A single amphioxus and sea urchin runt-gene suggests that runt-gene duplications occurred in early chordate evolution

A single amphioxus and sea urchin runt-gene suggests

that runt-gene duplications occurred in early

chordate evolution

S. Strickera,*, A.J. Poustkaa,*, U. Wiechaa, A. Stiegea, J. Hechta, G. Panopouloua,A. Vilcinskasb, S. Mundlosa,†, V. Seitza,†

aMax Planck Institute Molecular Genetics, Ihnestr. 73, 14195 Berlin, GermanybSystematic Zoology and Evolutionary Biology, University of Potsdam, Villa Liegnitz, Lennestr. 7a, 14417 Potsdam, Germany

Received 4 October 2002; revised 16 January 2003; accepted 28 January 2003

Abstract

Runt-homologous molecules are characterized by their DNA binding runt-domain which is highly conserved within

bilaterians. The three mammalian runt-genes are master regulators in cartilage/bone formation and hematopoiesis. Historically

these features evolved in Craniota and might have been promoted by runt-gene duplication events. The purpose of this study

was therefore to investigate how many runt-genes exist in the stem species of chordates, by analyzing the number of runt-genes

in what is likely to be the closest living relative of Craniota—amphioxus. To acquire further insight into the possible role of

runt-genes in early chordate evolution we have determined the number of runt-genes in sea urchins and have analyzed the runt-

expression pattern in this species.

Our findings demonstrate the presence of a single runt-gene in amphioxus and sea urchin, which makes it highly likely that

the stem species of chordates harbored only a single runt-gene. This suggests that runt-gene duplications occurred later in

chordate phylogeny, and are possibly also associated with the evolution of features such as hematopoiesis, cartilage and bone

development.

In sea urchin embryos runt-expression involves cells of endodermal, mesodermal and ectodermal origin. This complex

pattern of expression might reflect the multiple roles played by runt-genes in mammals. A strong runt-signal in the

gastrointestinal tract of the sea urchin is in line with runt-expression in the intestine of nematodes and in the murine

gastrointestinal tract, and seems to be one of the phylogenetically ancient runt-expression domains.

q 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Runt; Amphioxus; Sea urchin; Cartilage; Bone; Hematopoiesis; Gene duplication; Chordates

1. Introduction

Runt-homologous molecules are a small family of

transcription factors, characterized by a DNA binding

runt-domain, which is highly conserved in bilaterians.

0145-305X/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0145-305X(03)00037-5

Developmental and Comparative Immunology 27 (2003) 673–684

www.elsevier.com/locate/devcompimm

* Both authors contributed equally to this work.† Corresponding authors. Tel.: þ49-30-8413-1263; fax: þ49-30-

8413-1385.

E-mail addresses: [email protected] (V. Seitz);

[email protected] (S. Mundlos).

Abbreviations: UTR, untranslated region; bp, base pair;

WMISH, whole mount in situ hybridization; hr, hour; Gen-Dup,

gene duplication; P1, distal promoter; P2, proximal promoter.

Runt-transcription factors have been described,

among other species, in the fruit fly [1], in nematodes

[2], in mice [3] and in humans [4]. Interestingly, the 3

runt-genes characterized in mammals are related to

features, which evolved during early chordate evol-

ution, such as cartilage, bone and hematopoiesis

(Fig. 1). Runx-2 (also CBFA-1; PEBP2aA; AML-3) is

a master regulator of osteoblast differentiation [4].

Furthermore, runx-2 as well as runx-3 (also CBFA-3;

PEBPaC; AML-2) are both involved in cartilage

formation [5–7]. An important role in hematopoiesis

has been demonstrated for runx-1 (CBFA-2; PEB-

P2aB; AML-1) and runx-3 is also expressed in

hematopoietic cells [6,8–12]. However, runt-genes

are not only expressed in newly evolved chordate

organ structures but also in tissues common to all

bilaterians e.g. in neural cells [6,13]. Furthermore,

runx-3 has been shown to be expressed in the murine

gastrointestinal tract [14]. This is comparable to the

expression of runt in the intestine of the nematode

Caenorhabditis elegans [2].

To date there is still no information available on

the number and expression of runt-genes in the stem

species of the phylum chordates. As two runt-genes

have been reported in Drosophila melanogaster [15],

one in C. elegans [16] and three in mammals [4,12,17]

it can be assumed that in the stem species of chordates

one or two runt-genes were most likely present, and

were probably also involved in gut and nervous tissue

development.

