Expression patterns of FHL/SLIM family members suggest important functional roles in skeletal muscle...

Gene expression pattern

Expression patterns of FHL/SLIM family members suggest importantfunctional roles in skeletal muscle and cardiovascular system

Po-Hsien Chua,b, Pilar Ruiz-Lozanoa,b, Qiang Zhoua,b, Chenleng Caia,b, Ju Chena,b,*

aUCSD-Salk Program in Molecular Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0613, USAbDepartment of Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0613, USA

Received 4 February 2000; received in revised form 14 April 2000; accepted 14 April 2000

Abstract

LIM domain containing proteins play critical roles in animal development and cellular differentiation. Here, we describe the cloning and

expression patterns of three members of the four and a half LIM domain-only protein family, FHL1, 2, and 3, from mouse. A comparison of

embryonic expression patterns of these three highly-related genes indicates that they are expressed in an overlapping pattern in the

developing cardiovascular system, and skeletal muscle. In adult tissues, the three genes are expressed in a predominant and overlapping

manner in cardiac and skeletal muscle. Of the three genes, FHL2 appears to have the most restricted expression pattern during development,

in heart, blood vessels, and skeletal muscle. Expression in heart is highest in cardiac septa and in the region adjacent to the atrio-ventricular

ring, suggesting a potential role in septation or conduction system development. In the heart, FHL1expression was observed strongly in

developing out¯ow tract, and to a lesser extent in myocardium. FHL3 displays low and ubiquitous expression during mouse development.

Cardiac ventricular expression of FHL1, but not FHL2 or FHL3, was upregulated in two mouse models of cardiac hypertrophic and dilated

cardiomyopathy. Taken together, these data indicate the potential importance of this FHL family in the development and maintenance of the

cardiovascular system and striated muscle, and suggest that FHL1 may play a role in the development of heart disease. q 2000 Elsevier

Science Ireland Ltd. All rights reserved.

Keywords: Four and half LIM domains; SLIM; MLP; Cardiovasculature; Skeletal muscle; Cardiac hypertrophy

1. Results and discussion

LIM domains are protein-protein interaction domains that

are receiving increasing attention from cellular and mole-

cular biologists. Named after their initial discovery in two

Caenorhabdits elegans gene products, lin-11 and mec-3,

and the rat insulin enhancer binding protein, Islet-1 (Way

and Chal®e, 1988; Freyd et al., 1990; Karlsson et al., 1990),

LIM domains have since been identi®ed in many proteins

(Dawid et al., 1998). The LIM domain is a cysteine rich

motif having the consensus sequence CX2CX16±

23HX2CX2CX2CX16±21CX2(C, H, D) that coordinately binds

two zinc atoms and mediates protein-protein interactions

(Arber and Caroni, 1996). Previous studies have revealed

that some LIM domain containing proteins are nuclear

proteins involved in cell lineage determination and pattern

formation during development, whereas others encode cyto-

solic proteins associated with the cytoskeleton, and have

been found to play a role in adhesion plaque and actin

micro®lament organization (Zhou et al., 1980; Lumsden,

1995; Beckerle, 1997; Dawid et al., 1998).

A newly identi®ed group of LIM-only proteins with four

and half LIM domains (FHL) (Chan et al., 1998; Lee et al.,

1998) are enriched in striated muscle. This family consists

of four members, three of which are expressed in adult

striated muscle: FHL1/SLIM1, FHL2/SLIM3, and FHL3/

SLIM2 (SLIM is an acronym for skeletal muscle LIM,

Morgan and Madgwick, 1996). The fourth member of this

family, FHL4 (accession number: AA144955), may be

expressed exclusively in testes, as inferred from electronic

`Northern blot' analyses, utilizing the full-length FHL4

cDNA sequence to search the EST data base. This analysis

revealed that matching sequences were found only in ESTs

from testis cDNA libraries. In con®rmation of our search, a

recent publication has also shown that FHL4 is expressed in

testes (Morgan and Madgwick, 1999). An accumulating

body of evidence indicates that FHL proteins may play

important roles in both cardiac and striated muscle (Genini

et al., 1997; Hwang et al., 1997; Takahashi et al., 1998).

A search of the mouse EST database with the LIM

domain of MLP revealed multiple EST clones correspond-

Mechanisms of Development 95 (2000) 259±265

0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0925-4773(00)00341-5

www.elsevier.com/locate/modo

* Corresponding author. Tel.: 11-858-822-2452; fax: 11-858-534-2069.

