Journal of Histochemistry and Cytochemistry, Vol. 50, 107-112, January 2002, Copyright © 2002, The Histochemical Society, Inc.
Expression of the Gene Encoding the LIM Protein CRP2: A Developmental Profile
James R. Hendersona,
Doris Brownb,
James A. Richardsonc,
Eric N. Olsonb, and
Mary C. Beckerlea
a Department of Biology/Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah
b Department of Molecular Biology and Oncology, University of Texas Southwestern Medical Center, Dallas, Texas
c Department of Pathology, Molecular Cardiology Research Laboratories, University of Texas Southwestern Medical Center, Dallas, Texas
Correspondence to:
Mary C. Beckerle, Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84112-0840. E-mail: mary.beckerle@hci.utah.edu
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Summary |
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Members of the cysteine-rich protein (CRP) family are evolutionarily conserved Lin-11, Isl-1, Mac-3 (LIM) domain proteins that have been implicated in cell differentiation. Here we describe the expression pattern of the CRP family member CRP2 in mouse. Unlike other CRP family members, which are expressed primarily in muscle, CRP2 is more broadly expressed. In addition to expression in vascular smooth muscle cells, we also detect CRP2-specific transcripts in mesenchymal derivatives and several epithelia.
(J Histochem Cytochem 50:107111, 2002)
Key Words:
development, LIM domains, kidney, epithelia, mesenchyme, vascular smooth muscle, lamina propria, heart, heart valves
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Introduction |
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Members of the cysteine-rich protein (CRP) family are evolutionarily conserved proteins that exhibit two double zinc finger LIM domains. In vertebrates, three CRP family members, CRP1, CRP2, and CRP3, have been identified (Sadler et al. 1992
; Weiskirchen and Bister 1993
; Arber et al. 1994
; Crawford et al. 1994
; Weiskirchen et al. 1995
). We have described the gene encoding CRP1 (in vertebrates the gene family that encodes the CRPs is referred to as csrp) (Weiskirchen et al. 1995
) as a smooth muscle marker in the mouse (Henderson et al. 1999
). CRP3, also referred to as the muscle LIM protein (MLP), is found exclusively in striated muscle (Arber et al. 1997
; Louis et al. 1997
). Ablation of the csrp3/mlp gene in mouse is associated with a dilated cardiomyopathy that may result from irregularities in intercalated discs, sites of cell adhesion, and mechanical coupling in cardiac muscle (Arber et al. 1997
; Ehler et al. 2001
). These data have led to the hypothesis that CRPs function to maintain the stability of the contractile apparatus (Arber et al. 1997
; Pomies et al. 1997
).
Studies in both chick and rat have revealed that CRP2 (also referred to as SmLIM) is present at highest levels in arterial samples (Jain et al. 1996
; Louis et al. 1997
), and subsequent work has focused on a potential role for CRP2 in the vascular system (Jain et al. 1998
). Here we describe the pattern of csrp2 expression in the developing and adult mouse. We demonstrate that csrp2 is broadly expressed in mouse embryos and adults.
Plaque lifts of a mouse embryonic (E) 11.5 day 5' stretch
gt11 cDNA library (Clontech; Palo Alto, CA) were performed exactly as described (Henderson et al. 1999
) using the coding region of the chicken csrp2 cDNA (Weiskirchen et al. 1995
) as a probe. A screen of 500,00 pfu provided one positive phage (IC8) that was purified. The cDNA insert of IC8 was excised using EcoRI, cloned into pBluescript (ks-) (Stratagene; La Jolla, CA), and sequenced.
Northern analyses of a mouse embryo multiple tissue northern (MTN) blot and a mouse MTN blot (Clontech) were performed exactly as described previously (Henderson et al. 1999
), using the csrp2 3' UTR (nucleotides 705850 in Fig 1A) as a probe. According to the manufacturer's instructions (Clontech), MTN lanes contain approximately 1 µg of RNA, and a control experiment using gadph as a probe verified that all lanes of the mouse embryo MTN blot contained RNA.

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Figure 1.
(A) Nucleotide and deduced amino acid sequence of mouse csrp2. Cysteine and histidine residues that comprise the metal-coordinating core of the LIM domain are boxed. Glycine residues of the glycine repeats are circled. (B) Diagram of the CRP2 protein.