Fig. 1. Overview of runt-gene number and function in bilaterians mapped on a simplified phylogeny of bilaterians, showing a contentious

position for hagfish [42–44]. Although the presented phylogeny is widely accepted, alternative chordate phylogenies have been proposed [45,

46]. The step-wise evolution of cartilage [47], bone and hematopoiesis and the most likely time intervals of gene-duplications (Gen-Dup)

according to Holland [48] are indicated. As the position of the fossil Ostracodermi and Placodermi is still unclear, the time of the evolution of

desmal bone and the internal skeleton is also uncertain [42]. The exon/intron structure of the B. floridae runt-gene locus with estimations of its

intron lengths (in base pairs) is shown. P1: distal promoter; P2: proximal promoter.

S. Stricker et al. / Developmental and Comparative Immunology 27 (2003) 673–684674

To clarify this question we have searched for runt-

genes in the cephalochordate amphioxus, thought to

be the closest living relative of Craniota. We have also

analyzed the number of runt-genes, and the runt-

expression pattern in the sea urchin Strongylocentro-

tus purpuratus, a deuterostome more distantly related

to the phylum chordates.

2. Materials and methods

2.1. Materials

Adult specimens of Branchiostoma lanceolatum

(amphioxus) were obtained from the Biologische

Anstalt Helgoland (Germany) and kept in natural

seawater. Developmental stages of 1.5–3.5 day old

larvae were kindly provided by Prof. Denuce

(Catholic Univ. Nijmegen; The Netherlands) using

the facilities of the Biologische Anstalt Helgoland.

Mature specimens of another species of

amphioxus, B. floridae, were collected by shovel

and sieve in water of 1 m in depth in Tampa Bay,

Florida, during the summer breeding season [18].

Ripe adults of S. purpuratus were obtained from

Marinus Inc. Long Beach, CA. USA. Urchins were

kept at 10 8C in 100 l tanks in seawater obtained

from the Biologische Anstalt Helgoland up until

spawing. As a probe for the S. purpuratus runt-gene

clone 691REA_13B04 was used for antisense DIG

labelled RNA synthesis. This clone was identified in

a S. purpuratus EST project (Poustka et al. unpub-

lished results) and covers the entire 50-untranslated

region (UTR) and the first 105 bp of the coding

region.

2.2. RNA Isolation

Total RNA was isolated using Trizol peqGold

TriFast (peqLab Biotechnologie GmbH, Erlangen,

Germany) in accordance with the manufacturer’s

instructions from adult B. floridae and B. lanceolatum

as well as from pooled 1.5, 2.5 and 3.5 day old B.

lanceolatum larvae. This was followed by DNAse

treatment (RNase-Free DNase Set; Qiagen, Hilden,

Germany) and repurification with RNeasyw Mini-Kit

(Qiagen).

2.3. Amplification of amphioxus runt-genes

B. lanceolatum. A RT-PCR with the degenerated

runt-domain primers runt-up (CAC TGG CGG TSS

AAG AAG) and runt-low (ACG AAN CGC AGG

TCG TTR AA) was performed (GeneAmpw RNA-

PCR core kit PCR, Roche, Lewes, UK) in accordance

with the instruction manual. Cycle conditions were

96 8C 2 min; 5 £ (96 8C 15 s; 57 8C 30 s; 72 8C 30 s)

and 35 £ (96 8C 15 s; 55 8C 30 s; 72 8C 30 s). After

purification and sequencing of the PCR-product, the

30- and 50ends of the runt-cDNA were obtained by

RACE-PCR (Clontech Heidelberg, Germany) in

accordance with the manufacturer’s recommen-

dations, employing the gene specific primers:

BrGSP3-RACE (CAA GAC GCT CCC CGT GCC

TTT C), BrGSP3-nestRACE (GTC ATG GCA GGG

AAC GAC GAG), BrGSP5-RACE (CTC GTC GTT

CCC TGC CAT GAC) and BrGSP5-nestRACE (GAA

AGG CAC GGG GAG CGT CTT G).