E-mail address: [email protected] (J. Chen).

ing to cDNAs encoding FHL1, FHL2, and FHL3. EST

clones AA220340 (FHL1), AA023645 and AA419967

(FHL2) and AA016340 (FHL3) were purchased from

Genome Systems Inc. and fully sequenced. Alignment of

deduced amino acid sequences and their human orthologues

were performed utilizing the CLUSTAL program (http://

www.clustalw.genome.ad.jp) (Fig. 1).

All FHL family members contain four and a half LIM

domains and share high homology throughout their amino

acid sequences. Lengths of the LIM1, LIM2, and LIM3

domains (53, 53, 51 amino acids, respectively) are similar

among all FHL family members, as is the length of the half-

LIM domain (25 amino acids). However, the length of the

LIM4 domain varies among different FHL family members.

LIM4 consists of 56 amino acids in both human and mouse

FHL1, and 55 amino acids in human and mouse FHL2, and

human FHL3. Interestingly, when compared to human

FHL3, mouse FHL3 LIM4 has a 9 amino acid insertion

resulting in a 64 amino acid domain.

To ensure that the 9 amino acid insertion in mouse FHL3

was not a cloning artifact, we performed reverse-transcrip-

tion polymerase chain reaction (RT-PCR) analysis of adult

heart RNA utilizing two primers ¯anking the insertion. Gel

electrophoresis revealed one PCR product and sequencing

P.-H. Chu et al. / Mechanisms of Development 95 (2000) 259±265260

Fig. 1. Alignment of mouse and human FHL1, FHL2, and FHL3 amino acid sequences. Identical residues are indicated with an asterisk. Each LIM domain is

indicated in bold. The cDNA sequences of mouse FHL1, FHL2, and FHL3 were deposited in Genbank (accession numbers AF114380, AF114381, and

AF114382, respectively).

of the subcloned PCR product con®rmed the 9 amino acid

insertion. The functional consequences of this insertion

remain to be explored.

Results of Northern blot analyses (Fig. 2) demonstrated

that FHL1 is expressed predominantly in skeletal muscle

and lung, and to a lesser extent in heart, brain, and kidney.

FHL2 is expressed strongly in heart, and also detectable in

brain and skeletal muscle. FHL3 is highly expressed in

skeletal muscle, and to a lesser extent in heart, lung, and

kidney.

To exploit FHL1 and FHL2 as potential lineage markers, we

performed homologous recombination to introduce a LacZ

cDNA into the endogenous FHL1 and FHL2 loci (Chu and

Chen, manuscript in preparation). Assays for b-galactosidase

activity are very sensitive and give resolution at single cell

level, permitting a detailed examination of FHL1 and FHL2

expression during embryogenesis. We have veri®ed that the

pattern of expression obtained from the LacZ knock-in mice

truly re¯ects expression of endogenous FHL1 and FHL2 by

performing RNA in situ hybridization both by whole mount

analysis with non-radioactive probes, and by analysis of

sectioned embryos with radioactive probes. The whole-

mount RNA in situ with non-radioactive probes gives us a

three-dimensional image of the expression pattern, whereas

the radioactive RNA in situ with sections provides greater

sensitivity. In addition, section analysis is required for later

stages of embryonic development. We have also performed

RNA in situ analysis of FHL3 to compare its expression to that

of FHL1 and FHL2. For each gene, results obtained by the

various approaches gave us consistent patterns. Representa-

tive examples of each experimental approach are shown (Figs.

3±6).

Our analyses demonstrated that FHL1 mRNA could be

detected at embryonic day (E)8.5, with high expression in

the out¯ow tract (Fig. 3A). At E10.5, FHL1 mRNA was

observed in heart, somites, limb buds, neural tube, brain

and eye (Fig. 3B,C). At E14, strong expression of FHL1

was observed in neural tube and vasculature (aorta, pulmon-

ary trunk) and lower levels of expression were observed in

myocardium (Fig. 4A). Strong hybridization signals for

P.-H. Chu et al. / Mechanisms of Development 95 (2000) 259±265 261

Fig. 2. Northern blot analyses of FHL mRNAs in adult tissues. Each lane contains approximately 2 mg of poly(A)1 RNA from adult mouse heart (H), brain (B),

spleen (Sp), lung (Lu), liver (Li), skeletal muscle (Sk), kidney (K), and testis (T). Probes for each FHL mRNA were generated as detailed in Materials and

Methods. An EF-1a cDNA probe was used to normalize for RNA loading. The position of RNA molecular weight standards (in kb) is indicated on the right.

Fig. 3. X-gal staining of FHL1 LacZ/knock-in mice during embryonic development. X-gal staining of FHL1 LacZ/knock-in mice revealed that FHL1 is

expressed in the out¯ow tract (arrowheads) at the heart looping stage (E8.5) (A); and in the heart, somites, neural tubes and developing limb buds (B,C). (B) and

(C) are lateral and dorsal views, respectively. Symbols are: FL, forelimb; H, heart; HL, hindlimb, NT, neural tubes; S, somites.