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To perform in situ hybridization (ISH) analyses, the csrp2 3' UTR was cloned into pBluescript (ks-) (Stratagene) and verified by sequencing. Generation of antisense radiolabeled riboprobes and ISH of staged E9.5E16.5 mouse embryos and of adult mouse organs, was performed as described (Henderson et al. 1999
). Control experiments using sense riboprobes on whole-mount embryos did not produce a specific signal (data not shown). In situ hybridization of formalin-fixed, paraffin-embedded E7.0 embryos was performed using digoxigenin-labeled probe according to the manufacturer's protocol, followed by incubation with a mouse anti-digoxigenin antibody (Jackson ImmunoResearch; West Grove, PA) conjugated to alkaline phosphatase detected with BCIP/NBT (DAKO; Carpinteria, CA).
The sequence of mouse CRP2 (Fig 1A) is highly conserved, displaying 95.4% and 99.5% amino acid identity to chicken and human CRP2, respectively. Each LIM domain in CRP2 is followed by a short glycine-rich repeat (Fig 1A and Fig 1B). Northern blotting analysis of total RNA from mouse embryos was performed using the csrp 3' UTR as a probe. A single csrp2 transcript is detected during development (Fig 2A). The same blot probed for gadph transcripts indicates that all lanes contain RNA (Fig 2A). Analysis of RNA from adult organs (Fig 2B) indicates that csrp2 displays organ-specific regulation, with particularly high levels of expression observed in kidney. We characterized the expression pattern of csrp2 in several adult organs by ISH as described in Henderson et al. 1999
. csrp2 is highly expressed in epithelial cells of the kidney (Fig 2C, Fig 2C', and 2C''), lung (Fig 2D), small intestine (Fig 2E), and weakly in the germinal epithelium of the testis (data not shown). Strong expression occurs in the lamina propria of the bladder (Fig 2F). csrp2 expression is also observed in coronary artery smooth muscle cells (SMCs) (Fig 2G), and in the choroid plexus of the brain (data not shown). csrp2 transcripts were not detected in skeletal muscle (data not shown).

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Figure 2.
(A) Northern blot of total RNA from a series of murine embryonic stages, demonstrating a single 1-kb csrp2 transcript. The same blot probed for gadph transcripts indicates that all lanes contain RNA. (B) Northern blot of total RNA from murine adult organs. In situ hybridizations: (C) Adult kidney; expression is limited to the tubules of the cortex (Cr). (C',C'') Enlargement of C, viewed with brightfield or darkfield optics, respectively. There is no csrp2 expression in glomeruli (Gl). (D) Adult lung; weak expression in the epithelium (Ep) of the airways. (E) Adult small intestine; expression is limited to the epithelial cells (Ep) of the crypts and does not extend into the lamina propria or smooth muscle. (F) Adult bladder; strong expression in the lamina propria (Lp) but not in the smooth muscle layers (Sm). (G) Adult heart; expression detected in the smooth muscle of coronary arteries (Ca). Bars: CF = 1 mm; E = 0.5 mm; C',G = 0.1 mm.
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Consistent with the wide csrp2 expression pattern observed in adults, csrp2 transcripts were detected in a number of cell types during embryogenesis, including mesenchyme, vascular SMCs, and epithelial derivatives (Fig 3). In E7.0 embryos (Fig 3A), csrp2 transcripts are apparent in single cells within the embryonic mesoderm and neural ectoderm. As development proceeds, high levels of csrp2 transcripts are observed in populations of mesenchymal cells, notably in the head and in condensations surrounding developing bone (Fig 3B3G). In contrast to csrp1, the SMC-specific CRP family member (Henderson et al. 1999
), csrp2 transcripts were not detected in vascular SMCs of the dorsal aorta at E9.5 (Fig 3B), demonstrating that csrp2 is not an early marker of SMCs in the mouse. Instead, csrp2 expression was first observed in vascular SMCs at E11.5 (Fig 3C). Continued csrp2 expression was detected in both vascular and venous SMCs through development (Fig 3D, Fig 3E, Fig 3I, and Fig 3L), as indicated in a previous report (Jain et al. 1998
). csrp2 gut and stomach epithelium expression was first noted at E13.5 (Fig 3G) and continued at later stages (Fig 3I). Interestingly, although csrp2 transcripts are not observed in adult heart muscle (Fig 2G), significant csrp2 expression was detected in heart structures throughout development (Fig 3B3D and Fig 3J3L).