B. floridae. A cDNA clone identified in a randomly

sequenced cDNA library (neurula stage, 26 h; Panou-

pulou, unpublished) contained the complete 30-UTR

and 966 bp of the 30-coding region of the B. floridae

runt-gene. To obtain the full length coding region a

RT-PCR was performed with B. floridae cDNA using

a primer specific to the 50-UTR of B. lanceolatum

(ACA CAC CAG AGC AGC GAC AG) in conjunc-

tion with a 30-UTR primer (CAG TTT GCC TTG

ACT GAC ACC CAT).

The coding runt-gene sequence of B. floridae and

B. lanceolatum was confirmed by RT-PCR employing

proof-reading polymerase (Pwo DNA-polymerase,

Roche) and primers annealing within the 50- and 30-

UTRs.

2.4. DNA-isolation and Southern blot

DNA was isolated from one specimen of B.

floridae and the sea urchin S. purpuratus by grinding

approximately 250 mg of tissue in liquid nitrogen and

resolving the powder in 40 ml TEN 9 buffer (50 mM

Tris pH 9, 100 mM EDTA, 200 mM NaCl). After

addition of RNAse A (Roche; final concentration

100 mg/ml), proteinase K (Roche; final concentration

1 mg/ml) and SDS (Roth, Karlsruhe, Germany, final

concentration 1%) digestion followed by incubation

overnight at 50 8C. Thereafter 2 rounds of phenol

S. Stricker et al. / Developmental and Comparative Immunology 27 (2003) 673–684 675

purification were followed by phenol/chloroform/i-

soamylalcohol (25:24:1) and chloroform/isoamylal-

cohol (24:1) purification. The DNA was precipitated

with NaCl (final concentration 250 mM) and 0.7

volumes of isopropanol, washed in 70% ethanol and

resolved in 0.5% TE buffer. Southern blots were

prepared from digested DNA (B. floridae: EcoR1 and

Hind3; S. purpuratus: BamH1 and Hind3). To

determine the number of runt-related genes and

copy number, the blot was hybridized with a 32P-

labelled fragment of exon-2 that included a part of the

highly conserved runt-domain. (bp 21–326 B. lan-

ceolatum, Acc Nr. AY146615 and bp 489–821 S.

purpuratus, AccNr: U41512.1). The hybridization

was carried out at moderate stringency in Church

buffer (7% SDS, 0.5 M, Na2HPO4 pH 7.2, 1 mM

EDTA) with 0.1 mg/ml herring sperm overnight at

60 8C. Washes were performed with Church wash

buffer (25 mM Na2HPO4, 1% SDS) three times at

room temperature followed by another three washes at

60 8C for 30 min. The membrane was then exposed to

a phosphorimager screen.

2.5. Genomic walking

A universal genomic walking library (Clontech)

was constructed from B. floridae DNA in accordance

with the manufacturers instructions. Gene specific

primers were used to amplify runt-DNA fragments

containing the exon/intron borders, which were

determined by sequencing employing exon-specific

primers. In addition long distance PCRs were used to

determine the exon/intron structure and the size of the

amphioxus runt-gene locus.

2.6. Phylogenetic analysis

A maximum parsimonious phylogenetic tree was

constructed using the program PAUP (version 3.1.1)

in 100 rounds of heuristic random stepwise additions

[19] with runt-gene sequences of S. purpuratus, B.

lanceolatum, B. floridae, M. musculus and H. sapiens

using runt-genes of D. melanogaster and C. elegans as

an outgroup. Gaps in the alignment were treated as

inapplicable characters using ‘?’ [20]. Alignments

were constructed using Clustal W [21] from full

length runt-protein sequences and further with

highly conserved runt-cDNA-sequence portions,

which combined the 384 bp runt-gene domain with

a conserved 99 bp region beginning with the start

codon of exon-2, and with another 24 bp region

ending with the 30-VWRPY-motive.

2.7. Whole mount in situ hybridization (WMISH)

Sea urchins were induced to spawn by the injection

of 0.55 M KCl solution. Embryo cultures were grown

at 15 8C and fixed for WMISH for 2 h in 2.5%

glutaraldehyde in phosphate buffer or in 4% PFA in

MOPS buffer as described in Ref. [22]. Sea urchin

embryo WMISH was carried out according to the

protocol given in Ref. [23], with the following

changes. Hybridisation and post hybridisation washes

were carried out at 65 8C. The third posthybridization

wash was accomplished using 0.1 £ SSC instead of

1 £ SSC. Probe concentration was raised to 1 ng/ml,

while the anti-DIG-AP Antibody concentration was

decreased to a dilution of 1:3500. Staining was carried

out employing the BCIP/NBT system (Vector Lab-

oratories Inc., Burlingame, USA) according to

manufacturers instructions.

