Phylogenetic implications of the superfast myosin in extraocular muscles
Department of Cell Biology, Division of Physiology, Duke University Medical School, Durham, NC 27710, USA
* e-mail: f.schachat{at}cellbio.duke.edu
Accepted 13 May 2002
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Summary |
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Key words: extraocular myosin, MYH13, myosin, phylogeny, exon-intron organization, gene structure
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Introduction |
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EOMs arise from unsegmented head mesenchyme rather than somites, which give
rise to trunk and limb muscles (Noden et
al., 1999). They are innervated by cranial, rather than spinal,
motoneurons and are organized into motor units composed of fibers with
different molecular and ultrastructural properties
(Goldberg and Shall, 1999
).
Ultrastructural analyses have identified six fiber types in extraocular muscle
(Peachey, 1971
;
Spencer and Porter, 1988
), and
the majority of EOM fibers are singly-innervated and phasic, like limb and
trunk skeletal muscles, while a minority are multiply-innervated and tonic.
But, irrespective of their innervation, almost all are heterogeneous in myosin
expression (Jacoby et al.,
1990
; McLoon et al.,
1999
; Rubinstein and Hoh,
2000
) and none corresponds to the Type I and II fibers found in
other skeletal muscles. Most importantly, they exhibit distinctive
physiological properties, in particular, superfast contractions
(Close and Luff, 1974
;
Li et al., 2000
) that are
linked to the expression of a novel superfast myosin heavy chain.
In many ways, the superfast EOM myosin is a molecular reflection of EOM's
distinctive ontogenic, structural and physiological features. First, its
expression is tissue-restricted: aside from extraocular muscle, it is found
only in laryngeal muscles (Briggs and
Schachat, 2000; Lucas et al.,
1995
). Second, in many EOM fibers, it is co-expressed with other
myosins (Briggs and Schachat,
2001
; Rubinstein and Hoh,
2000
). And third, its localization to the central end-plate band
region where EOM fibers are innervated links its expression directly to
innervation (Briggs and Schachat,
2001
; Rubinstein and Hoh,
2000
). Here, the MYH13 gene that encodes this distinctive
molecular marker is analyzed by genomic mapping, sequence-based phylogenetic
techniques and phylogenetic footprinting to gain insights into the evolution
of diversity and specialization in striated muscle myosins and the factors
that probably account for the tissue-restricted expression of MYH13.
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The superfast extraocular myosin heavy chain gene is a member of the fast/developmental MYH gene cluster |
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The structure of the MYH13 gene implies it has been `insulated' from the other members of the fast/developmental cluster |
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The conservation of exon size and phase (reading frame) among members of the fast/developmental MYH cluster (Table 1) enables regions of homologous intronexon spacing to be identified. In Fig. 1B, these regions are indicated by common color; only two common patterns in the 5' region are present in MYH13. Because these common spatial patterns probably reflect lineage (e.g. gene duplications) and genetic exchanges between neighboring members of the cluster, both the size and the non-homologous intronexon organization of MYH13 indicate that it has been largely `insulated' from events that have shaped the current organization of the other members of the fast/developmental MYH gene cluster.
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The EO MYH occupies a pivotal position in the phylogeny of mammalian striated myosin heavy chains |
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Support for that ancient lineage is suggested by phylogenetic analysis of
the two functionally distinct domains of striated muscle myosin. Previous
studies have found that the sequences of other striated muscle myosin head or
motor domains and their coiled-coil rod or tail domains generate similar
phylogenetic relationships implying that the these functionally
distinct domains have evolved at similar rates
(Korn, 2000). However, as
shown in Fig. 4, the motor and
rod domains of MYH13 do not follow this relationship: the rod is more closely
related to the slow/cardiac MYHs than to the fast/developmental genes, while
the motor domain is more similar to the fast/developmental MYHs. Coupled with
the sequence phylogeny, this finding implies that the MYH13 gene arose before
the fast/developmental and cardiac MYH genes had significantly diverged,
possibly at a time when the precursors of the slow/cardiac and
fast/developmental gene clusters were linked.
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Independent support for the early divergence of the MYH13: exon organization |
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Distinctive features of full-length MYH13 transcripts suggest an early divergence and specialization |
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Phylogenetic footprinting suggests that transcription of MYH13 is regulated by a specialized network of transcriptional activators |
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Because its expression is restricted to extraocular and laryngeal muscle, regulation of the EO MYH must differ from that of the other fast/developmental and slow/cardiac MYHs. Minimally, this means that factors other than the myogenic transcription factors must also be involved in determining when and where MYH13 is transcribed. The co-expression of MYH13 with adult fast myosins in extraocular muscle and laryngeal muscle fibers, coupled with its absence from limb fibers expressing the adult fast myosins, serves to emphasize its requirement for regulatory factors other than those that drive the trunk and limb muscle MYH genes.
Insights into the nature of that regulation come from the identification of
conserved sequences in the 5' flanking and intronic regions of the human
MYH13 and mouse myh13 genes. This comparative genomic technique, known as
phylogenetic footprinting, is based on the observation that important
regulatory sequences are conserved across species and has been used to detect
muscle-specific regulatory sequences
(Wasserman and Fickett, 1998).
Using BLAST 2 (Tatusova and Madden,
1999
), several highly conserved regions were identified, including
a likely proximal promoter, which includes 250bp upstream of the transcription
start sites, as well as several possible enhancer or repressor regions
upstream and in the first two introns
(Fig. 7).
|
The potential transcription factor binding sites in these regions were
characterized with the matInspector 2.2
(Quandt et al., 1995) and
GeneQuest (DNAstar, Madison, WI, USA) programs, which compare a test sequence
with a large database of consensus binding sites. Many potential sites are
clustered in the conserved regions. Among the most frequent consensus
sequences present in both the mouse and human genomic sequences are those for
NF1, which directs aldolase expression in a subset of fast fibers
(Salminen et al., 1996
),
Pitx2, which is necessary for EOM embryonic development
(Kitamura et al., 1999
),
NKX2.5, which is a mammalian homolog of tinman, a regulator of cardiac
development (Schwartz and Olson,
1999
), TCF11, which binds a DNase-hypersensitive site in the
ß-globin cluster (Johnsen et al.,
1998
), and delta EF1, which inhibits myoD-promoted myogenesis
(Sekido et al., 1994
). Some
myogenic factor binding sites are also present, but they do not generally
occur in the clusters that allow for myogenic factor dimerization that is
critical for activating other MYH genes. The Pitx2, NKX2.5, TCF11 and delta
EF1 consensus binding sites are particularly interesting given the early
specialization of the EO MYH13 gene, its phylogenetic relationship to the
slow/cardiac MYH genes, whose transcription is not regulated by myogenic
factors, and the myoD-independent development of EOM revealed in studies on
the myoD/myf5 knockout mouse (Tajbakhsh et
al., 1997
). The presence of these conserved sites suggests that
MYH13 expression may be integrated into a broader more primitive developmental
program that directs the formation of EOMs or of the eyes themselves. This is
consistent with the finding that NKX2.5, which regulates cardiac development,
is expressed at high levels in extraocular muscle and the fact that mammalian
homologs of factors that control eye development in Drosophila,
including Dach2, Six1, Eya2 and Pax3, have been found to participate in a
regulatory network that directs myogenic factor expression in vertebrate
somites (Heanue et al.,
1999
).
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Indications of hierarchical regulation of MYH13 by chromatin structure |
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A model for the natural history of the skeletal muscle fast/developmental gene cluster |
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Acknowledgments |
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