Differential mesodermal expression of two amphioxus MyoD family

members (AmphiMRF1 and AmphiMRF2)

Michael Schuberta, Daniel Meulemansb, Marianne Bronner-Fraserb,Linda Z. Hollandc,*, Nicholas D. Hollandc

aEcole Normale Superieure de Lyon, Laboratoire de Biologie Moleculaire et Cellulaire, UMR 5665 CNRS, 46, Allee dItalie, 69364 Lyon, Cedex 07, FrancebDivision of Biology, 139-74, California Institute of Technology, Pasadena, CA 91125, USA

cMarine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093-0202, USA

Received 9 October 2002; received in revised form 11 December 2002; accepted 13 December 2002

Abstract

To explore the evolution of myogenic regulatory factors in chordates, we isolated two MyoD family genes (AmphiMRF1 and AmphiMRF2)

from amphioxus. AmphiMRF1 is first expressed at the late gastrula in the paraxial mesoderm. As the first somites form, expression is

restricted to their myotomal region. In the early larva, expression is strongest in the most anterior and most posterior somites. AmphiMRF2

transcription begins at mid/late gastrula in the paraxial mesoderm, but never spreads into its most anterior region. Through much of the

neurula stage, AmphiMRF2 expression is strong in the myotomal region of all somites except the most anterior pair; by late neurula

expression is downregulated except in the most posterior somites forming just rostral to the tail bud. These two MRF genes of amphioxus

have partly overlapping patterns of mesodermal expression and evidently duplicated independent of the diversification of the vertebrate MRF

family.

q 2003 Elsevier Science B.V. All rights reserved.

Keywords: BMD1; BMD2; Branchiostoma floridae; Cephalochordate; Independent gene duplication; Muscle; Myogenesis; Somite; Transcription factor;

MyoD family; MRF; MRF4; Myf5; MyoD; Myogenin

1. Results and discussion

Members of the vertebrate MyoD or myogenic regulatory

factor (MRF) gene family (namely MyoD, Myf5, MRF4, and

myogenin) encode transcription factors characterized by a

basic helix-loop-helix (bHLH) domain mediating DNA

binding and protein dimerization. These genes are expressed

in developing paraxial mesoderm, somites, derivatives of

somites (e.g. pre-muscle cells of visceral arches and limb

buds), and skeletal muscles (Rescan, 2001). During

vertebrate development, MyoD and Myf5 act early in

myogenic determination, whereas myogenin and MRF4 act

later, during muscle differentiation (Ordahl and Williams,

1998; Buckingham, 2001; Rescan, 2001). Within ver-

tebrates, differences in the expression patterns and functions

of MRF family genes have been interpreted as adaptations to

different patterns of body muscle formation.

Within the invertebrate deuterostomes, full-length MRF

genes have been isolated from sea urchins and tunicates,

while two PCR fragments have been cloned from

amphioxus (Araki et al., 1994, 1996; Meedel et al., 1997;

Beach et al., 1999). Gene expression studies have shown

that transcription of sea urchin MyoD is not restricted to

myogenic lineages (Beach et al., 1999), whereas expression

of tunicate MyoD is found in muscle cells flanking the larval

notochord (Meedel et al., 1997). Neither full-length clones

nor expression data have yet been published for any

amphioxus species.

Like vertebrates, amphioxus has segmentally arranged

somites that share many properties with vertebrate somites

including expression of alkali myosin light chain and

muscle actin (Holland et al., 1995; Kusakabe et al., 1997).

To better understand the evolutionary history of muscle

differentiation in the chordate lineage, we have isolated and

studied the developmental expression of two amphioxus

MRF family members (AmphiMRF1 and AmphiMRF2). The

AmphiMRF1 cDNA (accession number AY154744) is 1108

1567-133X/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S1567-133X(02)00099-6

Gene Expression Patterns 3 (2003) 199202

www.elsevier.com/locate/modgep

* Corresponding author. Tel.: 1-858-534-5283; fax: 1-858-534-7313.E-mail address: lzholland@ucsd.edu (L.Z. Holland).

bp long and encodes a putative protein of 259 amino acids,

while AmphiMRF2 cDNA (accession number AY154745) is

1248 bp long and encodes a putative protein of 229 amino

acids.