3. Results

3.1. Isolation of the B. lanceolatum

and B. floridae runt-gene

A protein sequence alignment of the isolated B.

lanceolatum and B. floridae runt-gene is given in

Fig. 2. RACE PCR in conjunction with genomic DNA

sequencing revealed two runt-gene variants. Whereas

one runt-gene variant begins with exon-1 (only in the

larvae), the other begins with exon-2 (expressed in

adults and larvae). This structure corresponds to the

murine and human runt-gene organization [12,17,24]

and strongly indicates the presence of two distinct

amphioxus runt-gene promoters. Identically, mam-

malian runx-1, -2 and -3 genes have two promoters.

These have been described as distal, and as proximal

promoters [12,17,24]. The distal promoter controls the

expression of mammalian runt-genes beginning with

exon-1, whereas the proximal promoter regulates

runt-gene expression from exon 2 [12,17,24].

The amphioxus runt-gene has 5 exons (Fig. 2). One

intron, within the highly conserved runt-domain,

S. Stricker et al. / Developmental and Comparative Immunology 27 (2003) 673–684676

Fig. 2. Runt-protein alignment (ClustalW; BioEdit: www.mbio.ncsu.edu/BioEdit/):B. floridae (B.f; AccNr: AY146617), B. lanceolatum (B.l;

AccNr. AY146615), and Mus musculus runx-1, -2- and 3 (M 1-3; AccNrs: BAA02960.1, BAA03485.1, AF155880_1). Exon/intron borders are

marked in yellow (in case only one amino acid is colored its codon is separated in the two exons. ‘Exon-6’ is shown in blue for runx-1 and runx-

2 and ‘exon-6b’ is shown in purple for runx-2. As ‘exon-6’ is missing in runx-3 [35] the alignment has been adjusted manually for single amino

acids that aligned within this region. The first line shows the amino-terminal part of the protein derived from promoter P1 (M.1-3: AF193030.1,

AF053955.1, XM_144093.1; B.f. AY146618; B.l. AY146616) which differs from the P2-derived form.

S. Stricker et al. / Developmental and Comparative Immunology 27 (2003) 673–684 677

which is present in murine and human runx1-3 genes,

is missing in the amphioxus runt-gene, whereas the

other exon/intron boundaries are at similar locations

(Fig. 2). The amphioxus runt-gene locus comprises

approximately 22 300 bp (Fig. 1). As the introns are

highly polymorphic with numerous deletions and

insertions their sizes can only be estimated.

3.2. Number of runt-genes in amphioxus

and sea urchin

A Southern blot with DNA from one B. floridae

and a S. purpuratus, revealed a single fragment in

the BamH1 digestion of the S. purpuratus DNA and

in the other three digestions two fragments of equal

intensity were detected (Fig. 3). These corresponded

to two alleles of one single runt-gene from B.

floridae and S. purpuratus. The presence of two

runt-gene alleles in B. floridae was confirmed by

genomic walking—using DNA from the same

individual as that for the Southern blot. Sequence

analysis of the proximal promoter (unpublished

data) revealed a Hind3 site approximately 3800 bp

upstream of the exon-2, which was only present in

one of the two runt-alleles.

3.3. Phylogenetic analysis

The phylogenetic analysis (Fig. 4(a)) with full

length runt-protein sequences of D. melanogaster,

C. elegans, S. purpuratus, B lanceolatum, B.

floridae, M. musculus and H. sapiens is in harmony

with the species tree (Fig. 1), if two runt-gene

duplications are postulated in chordate evolution.

The molecular phylogeny suggests runx-3 to be the

sister-clade to runx-1 and -2, this is however, only

weakly supported by a bootstrap value of 53. In Fig.

4(b) the phylogenetic tree is based on a DNA

alignment of conserved runt-gene regions. It shows

the same result as Fig. 4(a), however the clades of

human and murine runx-1, -2 and -3 genes are not

resolved.

Fig. 2 (continued )

Fig. 3. Southern blot: B. floridae DNA digested with Hind3 (lane 1)

and EcoR1 (lane 2); S. purpuratus DNA digested with BamH1 (lane

3) and Hind3 (lane 4). The lambda/Hind3 bp marker is shown. In

both species only a single runt-gene is present with two distinct

alleles.

