatc1+/+ F the standard deviation. (B) Transient transfection assays. Expression of DSCR1(e4)/lacZ in the developing heart C2C12 myoblasts were transfected with the DSCR1(e4)-luciferase reporter valves is similar to expression of NFATc1, which is required in combination with pBJ5 control vector, wt-Nfatc1, or ca-Nfatc1 expression for normal valvuloseptal formation (de la Pompa et al., vectors. The reporter plasmid is activated by wt-Nfatc1 (5.01 fold F1.43) F 1998; Ranger et al., 1998). NFATc1 is expressed in the and ca-Nfatc1 (8.02 fold 3.54) in comparison to basal expression with the vector control. The graph represents the average fold activation F the endocardium of the AV and semilunar valve primordia at standard deviation from three independent transfections performed in E13.5 (Figs. 3B, D). Comparison to DSCR1(e4)/lacZ ex- triplicate. Asterisks represent statistical significance as determined by pression reveals that the transgene is expressed in the same Student’s t test ( P < 0.05). A.W. Lange et al. / Developmental Biology 266 (2004) 346–360 353 lacZ expression may represent the cells in which DSCR1 is the activation of transgene expression in the developing regulated by activated NFATc1. valve endocardium. To determine the dependence of DSCR1(e4)/lacZ expres- The colocalization of NFATc1 and DSCR1(e4)/lacZ sion on NFATc1 function in the developing heart, transgene expression in the embryonic valve endocardium DSCR1(e4)/lacZ transgenic mice were crossed with NFATc1 was confirmed by confocal microscopy. Double-labeled mutant mice (Ranger et al., 1998). NFATc1 mutant embryos immunofluorescence for NFATc1 and h-galactosidase pro- carrying the DSCR1(e4)/lacZ transgene were examined at tein was performed on sections from E11.5 DSCR1(e4)/lacZ E12.5 when the AV valve primordia are apparent and the transgenic embryos. NFATc1 is detected in the endocardial NFATc1À/À embryos are still viable. In DSCR1(e4)/lacZ; cells of the OT and AV valvuloseptal primordia, which is NFATc1+/+ embryos, the transgene is expressed predomi- consistent with the observed gene expression pattern (Figs. nantly in the developing mitral valve (Fig. 3E). However, 4A, D).Likewise,h-gal expression is restricted to the mitral valve expression of DSCR1(e4)/lacZ is visibly re- developing OT and AV valvuloseptal endocardium (Figs. duced or absent in transgenic embryos that are null for 4B, E), and transgenic h-gal specifically colocalizes with a NFATc1 (Fig. 3F). The loss of DSCR1(e4)/lacZ transgene subset of NFATc1-expressing cells (Figs. 4C, F).The expression is not due to absence of the endocardium in coincident expression of NFATc1 and h-gal in the primitive NFATc1À/À embryos, as determined by PECAM-1 staining valves and dependence of DSCR1(e4)/lacZ expression on of endothelial cells (de la Pompa et al., 1998). In addition, NFATc1 is consistent with regulation of DSCR1 gene DSCR1(e4)/lacZ expression was not observed in the severe- expression in the developing valve endocardium by NFATc1 ly hypomorphic semilunar valves of NFATc1À/À embryos through the NFAT-rich regulatory sequences. (data not shown) (de la Pompa et al., 1998; Ranger et al., The potential direct regulatory interaction between 1998). The overlapping expression of NFATc1 and NFATc1 and DSCR1 gene expression controlled by the DSCR1(e4)/lacZ suggests that NFATc1 may contribute to intragenic promoter element was examined in mutant mice
Fig. 6. DSCR1(e4)/lacZ is expressed in developing organs affected in Down syndrome and in NFATc1 null embryos. Embryos were analyzed between E9.5 and E13.5, and h-gal activity was detected by overnight X-gal staining. Expression is detected in developing organ systems associated with Down syndrome phenotypic traits including the eye (arrow, E), ear (arrowhead, E), pharyngeal arches (black arrow, B), brain (white arrow, B), limbs (arrows, D), and heart (arrow, A). (C, F) E11.5 DSCR1(e4)/lacZ;Nfatc1À/À embryos exhibited transgene expression in developing organs comparable to DSCR1(e4)/lacZ;Nfatc1+/+ littermates with apparent decreased expression in the face (arrowheads) and ear (arrows). 