The phylogenetic tree (Fig. 1) is based on MRF proteins

from the major deuterostome groups plus two protostomes

(Drosophila and Caenorhabditis). The tree supports the

hypothesis of Araki et al. (1996) that the duplication of the

amphioxus MRF locus was probably lineage-specific and

thus independent of the diversification of the vertebrate

MRF family. The analysis also confirms the results of

Atchley et al. (1994), who showed that the family of

vertebrate myogenic regulatory factors branches, respect-

ively, into a MyoD1/Myf5 and a MRF4/myogenin clade. In

Fig. 1, the grouping of tunicate AMD1 with vertebrate

myogenins may represent a tree-building artifact, because

the position of AMD1 is not well supported by boot-

strapping or by statistical analysis (method of Kishino and

Hasegawa, 1989). In addition, comparisons of

AmphiMRF1, AmphiMRF2, and AMD1 with Ciona

genome sequences (http://www.jgi:doe.gov/programs/

ciona/ciona_mainpage.html) indicate that tunicates have

only one bHLH myogenic factor.

The patterns of AmphiMRF1 and AmphiMRF2 gene

expression were examined from mid-gastrulation through

the early larval stage. The two transcription factors display

distinct but partly overlapping distributions. AmphiMRF1 is

first expressed in the paraxial (dorsolateral) mesoderm on

either side of the late gastrula embryo (Fig. 2A). At the

neurula stage (Fig. 2B,C), this gene continues to be

expressed in all the somites, including the newly formed

ones arising from the tail bud. Transverse sections (Fig. 2D)

reveal that expression is limited to the myotomal component

of each somite. In the early larva (Fig. 2E), transcription is

detectable in all the somites, but is most conspicuous

anteriorly in the first two pairs and posteriorly in the newly

formed ones just anterior to the tail bud. AmphiMRF1

expression is not detectable in later larvae.

In contrast to AmphiMRF1, AmphiMRF2 is expressed

slightly earlier, in the paraxial mesoderm of the mid/late

gastrula (Fig. 2F,G). In the early and mid neurula stages

(Fig. 2HJ), expression is in the myotomal region of the

somites (except the first). The undetectable expression of

AmphiMRF2 in the first somite mirrors similar expression

patterns for AmphiWnt8 and AmphiWnt11, which mark the

rest of the paraxial mesoderm in developing amphioxus

(Schubert et al., 2000a,b). By the late neurula stage (Fig.

2K), AmphiMRF2 expression becomes limited to the most

posterior somites just rostral to the tail bud. No AmphiMRF2

expression is detectable during subsequent development.

Neither of the amphioxus MRF genes is expressed in the

developing notochord, which is a modified muscle expres-

sing many muscle-specific genes (Suzuki and Satoh, 2000).

Because the notochord and somitic muscles express

different actins, it is possible that amphioxus has a

notochord-specific MRF gene that has yet to be discovered.

2. Materials and methods

2.1. Cloning of myogenic regulatory factor cDNAs

AmphiMRF1 was obtained by screening a cDNA library

in Lambda ZAP II (Stratagene) from 8 to 18 h Branchios-

toma floridae embryos. A probe recognizing bHLH domains

was used for this low stringency screening, which resulted

in the isolation of a full-length cDNA for AmphiMRF1. The

primer for the isolation of the 30 end of AmphiMRF2 wasdesigned using the BMD2 PCR fragment previously

described by Araki et al. (1996). The 30 end of AmphiMRF2was then isolated by PCR with the gene- and a vector-

specific primer using the 8 to 18 h cDNA library as a

template. The 50 end of AmphiMRF2 was subsequentlyobtained by 50 RACE with the GeneRacer Kit (Invitrogen).