S. Stricker et al. / Developmental and Comparative Immunology 27 (2003) 673–684678

3.4. Expression pattern of runt in sea urchin embryos

Runt-gene expression was analyzed throughout sea

urchin embryogenesis by WMISH. Runt-RNA was

expressed ubiquitously up until the mesenchymal-

blastula stage (20 h). We speculate that this

ubiquitous distribution might correlate with maternal

Runt-mRNA [25]. Zygotic runt-transcription has been

shown to start at around 20 h of development [25].

These zygotically produced runt-transcripts were

found to be enriched in the endo-mesodermal domain

in the vegetal plate until the point where endo-

mesoderm invagination occurred (30 h and 35 h

embryos in Fig. 5(a) and (b)). During gastrulation

runt-gene expression continued to be prominent

within the invaginating gut. In addition to the gut

expression, in a late gastrula stage (45 h of develop-

ment) we found higher levels of runt-gene expression

in the oral ectoderm (Fig. 5c1). At this stage of

development some single cells in the aboral ectoderm

were stained, this most likely represented secondary

mesenchyme cells (arrowheads in Fig. 5c1). In the

prism and pluteus stages embryos (60–70 h) runt-

gene expression was found at the highest level in the

elongating arms (arrows in Fig. 5d2) and also

continued to be prominent throughout the gut, oral

ectoderm and ciliated band, while expression in single

aboral ectoderm cells was apparently lost. (Fig. 5d).

Within the ciliated band some cells showed higher

levels of expression than others (arrow in Fig. 5d3).

4. Discussion

In ancestral chordates either multiple single gene

duplications or complete genome duplications

resulted in an increased number of genes in ver-

tebrates [26–30]. In contrast to homologous genes—

which evolved in the speciation process (orthologous

genes) and often maintain a similar function during

evolution—it has been postulated that after gene

duplication the duplicated genes (paralogous genes)

Fig. 4. Phylogenetic analyses: The most parsimonious phylogenetic tree of runt-genes based on ClustalW alignments with; (a) full length runt-

protein sequences, and; (b) highly conserved parts of the runt-gene cDNA sequence (the branch length in ‘a’ indicates the number of

apomorphies). The tree was constructed by random stepwise addition with 100 replicates taking D. melanogaster and C. elegans runt-genes as

an outgroup. The values at the branching points are bootstrap percentages (100 replicates). The Acc. Nr of the runt-genes used are D.

melanogaster runt, NM_078700.1 (D.r); D. melanogaster lozenge, NM_078544.1 (D.l); C. elegans rnt, AB027412.1 (C.r); S. purpuratus

SpRunt-1, U41512.(S.p); Mus musculus runx-1-3, D13802.1, D14636, AF155880 (M.1-3); Homo sapiens runx 1-3, Q01196, NM_004348,

X79550.2 (H.1-3).

S. Stricker et al. / Developmental and Comparative Immunology 27 (2003) 673–684 679

Fig. 5. Expression pattern of runt-RNA in S. purpuratus embryos: (a) and (b) 30 h and 35 h embryo. Early gastrula embryo, transcripts are

enriched in the vegetal plate and invaginating endomesodermal domain. Vegetal sides of the embryos are located toward the bottom of the

pictures. (c) WMISH in 45 h embryo (late gastrula stage). Side (c1), oral (c2) and vegetal (c3) view, respectively. In (c1) and (c3) the oral side of

the embryo is located to the right. In (c1) and (c2) the vegetal side of the embryo is located to the bottom of the picture. Expression is detected at

the highest levels in the gut and the oral ectoderm. The aboral ectoderm remains largely unstained. Only some single cells in the aboral ectoderm

express runt-RNA (arrowhead in c1). Arrow in c1 points to an enhanced expression domain around the stomodeum. (d) Expression of runt-RNA

in 70 h (pluteus stage) embryos. Side (d1 oral is oriented to the right) and oral view (d2, d3) in two different angles respectively. Expression is

apparent in the ciliated band of the oral ectoderm. Some further uncharacterized cells within the ciliated band show higher expression levels of

runt than others, some of which are depicted with arrows in 5d3. Arrows in 5d2 show the highest expression levels in elongating arms.

Expression remains high in the entire gut, with the highest levels most probably in the foregut (5d1). Cells in the aboral ectoderm did not

continue to express runt-RNA in 70 h embryos (5d1).