FHL1 at E14 were also observed in tongue (Fig. 4B), Limb

buds, abdominal muscle (Fig. 4C,D), intercostal muscle,

and thoracic muscle (Fig. 4D).

Previous analysis performed by whole mount in situ with

digoxygenin-labeled probes indicated that early embryonic

expression of FHL1 is restricted to the out¯ow tract (Brown

et al., 1999). This result is consistent with our data obtained

in the same manner. However, our data utilizing more sensi-

tive detection techniques demonstrated that FHL1 is not

only expressed in out¯ow tract but also at lower levels in

embryonic myocardium (Fig. 3A,B).

Of the three genes, FHL2 exhibited the most restricted

expression pattern, being expressed predominantly in the

cardiovascular system and in skeletal muscle. At E8.0±

E8.5, FHL2 was expressed predominately in heart (Fig.

5A,B). At E10, FHL2 was strikingly expressed in develop-

ing vasculature, including the aortic arch arteries, dorsal

aorta, and intersegmental arteries (Fig. 5C). At E14, FHL2

continued to be expressed in all compartments of the heart,

with highest levels in the ventricular septum and at the

junctional region of atria and ventricle (Fig. 6A,D). FHL2

was also detected in other striated muscle (tongue) (Fig.

6B), smooth muscle (midgut) (Fig. 6C), vasculature (umbi-

lical vein), and the area surrounding Rathke's pouch within

the pituitary primordium (Fig. 6B).

Our data represent the ®rst analysis of FHL2 expression in

developing embryos. Interestingly, expression of FHL2 in

embryonic heart is highest in the ventricular septum and

areas adjacent to the atrio-ventricular ring (Fig. 6). This

expression pattern suggests that FHL2 may play a role in the

development and function of cardiac septa, and in the cardiac

conduction system. The striking expression of FHL2 in the

developing vasculature suggests that it also play an important

role in the development of the circulatory system.


Fig. 4. Radioactive in situ hybridization analyses of FHL1 mRNA during

embryonic development. At E14, FHL1 expression is enhanced in the

ventricular out¯ow tract and great vessels (A); neural tube (B) (see arrow-

head); limb and abdominal muscles (C); tongue and intercostal muscles

(D). Symbols are: aa, aorta; ab-m, abdominal muscle; icm, intercostal

muscles; lb, limb; lg, lung; nt, neural tube; pt, pulmonary trunk; sp, inter-

ventricular septum; tn, tongue; tr-m, thoracic muscle; v, ventricle.

Fig. 5. X-gal staining of FHL2 LacZ/knock-mice during early embryonic development. X-gal staining of FHL2 LacZ/knock-in mice revealed speci®c staining

for heart and developing vasculature. The LacZ positive staining was observed in the fused heart tube at E8.0 (A); the looping heart at E8.5 (B); heart, dorsal

aorta, and arteries at E10±10.5 (C). Symbols are: Ao, aorta; H, heart.

In contrast to the restricted expression of FLH1 and FLH2,

FHL3 displayed a low and ubiquitous expression pattern

during mouse development (data not shown). However, in

adult tissues, FHL3 expression is more restricted and appears

to be inverse of that seen for FHL2, that is, extremely high

levels in skeletal muscle, and lower levels in heart (Fig. 2). As

in the case of FHL2, other adult tissues examined did not

express detectable levels of FHL3 mRNA.

To determine whether FHL1, FHL2, and FHL3 gene

expression is altered during cardiomyopathy, we exam-

ined mRNA expression in two mouse models. One of

these is a model of hypertrophy, induced by thoracic

aortic constriction (TAC). The other is a model of dilated

cardiomyopathy, generated by homozygous knockout of a

muscle LIM protein (MLP). Northern blot analysis of

RNA extracted from hypertrophic or dilated hearts indi-

cated that FHL1 mRNA expression was signi®cantly

upregulated in both, relative to control normal hearts. In

contrast, expression of either FHL2 or FHL3 mRNAs did

not change (Fig. 7A,B).

These results are consistent with the observation that

FHL1 mRNA is increased dramatically in hypertrophic

and failing hearts from human patients (Hwang et al.,

1997). In contrast, a recent study demonstrated that FHL1

mRNA levels decreased in patients with dilated cardiomyo-

pathy (Zimmermann et al., 1999). The different results may

be due to the different types and etiologies of cardiomyo-

pathy in these studies.