354 A.W. Lange et al. / Developmental Biology 266 (2004) 346–360 and cultured cells with decreased or increased NFATc1, dent on NFATc1 through a conserved intragenic promoter respectively. The dependence of the endogenous DSCR1 containing multiple consensus NFAT sites. intragenic promoter on NFATc1 was examined by quan- titative real-time RT-PCR analysis of DSCR(e1) and DSCR1(e4)/lacZ is expressed in developing organs affected DSCR1(e4) expression in Nfatc1 gene targeted mice. In in human Down syndrome and mouse trisomy 16 cultured cells and adult hearts, DSCR1(e4) expression is responsive to calcineurin signaling, whereas expression of DSCR1(e4)/lacZ transgenic embryos were examined for the DSCR1(e1) splice variant is insensitive to such stimuli developmental expression in organ systems other than the (Yang et al., 2000). Endogenous DSCR1(e4) vs. (e1) heart by whole mount X-gal staining. Transgene expression transcript levels were examined in OT of Nfatc1 mutant mice, with major defects in semilunar valve formation (de la Pompa et al., 1998; Ranger et al., 1998). DSCR1(e4) expression levels were significantly reduced in Nfatc1À/À OT in comparison to OT from Nfatc1+/+ hearts (Fig. 5A). DSCR1(e4) expression is also decreased in Nfatc1+/À OTs, but the reduction is not statistically significant in comparison to Nfatc1+/+ levels (Fig. 5A). In contrast, DSCR1(e1) ex- pression is relatively unaffected by loss of NFATc1 (Fig. 5A). These results are consistent with previous reports of differential gene regulation of DSCR1(e1) and DSCR1(e4) isoforms in response to calcineurin/NFAT signaling. The ability of NFATc1 to trans-activate the DSCR1(e4) regulatory element also was assessed in transfected C2C12 myogenic cells. The approximately 600 bp DSCR1(e4) intragenic sequence was cloned into a luciferase vector to generate a DSCR1(e4)-luciferase reporter construct. In co-transfection assays, the DSCR1(e4) regulatory element is activated by wt- NFATc1 (5.01 F 1.43-fold) and constitutively active (ca)- NFATc1 (8.02 F 3.54-fold) in comparison to the control vector (Fig. 5B). These data provide further evidence for direct gene regulation. Together, these results demonstrate that in vivo and cell culture expression of the calcineurin- sensitive DSCR1(e4) isoform, but not DSCR1(e1), is depen-
Fig. 7. Cardiac expression of endogenous DSCR1 and transgenic DSCR1(e4)/lacZ colocalizes with anomalous development of Ts16 hearts. (A, C, E, G) Disomic embryonic hearts. (B, D, F, H) Trisomy 16 embryonic hearts. (A–D) In situ hybridization for DSCR1 was performed on bisected E12.5 (A, B) and cryosectioned (14 Am) E13.5 (C, D) hearts from intercrosses between Rb(6.16)24Lub/Rb(16.17)7BnrF1 males and non- transgenic females. Endogenous DSCR1 gene expression is detected in the interventricular septum (arrowheads, A–D) and endothelium of the developing endocardial cushions/valves (ec) (arrows, B– D) in both disomic and trisomic hearts. (D) Note the abnormal atrioventricular endocardial cushions and malalignment of the ventricular septum (asterisk) in correlation with DSCR1 expression in the heart of a Ts16 embryo. (E–H) Cryosections (10 Am) of X-gal stained hearts stained from intercrosses between Rb(6.16)24Lub/Rb(16.17)7BnrF1 males and DSCR1(e4)/lacZ transgenic females. (E) At E12.5, a DSCR1(e4)/lacZ;Ds16 heart has normal transgene expression in the developing mitral valve (arrow). (F) h-gal is detected in the abnormally developing atrioventricular endocardial cushions of an E12.5 DSCR1(e4)/lacZ;Ts16 heart (arrows). (G) By E14.5, transgene expression is reduced in the maturing mitral and tricuspid valves of a DSCR1(e4)/lacZ;Ds16 heart (arrows). (H) h-gal activity persists in the malaligned endocardial cushions of an E14.5 DSCR1(e4)/lacZ;Ts16 heart with delayed valvuloseptal development. Quantitative assays reveal average h-gal activity in E13.5 DSCR1(e4)/lacZ;Ts16 whole hearts (n =5)is approximately 2.5Â greater than in DSCR1(e4)/lacZ;Ds16 hearts (n = 13). A.W. Lange et al. / Developmental Biology 266 (2004) 346–360 355 in the embryo was first observed at E8.5 in the neural tube ultimately affected in human Down syndrome. Progeny of and at E9.5 in the outflow tract of the heart, consistent with male mice bearing metacentric Robertsonian chromosome the onset of endogenous DSCR1 expression (data not shown 6/16 and 16/17 fusions (Rb(6.16)24Lub/Rb(16.17)7BnrF1) and Fig. 6A). Between E10.5 and E13.5 DSCR1(e4)/lacZ is are trisomic for chromosome 16, have developmental expressed in several developing craniofacial structures in- anomalies, and