S. Stricker et al. / Developmental and Comparative Immunology 27 (2003) 673–684680

could fulfill some distinct new functions [31,32]. It

needs to be further defined in which time period after

gene duplication different gene functions could

evolve. Furthermore it is of interest to see if gene

duplication events have a causal relationship to the

evolution of new organ structures such as cartilage

and bone. Runt-genes are ideal in helping to find

answers to these questions, as they comprise of a

small family of transcription factors (#3 per species)

and are key players in features newly evolved within

the chordates, such as bone and hematopoiesis.

To analyze the early runt-gene evolution in

chordate phylogeny, we searched for runt-genes in

amphioxus—a chordate deuterostome—to determine

the runt-gene number and exon/intron structure. In

addition, we analyzed the number and expression of

runt-genes in sea urchin embryos, a deuterostome

more distantly related to the chordates.

The number and function of runt-genes in bilater-

ians are shown in a simplified phylogenetic tree (Fig.

1). Our finding of only one runt-gene in the sea urchin

and amphioxus (Fig. 3) and the presence of a single

runt-gene in C. elegans leads us to the conclusion that

the stem species of chordates harbored only a single

runt-gene.

Comparison of murine and human runx-1, -2 and -

3 structures [17,33,34] with the amphioxus runt-gene

reveals a strong similarity between runt-genes in

respect of their genomic organization, DNA sequence

and transcriptional regulation by two distinct promo-

ters. Runx-1 is the largest in the runt-gene family and

spans approximately 260 kb with 11 exons. Runx-2

comprises approximately 222 kb and has 8 exons [35]

whereas runx-3 spans only approximately 67 kb and

has only 6 exons [12]. Beside runx-3 being the

smallest of the three mammalian runt-genes it

contains the highest level of the ancient mammalian

repeat Mir [12]. It was therefore speculated, that runx-

1 and runx-2 underwent mammalian-specific

sequence expansion, during which the MIRs were

replaced with more recent retroposon insertions [12].

The notion that runx-1 and runx-2 are derived,

whereas runx-3 shares more plesiomorphic features

with the hypothetical runt-gene of the chordate stem

species, is in agreement with the small size of the

amphioxus runt-gene locus (approximately 22.3 kb),

comprising only 5 exons. Compared to the 6 exons of

runx-3 there is one exon less in amphioxus, since

the first runt-domain intron of murine and human

runx-1-3 genes is missing in the amphioxus runt-gene

(Fig. 2). As the borders for this intron are conserved

(FK/VV; Fig. 2) for all murine and human runx-1-3

genes, we conclude that this intron was most likely

introduced before runt-gene duplication in chordate

phylogeny. It is also of relevance for the elucidation of

runt-gene evolution, that within the last exon of the

amphioxus runt-gene an equivalent of ‘exon-6’—

which is present in runx-1 and -2 but not in runx-3—

can be found (Fig. 2). We thus speculate that after

runt-gene duplication the domain of ‘exon-6’ was lost

over time in the runx-3 orthologous gene, and

maintained in the runx-1/2 orthologous gene. In this

scenario the newly duplicated runt-genes were both

functional and split their functions after duplication.

Our analysis of the amphioxus runt-exon/intron

structure, in conjunction with the 50-RACE exper-

iments, makes the presence of a distal and a proximal

runt-promoter highly likely. The finding of a runt-

gene variant—beginning with exon-1—in larvae and

adults and another runt-gene variant—beginning with

exon-2—in adults only, suggests that the distal and

proximal promoter are both active in early larval

development, whereas in adulthood runt-gene

expression is driven by the proximal promoter.

Since a distal and proximal runt-promoter is present

in all three mammalian runt-genes [12,17,24], a

duplication of the entire runt-gene locus appears

most likely. Changes in the role of runt-molecules

during chordate phylogeny might thus have either

been driven by alterations in the runt-promoter

elements, or in the runt-protein coding region.

Analysis of runt-gene promoters, runt-gene numbers

and runt-gene expression in hagfish, lamprey and

shark is needed in order to clarify how the evolution of

cartilage, bone and hematopoiesis is related to runt-

gene evolution in particular.