2.2. Phylogenetic analysis

The phylogenetic analysis of 15 representative members

of the MRF family was based on a total of 103 amino acid

sites including the bHLH domain, the regions flanking the

bHLH sequence, and the C-terminal Domain III. The

alignment includes the AmphiMRF1 and AmphiMRF2

sequences from Branchiostoma floridae as well as the MDF

sequence from Branchiostoma belcheri. The tree was

Fig. 1. Phylogenetic tree (PAUP 3.1.1) of the MRF family based on

representatives from the major deuterostome lineages as well as outgroup

sequences from Drosophila and Caenorhabditis; support values are

bootstrap percentages. The arrow indicates the clade with amphioxus

MRF sequences (bold). The GenBank accession numbers are: CeMyoD

Caenorhabditis (M59940), Dmyd Drosophila (M68897), SUM1 sea urchin

(AF143808), AMD1 tunicate (D13507), myogenin mouse (NM_031189),

myogenin human (AF050501), MRF4 mouse (NM_008657), MRF4 human

(NM_002469), Myf5 mouse (NM_008656), Myf5 human (NM_005593),

MyoD1 mouse (NM_010866), MyoD1 human (XM_036339), MDF

Branchiostoma belcheri (AY066009), AmphiMRF1 (AY154744),

AmphiMRF2 (AY154745).

M. Schubert et al. / Gene Expression Patterns 3 (2003) 199202200

Fig. 2. Developmental expression of AmphiMRF1 (AE) and AmphiMRF2 (FK) in whole mounts (with 50 mm scale lines) and sections (counterstained pink

with 25 mm scale lines). For whole mounts in side or dorsal views, anterior is toward the left. (A) Dorsal view of late gastrula with AmphiMRF1 signal in the

paraxial mesoderm; the arrow indicates the blastopore. (B) Dorsal view of early neurula with expression in the paraxial mesoderm. (C) Dorsal view of late

neurula with expression in the myotomal region of all the somites. (D) Cross-section through plane indicated by arrows in (C) with expression in the somitic

myotomes (arrowed); the neural tube (nt) and notochord (no) are indicated. (E) Side view of a 2 day larva with transcripts in the myotomes of all the somites,

most conspicuously anteriorly and posteriorly. (F) Side view of mid/late gastrula (blastopore toward right) with AmphiMRF2 expression in the early paraxial

mesoderm. (G) Transverse view of (F) showing signal in the paraxial mesoderm. (H) Dorsal view of an early neurula with expression in the myotomal region of

the forming somites posterior to the first one. (I) Dorsal view of a mid neurula with expression in the myotomes of all somites posterior to the first. (J) Cross-

section through plane indicated by arrows in (I) showing expression in somitic myotomes (arrowed); the neural plate (np) and nascent notochord (no) are

indicated. (K) Side view of a late neurula with AmphiMRF2 expression limited to the most posterior somites.

M. Schubert et al. / Gene Expression Patterns 3 (2003) 199202 201

calculated with PAUP 3.1.1 in 100 random stepwise

additions and bootstrap percentages were calculated in

1000 replicates with ten random stepwise additions per

bootstrap cycle. Only one most parsimonious tree (length

224) was retained in the analysis. The Caenorhabditis

CeMyoD sequence was used as the outgroup. GenBank

accession numbers are given in the caption to Fig. 1.

2.3. Embryo collection, in situ hybridization, and histology

Ripe animals of the Florida amphioxus (Branchiostoma

floridae) were collected in Old Tampa Bay, Florida, and

gametes were obtained by electrical stimulation. Embryos

and larvae were raised at 22.5 8C according to Holland andHolland (1993). In situ hybridization and histology were

performed according to Holland et al. (1996).

Acknowledgements

The authors are indebted to John M. Lawrence at the

University of South Florida for laboratory facilities, to Jim

A. Langeland for the amphioxus cDNA library and to Marc

Robinson-Rechavi for invaluable statistical advice. In

addition, the manuscript was improved by the constructive

criticisms of Vincent Laudet, Hector Escriva Garcia,

Beatrice Horard, and Pierre-Luc Bardet. This work was

supported by a postdoctoral fellowship from the German

Academic Exchange Service (DAAD) to M.S., by NSF

grant IBN0078599 to N.D.H. and L.Z.H., and by NASA

grant 98-HEDS-02 to M.B.F. and NAG2-1376 to L.Z.H.

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Differential mesodermal expression of two amphioxus MyoD family members (AmphiMRF1 and AmphiMRF2)Results and discussionMaterials and methodsCloning of myogenic regulatory factor cDNAsPhylogenetic analysisEmbryo collection, in situ hybridization, and histology

AcknowledgementsReferences