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Figure 3.
(A) E7.0 sagittal; weak CSRP2 expression is detected in embryonic mesoderm (EM) and neural ectoderm (NE) cells. CSRP2 transcripts were also observed in extra-embryonic areas, including the ectodermal component of the chorion and the endometrial tissue surrounding the embryo (data not shown). (B) E9.5 sagittal; CSRP2 is expressed in the aortic sac (As) and atrium (At) and displays a generalized mesenchymal signal. (C) E11.5 transverse; transcripts in the right atrium (At) the ventral body wall (Bw), and SMCs of the dorsal aorta (Da), mesenchyme (M), and umbilicus (Um). (D) E12.5 sagittal; strong expression in the atrium (At) and atrioventricular valves (enlargement in J). CSRP2 transcripts are also present in the ventricle (V), head mesenchyme (M), perichondrial mesenchyme (PcM) condensations surrounding the vertebrae, and in SMCs of vessels (Vs). (E) E12.5 transverse; expression in SMCs of the dorsal aorta (Da) and umbilical arteries (Ua), perichondrial mesenchyme (PcM), gut epithelium (G), and the spinal cord (Sc). (F) E13.5 sagittal; CSRP2 is observed in head mesenchyme (M), the submucosa of the lips (Sb), and body wall (Bw). (G) E13.5 transverse; transcripts in perichondrial mesenchyme (PcM), gut (G) and stomach (S) epithelium, spinal cord (Sc), urogenital sinus (Us), and lung parenchyma (Lp). (H) E15.5 sagittal; expression in the neocortex (Nc). (I) E16.5 sagittal; transcripts in SMCs of the dorsal aorta (Da) and posterior vena cava (Vc), in the lamina propria of the bladder (B), and basal epithelial cells of the gut (G). (JL) Strong CSRP2 expression is detected in the atria and atrioventricular valves during development. (J) Enlargement of D showing CSRP2 transcripts in the atria (At) and atrioventricular valves (AtV) at E12.5. (K) CSRP2 expression remains strong in the atria (At) and atrioventricular valves (AtV) at E13.5. (L) E16.5. CSRP2 transcripts are detected in the atria (At) and ventricle (V), and in SMCs of the dorsal aorta (Da), pulmonary arteries (Pa), and hepatic arteries (Ha). Bars: A = 50 µm; B,C,E,F,K = 0.5 mm; D,GJ,L = 1 mm.
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CRP family members are small, actin cytoskeleton-associated proteins that are enriched in muscle (Arber et al. 1994
; Louis et al. 1997
; Henderson et al. 1999
). In this study we have analyzed the expression of the family member csrp2 in mouse. Consistent with a previous report, we determined that csrp2 is expressed in vascular, but not visceral SMCs (Louis et al. 1997
). However, we also detected high levels of csrp2 transcripts in mesenchyme and epithelia during embryogenesis and in adult tissues. These data indicate that csrp2 is the most widely expressed CRP family member in mouse.
During embryogenesis, csrp2 expression was prominently associated with mesenchyme and epithelia. High levels of csrp2 transcripts were observed in a generalized mesenchymal pattern at E9.5, with the mesenchymal signal becoming restricted to cells in the head and in cell condensations surrounding developing bone as embryogenesis progressed. In adults, csrp2 transcripts remained associated with mesenchymal derivatives, including connective tissue in the bladder. csrp2 was also observed in epithelia during development and in adults, with significant levels observed in both the gut and kidney. In adult mouse, csrp2 expression in the kidney was the highest of any organ investigated.