Functional conservation throughout chordate evol-

ution also needs to be determined for the hemato-

poietic factors ‘runt’ and ‘GATA’. We think that this

is a likely scenario, because in D. melanogaster, the

GATA-factor serpent and the runt-homolog lozenge

are key-players in hematopoiesis, whereas in ver-

tebrates GATA-2 and runx-1 are required for

definitive hematopoiesis [36]. Presently it is only

known that runt is involved in the innate immunity of

the sea urchin, as the sea urchin runt-gene was

S. Stricker et al. / Developmental and Comparative Immunology 27 (2003) 673–684 681

reported to be upregulated in coelomocytes upon

bacterial challenge together with a sea urchin GATA-

2/3-homolog [37]. It is not known if the amphioxus

runt-gene is also expressed in coelomocytes. These

coelomocytes were reported to be capable of phago-

cytising bacteria and therefore they most probably

have a crucial role in the innate immunity of

cephalochordata [38].

Whereas the role of runt-genes in hematopoiesis is

well established, the tasks of runt-genes are diverse

and our analysis of runt-expression in sea urchin

embryos revealed a complex pattern involving

ectodermal, mesodermal and endodermal derived

cells types. The sea urchin runt-gene is expressed at

the highest levels in the presumptive endo-mesoderm

domain of the blastulastage embryo and gets pro-

gressively restricted to the gut and the oral ectoderm

of gastrula and prism stage embryos. These cells have

been shown to be the most mitotically active cells

during development [39].

The strongest expression of runt in the gut at

gastrulation indicates an important role of runt in the

organization of the gastrointestinal tract, which is in

line with the report of runt-expression in the intestine

of Nematodes [2] and in the murine gastrointestinal

tract [14], and seems to be one of the phylogenetically

ancient expression domains of runt-genes. Further-

more, the arrow in Fig. 5c1 shows that there is a

slightly enhanced expression detectable around the

stomodeum in the late gastrula embryo, where the

mouth will form. In pluteus embryos, as the arrows

indicate in Fig. 5d3, single cells within the ciliated

band show a higher expression of runt RNA. In the sea

urchin embryo early neurons or neuron like cells are

associated with the mouth and ciliated band in

particular, and the thicker epithelial oral ectoderm is

also thought to be richer in neuron or neuron like cell

content than the aboral ectoderm [40]. We therefore

speculate that expression of runt in the oral ectoderm,

around the mouth and ciliated band, might in fact

reflect expression in neuron cells and correlate to a

second function of runt in neuron cell development.

The latter could also be a conserved runt-expression

domain as the D. melanogaster runt-protein is

involved in neurogenesis [15] and murine runx-1

and runx-3 are expressed in neural cells [6]. Finally,

during gastrulation the sea urchin runt-gene is

expressed in a subpopulation of secondary

mesenchymal cells (arrowheads in Fig. 5c1), which

points to an early role in mesodermal cells as well.

This complex expression pattern in sea urchin might

reflect the multiple roles of runt-genes in mammals, as

for example their involvement in epithelial-mesench-

ymal patterning [6].

In conclusion the chordate stem species most likely

only harbored a single runt-gene, which fulfilled

multiple functions and was most similar to mamma-

lian runx-3 in its exon/intron structure. Runt-gene

duplications occurred after the introduction of a new

intron into the runt-domain. Further research is

needed to analyze how these duplications are related

to the appearance of novel tissues such as cartilage

and bone and the evolution of vertebrate

hematopoiesis.

4.1. Note added during the final preparation

of the manuscript

In the period up to the final preparation of this

manuscript Robertson et al. have published an

expression note [41] showing a similar expression

pattern as reported by us for the sea urchin runt-gene.

In agreement with our amphioxus data they have

found that within the sea urchin runt-domain the first

intron—present in human and murine runt-genes—is

also missing in the sea urchin runt-gene. As we also

found this intron to be missing in the amphioxus runt-

gene locus, our conclusion that this intron was not

present in the runt-gene of the chordate stem species,

is supported by Robertson et al.’s findings.

Acknowledgements

We acknowledge Prof. Denuce who kindly pro-

vided us with B. lanceolatum larve and gave us

helpful comments on a preliminary version of the

manuscript. We also want to thank the staff at the

Biologische Anstalt Helgoland, (Germany) for their

support. Furthermore we would like to thank Nadine

Jurrmann for help with the experiments and Alex-

ander Kuhn for optimising a sea urchin WMISH

protocol and L. Udvarhelyi for his editorial help.

S. Stricker et al. / Developmental and Comparative Immunology 27 (2003) 673–684682

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