do not survive to birth (Epstein and Veke- cluding the brain, eye, pharyngeal arches, and ear (Fig. 6). mans, 1990; Epstein et al., 1985; Miyabara et al., 1982). Transgene expression is also apparent in the condensing Rb(6.16)24Lub/Rb(16.17)7BnrF1 male mice were bred with cartilage of the developing limbs (Fig. 6D). Whole mount euploid nontransgenic or DSCR1(e4)/lacZ females to pro- DSCR1(e4)/lacZ expression in Nfatc1À/À embryos carrying duce litters with a Mendelian ratio of 1/4 embryos with the transgene was also examined to assess if transgene trisomy 16. Embryos were examined at E12.5–E14.5 and expression was altered in any developing organs other than Ts16 embryos were confirmed by karyotype analysis. Sev- the heart. Expression of the transgene outside of the heart is eral obvious morphological defects are apparent in the Ts16 detected, but apparently reduced in some structures, includ- embryos beginning at E12.5 including edema, delayed rib ing the face and ear, of DSCR1(e4)/lacZ;Nfatc1À/À embryos cage development, abnormal head formation, and small in comparison to DSCR1(e4)/lacZ;Nfatc1+/+ littermates stature. (Figs. 6C, F). Together, these results show that embryonic Endogenous DSCR1 gene expression was examined in DSCR1(e4)/lacZ expression is regulated by complex Ts16 and Disomy 16 (Ds16) hearts by in situ hybridization. NFATc1-dependent and independent mechanisms and that DSCR1 is expressed in the IVS, AVC, ventricular myocar- the embryonic organ systems expressing in DSCR1(e4)/lacZ dium, and the AV endocardial cushion endothelium of correlate with the most prevalent developmental anomalies E12.5–E13.5 disomic and trisomic embryo hearts (Figs. characteristic of human Down syndrome. 7A–D). Expression regulated by the DSCR1(e4) NFAT-rich The expression of endogenous DSCR1 and the regulatory element was also evaluated in Ts16 embryos with DSCR1(e4)/lacZ transgene were examined in Ts16 mouse congenital heart malformations. Like endogenous DSCR1, embryos to assess gene expression in embryonic structures the DSCR1(e4)/lacZ transgene is expressed in endocardial
Fig. 8. DSCR1(e4)/lacZ is expressed in abnormally developing structures of Ts16 embryos. DSCR1(e4)/lacZ transgenic females were crossed with
Rb(6.16)24Lub/Rb(16.17)7BnrF1 males and h-galactosidase expression was detected by X-gal staining. (A) Whole mount DSCR1(e4)/lacZ transgene expression in an E14.5 disomic embryo. (B) In comparison, DSCR1(e4)/lacZ transgene expression is reduced in the abnormally developing eye (black arrow), face (asterisks), neural tube (white arrow), limbs (black arrowheads), and cranial structures (white arrowheads) of a trisomic littermate. (C–F) Cryosections (25 Am) of E14.5 heads stained with X-gal. DSCR1(e4)/lacZ is expressed in the neural retina (re, arrows) and near the eyelid (e, arrowheads) in both disomic (C) and trisomic (D) embryos. (E–F) h-gal is detected in the developing skull (sk, arrows) and striatum (st, arrowheads) of disomic (E) and trisomic (F) embryos. DSCR1(e4)/lacZ expression is reduced during abnormal skull development in Ts16 embryos (F, arrowhead). 356 A.W. Lange et al. / Developmental Biology 266 (2004) 346–360 cells of the developing valves of the heart in Ds16 and Ts16 NFATc1 deficiency or Ts16. DSCR1 is expressed in devel- embryos (Fig. 7). The abnormally developing endocardial oping valvuloseptal structures in the embryonic heart in- cushions and malalignment of the interventricular septum cluding the AVand semilunar valve primordia as well as the are evident in the Ts16 hearts and these structures express interventricular septum and AV canal myocardium. In DSCR1(e4)/lacZ and/or endogenous DSCR1, supporting the transgenic mice, conserved DSCR1 intragenic NFAT-rich association between DSCR1 expression and abnormal de- regulatory sequences control lacZ transgene expression in velopment of valve and septal precursors (Figs. 7B, D, F, several embryonic organ systems affected in Down syn- H).AtE14.5,DSCR1(e4)/lacZ transgene expression drome. In the developing heart, DSCR1(e4)/lacZ transgene remains strong in abnormal malaligned endocardial cush- expression colocalizes with NFATc1 in the endothelial layer ions of the Ts16 hearts but is down-regulated in the of the valve primordia and the DSCR1(e4) intragenic maturing AV valves of the normal Ds16 hearts (Figs. 7G, promoter