2. Material and methods

2.1. Genbank and EST database search

We searched Genbank and expressed sequence tag

(EST) databases for LIM domain containing proteins

using the BLAST search program (Altschul et al., 1997).

Based on our results, we subsequently focused our efforts

on the FHL family of proteins that is expressed in striated

muscle. Positive EST clones were purchased from Genome

Systems Inc.

2.2. RNA isolation and Northern blot analysis

To determine the RNA expression pattern of FHL genes

in adult mouse tissues, Northern blot analysis was

performed using an adult mouse multiple tissue Northern

blot (Clontech, #7762-1) containing 2 mg of poly(A)1 RNA

per lane. This blot was probed alternatively with a 425 bp

cDNA encoding amino acids 1 to 140 plus 3 bp of 5 0

untranslated sequence of FHL1; a 237 bp cDNA sequence

encoding amino acids 1±77 plus 6 bp of 5 0 untranslated

sequence of FHL2; and a 410 bp cDNA sequence encoding

amino acids 1±103 of FHL3.

In the transverse aortic constriction (TAC) experiment

(described below), total RNA was isolated from left ventri-

cles of both TAC and sham-operated mice using a polytron

with RNAzol B (Tel-Test). Total RNA was also isolated in a


Fig. 6. In situ hybridization analyses of FHL2 mRNA during late embryonic development. At E14, FHL2 is strongly expressed in myocardium (A), pituitary

gland and tongue (B), umbilical veins and gut (C). Within the heart, expression is very strong in both atrial and ventricular chambers (A,D) with highest level in

the ventricular septum (D) and areas adjacent to the atrio-ventricular ring (arrowheads in D). Expression of FHL2 mRNA is also observed in blood vessels, and

intestinal smooth muscle (arrowheads in A,C). Symbols are: a, cardiac atria; gt, gut; h, heart; lv, liver; nt, neural tube; pit, pituitary gland; sp, cardiac septum; tn,

tongue; uv, umbilical vein; v, ventricle.

similar manner from ventricles of wild type and homozy-

gous null MLP mutant mice. Total RNA (10 mg) was elec-

trophoresed on a 1% agarose gel and transferred to a nylon

membrane, which was hybridized subsequently with

[a-32P]dATP-labeled FHL1, 2, 3, or GAPDH cDNA probes

utilizing QuickHyb solution (Stratagene) as described by

the company.

2.3. In situ hybridization

Radioactive in situ hybridization of mouse tissues was

performed on 7±10 mm paraf®n sections as described

(Ruiz-Lozano et al., 1997). Probes were designed as

follows: a 470 bp BamH I-Hind III fragment of FHL1

cDNA (encoding AA 1±140 plus 50 bp of 5 0 untranslated

region), a 490 bp EcoR I- Pst I fragment of FHL2 cDNA

(encoding AA 1±160 plus 10 bp of untranslated region), and

a 362 bp Pst I fragment of FHL3 cDNA (encoding AA 165±

285) were each subcloned into pBluescript II KS (Strata-

gene) to generate templates for sense and antisense 35S-

UTP- or digoxigenin-labeled probes. To evaluate expression

patterns of FHL mRNAs during mouse development, both

radioactive and non-radioactive RNA in situ hybridization

were performed. Non-radioactive in situ hybridization of

whole-mount embryos was performed using digoxigenin-

labeled synthetic oligo-DNA probes following standard

techniques (Faerman and Shani, 1997).

2.4. Whole-mount X-gal staining of FHL1 and FHL2

transgenic embryos

For whole-mount LacZ expression analysis, transgenic

embryos were ®xed and stained using standard methods

(Ross et al., 1996). The duration of staining was dependent

on color development and ranged from 4 to 12 h at room

temperature or 308C. Whole embryos or tissues were

analyzed and photographed on a Zeiss SV-6 dissecting

microscope with a Nikon C-mount 35mm camera.

2.5. Transverse aortic constriction (TAC)

Pressure overload hypertrophy was induced in mice by

using a microsurgical technique involving transverse aortic

constriction (TAC) as previously described (Rockman et al.,

1991). Sham-operated animals underwent the same opera-

tion with no aortic constriction. Transthoracic echocardio-

graphy and histological studies were utilized to ensure that

ventricular hypertrophic had occurred (data not shown).

Acknowledgements

We thank Dr Kenneth R. Chien for his enthusiastic

support throughout the project, and Drs Sylvia Evans,

Tara R. St Amand and Raymond Clark for critical reading

of the manuscript. The authors would like to thank Julie

Anderson and Wendy Bardwell for expert technical assis-

tance. Dr Chu was supported by a grant from the Chung

Gang Memorial Hospital at Taiwan.

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