Interestingly, csrp2 expression overlaps with that of csrp1 and csrp3/mlp in only a few areas. csrp2 and csrp1 transcripts are both detected in vascular smooth muscle, and even though csrp1 and csrp2 transcripts are not observed in adult heart muscle, all three csrp genes are expressed in the developing heart (Arber et al. 1997
; Henderson et al. 1999
; and this study). Targeted disruption of csrp3/mlp in the mouse results in cardiac hypertrophy and disorganization of the contractile apparatus. However, these phenotypes are postnatal manifestations. Expression of csrp1 (Henderson et al. 1999
) and csrp2 (this study) in the embryonic heart suggests that CRP1 and/or CRP2 may be in a position to compensate for the loss of CRP3/MLP during heart development.
In summary, we have detected csrp2 transcripts in both muscle and non-muscle cells, indicating that csrp2 is the most widely expressed CRP family member. The non-muscle csrp2 expression is of particular interest because these are the first data suggesting that CRPs may function outside of striated and smooth muscle. As a result, further characterization of this highly related protein family may offer insight on the cytoskeletal organization of a variety of cell types.
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Acknowledgments |
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Supported by NIH grant HL60591 (to MCB) and by the Huntsman Cancer Foundation.
We thank John M. Shelton and the Molecular Pathology Core laboratory at UTSW and Kelley Murphy at the HCI Tissue Access and Imaging Core facility for expert technical assistance, and the University of Utah Oligonucleotide Synthesis and Sequencing Core facilities. JRH (NIH Developmental Biology Training Grant 5T32 HD07491) thanks the Beckerle lab and T. Wallow for helpful discussions.
Received for publication May 23, 2001; accepted September 12, 2001.
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Literature Cited |
---|
Arber S, Halder G, Caroni P (1994) Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation. Cell 79:221-231[Medline]
Arber S, Hunter J, Ross J, Hongo M, Sansig G, Borg J, Perriard J-C, Chien K, Caroni P (1997) MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell 88:393-403[Medline]
Crawford A, Pino J, Beckerle M (1994) Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton. J Cell Biol 124:117-127[Abstract]
Ehler E, Horowits R, Zuppinger C, Price R, Perriard E, Leu M, Caroni P, Sussman M, Eppenberger H, Perriard J (2001) Alterations at the intercalated disk associated with the absence of muscle LIM protein. J Cell Biol 153:763-772[Abstract/Free Full Text]
Henderson J, Macalma T, Brown D, Richardson J, Olson E, Beckerle M (1999) The LIM protein, CRP1, is a smooth muscle marker. Dev Dyn 214:229-238[Medline]
Jain M, Fujita K, Hsieh C-M, Endege W, Sibinga N, Yet S-F, Kashiki S, Lee W-S, Perrella M, Haber E, Lee M-E (1996) Molecular cloning and characterization of SmLIM, a developmentally regulated LIM protein preferentially expressed in aortic smooth muscle cells. J Biol Chem 271:10194-10199[Abstract/Free Full Text]
Jain M, Kashiki S, Hsieh C-M, Layne M, Yet S-F, Sibinga N, Chin M, Feinberg M, Woo I, Maas R, Haber E, Lee M-E (1998) Embryonic expression suggests an important role for CRP2/SmLIM in the developing cardiovascular system. Circ Res 83:980-985[Abstract/Free Full Text]
Louis H, Pino J, Schmeichel K, Pomiès P, Beckerle M (1997) Comparison of three members of the cysteine-rich protein family reveals functional conservation and divergent patterns of gene expression. J Biol Chem 272:27484-27491[Abstract/Free Full Text]
Pomiès P, Louis H, Beckerle M (1997) CRP1, a LIM domain protein implicated in muscle differentiation, interacts with
-actinin. J Cell Biol 139:157-168[Abstract/Free Full Text]
Sadler I, Crawford A, Michelsen J, Beckerle M (1992) Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton. J Cell Biol 119:1573-1587[Abstract]
Weiskirchen R, Bister K (1993) Suppression in transformed avian fibroblasts of a gene (crp) encoding a cysteine-rich protein containing LIM domains. Oncogene 8:2317-2324[Medline]
Weiskirchen R, Pino JD, Macalma T, Bister K, Beckerle M (1995) The cysteine-rich protein family of highly related LIM domain proteins. J Biol Chem 270:28946-28954[Abstract/Free Full Text]