is activated by NFATc1 in vitro. In addition, H). Analysis of transgene expression levels by quantitative expression of endogenous DSCR1(e4) and DSCR1(e4)/lacZ h-gal enzymatic assay reveals approximately 2.5-fold more is affected in NFATc1À/À hearts that exhibit valvuloseptal h-gal activity in Ts16 whole hearts compared to Ds16 hearts maturation defects, further supporting the regulation of (data not shown). The increased transgene expression in the DSCR1(e4) gene expression in the valvuloseptal endocardi- Ts16 hearts could be due to altered NFAT activity, but also um by activated NFATs. Expression of endogenous DSCR1 may be the result of delayed transgene down-regulation due and the DSCR1(e4)/lacZ transgene is also observed in to abnormal valve maturation and cushion remodeling pro- several embryonic structures that develop abnormally in cesses in these embryos. Together, the expression of endog- Ts16 mice. Together, these results provide evidence for enous DSCR1 and DSCR1(e4)/lacZ correlates with regions DSCR1 as a target gene for NFATc1 in valvuloseptal of the heart undergoing anomalous valvuloseptal develop- development and support a role for trisomic DSCR1 in ment in Ts16 embryos. Down syndrome-related developmental anomalies. DSCR1(e4)/lacZ expression was examined in other de- veloping organ systems affected in Ts16 embryos (Fig. 8). DSCR1(e4) expression is dependent on NFATc1 during In Ts16 embryos, DSCR1(e4)/lacZ expression in the poste- valvuloseptal development rior neural tube, eye, and face is reduced and expression in the developing skull appears abnormal relative to Ds16 The expression of endogenous DSCR1 and the embryos (Figs. 8A, B). Reduced h-gal expression is appar- DSCR1(e4)/lacZ transgene in the developing heart is con- ent on the surface of the face near the eyelids and nasal sistent with a role in valvuloseptal formation. The onset of structures of trisomic embryos in comparison to disomic DSCR1 expression in the OT and AV junction of the heart at littermates (Figs. 8A, B, indicated by asterisks). Frozen E9.5 is concurrent with the initiation of endocardial cushion sections (25 Am) of E14.5 heads revealed transgene expres- formation. In the endocardial cushions, expression of both sion in the neural retina of the developing eye in both DSCR1 and DSCR1(e4)/lacZ is limited to the endothelial disomic and trisomic embryos (Figs. 8C, D). DSCR1(e4)/ cell layer and is not observed in the cushion mesenchyme. lacZ expression in the inner surface of the retinal epithelium Notably, where DSCR1(e4)/lacZ expression in the heart is is consistent with expression of endogenous DSCR1 in the limited to the endocardium of the valvuloseptal precursors, developing eye (data not shown). However, where endog- endogenous DSCR1 expression is also apparent in the enous DSCR1 is also strongly expressed throughout the ventricular myocardium and muscular IVS. The non-over- developing lens, the DSCR1(e4)/lacZ transgene only shows lapping regions of DSCR1(e4)/lacZ and endogenous DSCR1 punctate expression in this region (Figs. 8C, D and data not gene expression in the developing heart may represent shown). Transgene expression in Ts16 embryos also is more expression regulated by other DSCR1 promoters, expression diffuse in the developing skull and is apparent in the regulated by additional intragenic regulatory sequences developing striatum of the forebrain (Figs. 8A, B, E, F). outside of the DSCR1(e4) proximal 700 bp, or expression DSCR1(e4)/lacZ transgene expression observed in the de- of alternative DSCR1 splice isoforms. Transcripts originat- veloping striatum is consistent with reports of endogenous ing from exon 1 and exon 4 of human DSCR1 are coex- DSCR1 gene expression in the embryonic and postnatal pressed in several adult tissues including the brain and heart, forebrain (Casas et al., 2001). Thus, DSCR1(e4)/lacZ is and selective induction of DSCR1(e4) vs. DSCR1(e1) has expressed in several embryonic organ systems with devel- been demonstrated in transgenic mice expressing constitu- opmental anomalies that result from trisomy 16 in mice. tively active calcineurin in the heart (Ermak et al., 2002; Fuentes et al., 1997; Yang et al., 2000). Therefore, differ- ential expression of mouse DSCR1 isoforms in the devel- Discussion oping heart could also be regulated by multiple promoters. Both endogenous DSCR1 and DSCR1(e4)/lacZ are The regulation of DSCR1 gene expression was examined expressed in the endothelial cell layer with NFATc1 in the at critical stages of normal valvuloseptal development as AV and semilunar valve primordia during valvuloseptal well as in mice with cardiac malformations resulting from maturation and remodeling. The expression of the A.W. Lange et al. / Developmental Biology 266 (2004) 346–360 357
DSCR1(e4)/lacZ transgene is significantly reduced or absent (Vega et al., 2003). However, it remains possible that other in the endothelium of the valve primordia in the absence of members of the DSCR1 family of proteins (DSCR1L1/ NFATc1. This loss of transgene expression is corroborated MCIP2 and DSCR1L2/MCIP3), which are expressed in by a significant decrease in endogenous DSCR1(e4) expres- the heart and are capable of interacting with calcineurin, sion in the OT of Nfatc1À/À hearts. Notably, DSCR1(e1) may be sufficient to functionally compensate for loss of expression was not affected in the OT of Nfatc1 mutant DSCR1 during embryonic development (Miyazaki et al., hearts, supporting differential sensitivity of DSCR1 splice 1996; Rothermel et al., 2000; Strippoli et al., 2000).In variants to altered calcineurin/NFAT signaling (Yang et al., addition, transgenic expression of DSCR1/MCIP1 in the 2000). Although the regulatory interaction between NFATc1 myocardium using the a-MyHC promoter is not overtly and the DSCR1(e4) intragenic promoter could occur by an detrimental to normal cardiac development and function, but indirect mechanism, several lines of evidence support endothelial expression of DSCR1 was not affected in these DSCR1(e4) as a target gene directly regulated by NFATc1 embryos (Rothermel et al., 2001). Currently, DSCR1 is in valvuloseptal development. These include colocalized thought to have dual roles in regulation of calcineurin endocardial expression of NFATc1 and DSCR1(e4)/lacZ, activity. Overexpression studies of DSCR1/MCIP1 both in the presence of conserved NFAT consensus binding sequen- vivo and in cultured cells suggest DSCR1 functions as a ces in the DSCR1(e4) promoter, in vitro activation of the feedback inhibitor of calcineurin. Analysis of DSCR1/ DSCR1(e4) promoter by NFATc1, and loss of transgene and MCIP1À/À mice, as well as loss of function mutations in endogenous DSCR1(e4) gene expression in NFATc1 mutant yeast or C. neoformans fungal homologues (Rcn1p and hearts. Together, the temporal and spatial expression of CBP1, respectively), suggest that DSCR1 is also required DSCR1 in the endocardial cushions and primitive valves for maximal calcineurin activity under certain conditions along with the dependence of DSCR1(e4) cardiac expres- (Rothermel et al., 2003; Vega et al., 2003). Together, these sion on NFATc1 support a role for DSCR1 in the endocar- findings support a model in which DSCR1 proteins can dium during valvuloseptal development. potentiate calcineurin activity, possibly by localizing calci- The identification of transcriptional targets for NFATc1 in neurin to the appropriate target substrates, while overex- the AV canal and outflow tract endocardium is in the early pression of DSCR1 may result in competition with stages of investigation. In this study, we show that DSCR1 is a downstream targets for interaction with calcineurin to likely target of NFATc1 in valvuloseptal remodeling. In effectively inhibit signal transduction (Rothermel et al., addition, NFATc1 was recently shown to cooperate with 2003). Thus, additional experiments are necessary to ad- GATA-5 to synergistically activate endothelin-1 gene expres- dress the specific contributions of DSCR1 to the calci- sion in cultured TC-13 endothelial cells (Nemer and Nemer, neurin/NFAT pathway during normal and abnormal 2002). Recent studies have also provided novel insights into valvuloseptal development. the signaling events that control NFATc1 activation in adult valves as well as during valvuloseptal development. In- Developmental regulation of DSCR1 is consistent with a creased VEGF signaling in adult human pulmonary valve role in Down syndrome-related congenital anomalies endothelial cells induces nuclear localization of activated NFATc1 as well as cell proliferation (Johnson et al., 2003). Congenital heart defects associated with Down syndrome VEGF also has a critical role in embryonic valve maturation, include high incidence of AV septal defects, ventricular however, the potential relationship of VEGF to NFATc1 septal defects, tetralogy of Fallot, and related valvuloseptal activation and function in the developing valves has not yet abnormalities (Freeman et al., 1998). Similar defects are been established (Carmeliet et al., 1996; Dor et al., 2001, seen in Ts16 mice and are attributed to cushion malalign- 2003; Ferrara et al., 1996). Additionally, endothelial-specific ment and defects in later chamber formation (Epstein et al., loss of NF1 (neurofibromatosis type 1), which encodes a ras 1985; Webb et al., 1997). In this study, we report that inhibitor, results in increased MAPK activation, premature endogenous DSCR1 and DSCR1(e4)/lacZ are expressed in nuclear localization of NFATc1, and enlarged endocardial cardiac structures affected in Ts16 embryos, supporting a cushions (Gitler et al., 2003; Lakkis and Epstein, 1998). possible contribution of DSCR1 to congenital heart defects Together, these studies suggest that intersecting signal trans- associated with Down syndrome. The spectrum of congen- duction pathways contribute toward nuclear translocation of ital anomalies associated with Down syndrome is thought to NFATc1 in developing valvuloseptal structures. Investigation result from disruption of normal developmental processes of the relationship between these signaling pathways and by overexpression of trisomic genes (Reeves et al., 2001). NFATc1 target genes in the valvuloseptal endocardium may Initial evidence for increased DSCR1 expression with triso- provide insight into the molecular mechanisms of cardiac my of the gene locus is provided by studies of gene valve and septa development. expression in the brain of Down syndrome fetuses (Fuentes The function of DSCR1 in valvuloseptal development is et al., 2000). The levels of endogenous DSCR1 expression not known. Notably, DSCR1 is not absolutely required for in Ts16 hearts reported herein appeared higher in the AV normal formation of the chambered heart as DSCR1À/À cushion endocardium but more quantitative methods of mice are viable with no obvious cardiac malformations evaluating gene expression are necessary to confirm that 358 A.W. Lange et al. / Developmental Biology 266 (2004) 346–360 trisomic DSCR1 expression is increased in the developing Acknowledgments heart. The identification of the specific consequences of elevated DSCR1 in valvuloseptal precursors may provide NFATc1 mutant mice were obtained from Dr. Laurie further clues into the molecular basis of Down syndrome- Glimcher and Rb(6.16)24Lub/Rb(16.17)7BnrF1 male mice related congenital heart defects. were purchased from Jackson Labs through the NICHD DSCR1 and the DSCR1(e4)/lacZ transgene are expressed sponsored program for Distribution of Mouse Models for in several organ systems of the embryo affected by Ts16 in Chromosomal Disorders. Initial karyotypes were performed mice and Down syndrome/Ts21 in humans. The with the guidance of Dr. Ruthann Blough-Pfau. Plasmids for DSCR1(e4)/lacZ transgene expression in the pharyngeal gene expression analysis were obtained from Dr. R. S. arches that contribute to the face is consistent with charac- Williams and Dr. N. Clipstone. Technical support was teristic facial abnormalities associated with Ts21 (Reeves et provided by Christine Liberatore and initial mouse husban- al., 2001). Further, expression of DSCR1(e4)/lacZ in the dry was provided by Ronald Waclaw. A.W.L is supported developing retina corresponds with vision defects common by an AHA-Ohio Valley Affiliate predoctoral fellowship in individuals with Down syndrome. Transgene expression and NIH training grant HL07752. Additional research surrounding the eye is also consistent with defective eyelid support was provided by NIH grant HL069779. closure in Ts16 embryos, as well as characteristic upslanting fissures and epicanthal folds associated with Down syn- drome (da Cunha and Moreira, 1996; Lipski and Bersu, 1990). DSCR1(e4)/lacZ expression in the otic vesicle and References developing ear also correlates with Down syndrome-related anomalies including small, malpositioned ears and conduc- Beals, C.R., Clipstone, N.A., Ho, S.N., Crabtree, G.R., 1997. 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