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The transcription factor Six1a plays an essential role in the craniofacial myogenesis
2
of zebra
fish
3
Cheng-Yung Lin, Wei-Ta Chen, Hung-Chieh Lee, Ping-Hsi Yang, Hsin-Jung Yang, Huai-Jen Tsai
⁎
4 Institute of Molecular and Cellular Biology, National Taiwan University, Room 307, Fisheries Science Building, No. 1, Section 4, Roosevelt Road, Taipei, 106, Taiwan5
6
a b s t r a c t
a r t i c l e i n f o
7 Article history:8 Received for publication 14 November 2008
9 Revised 15 April 2009 10 Accepted 24 April 2009 11 Available online xxxx 12 13 14 15 Keywords: 16 Six1a 17 Myf5 18 Myod 19 Cranial myogenesis 20 Zebrafish 21 Transcription factor Six1a plays important roles in morphogenesis, organogenesis, and cell differentiation.
22 However, the role of Six1a during zebrafish cranial muscle development is still unclear. Here, we
23 demonstrated that Six1a was required for sternohyoideus, medial rectus, inferior rectus, and all pharyngeal
24 arch muscle development. Although Six1a was also necessary for myod and myogenin expression in head
25 muscles, it did not affect myf5 expression in cranial muscles that originate from head mesoderm.
26 Overexpression of myod enabled embryos to rescue all the defects in cranial muscles induced by injection of
27 six1a-morpholino (MO), suggesting that myod is directly downstream of six1a in controlling craniofacial
28 myogenesis. However, overexpression of six1a was unable to rescue arch muscle defects in the tbx1- and
29 myf5-morphants, suggesting that six1a is only involved in myogenic maintenance, not its initiation, during
30 arch muscle myogenesis. Although the craniofacial muscle defects caused by pax3-MO phenocopied those
31 induced by six1a-MO, injection of six1a, myod or myf5 mRNA did not rescue the cranial muscle defects in
32 pax3 morphants, suggesting that six1a and pax3 do not function in the same regulatory network. Therefore,
33 we proposed four putative regulatory pathways to understand how six1a distinctly interacts with either
34 myf5 or myod during zebrafish craniofacial muscle development.
35 © 2009 Published by Elsevier Inc.
36 37
38 39
40 Introduction
41 During embryogenesis, the trunk and limb muscles originate from
42 somites, which are epithelial structures in the mesodermflanking the
43 neural tube, whereas head muscles mostly originate from cranial
44 mesoderm (Noden and Francis-West, 2006). Three groups contribute
45 to the craniofacial skeletal muscles: (1) branchial arch muscles, which
46 are derived from the unsegmented head mesoderm and operate the
47 jaw, facial expression, pharyngeal, laryngeal and gill function; (2)
48 most extraocular muscles, which develop from the prechordal
49 mesoderm and control eye movement; and (3) those muscles derived
50 from progenitor cells in the occipital somites of trunk, which move
51 into the head and give rise to muscles of the tongue and neck (Noden 52 and Francis-West, 2006; Chai and Maxson, 2006; Shih et al., 2007).
53 Despite the varied origins of these muscles, all of them are
54 controlled by myogenic regulatory factors (MRFs) (Buckingham, 55 2006). Proliferative myoblasts, which have undergone initial
myo-56 genic commitment, are marked by the expressions of Myf5 and MyoD,
57 while later myogenic differentiation is marked by Myogenin and
58 MRF4. However, because of the different origins of these muscle cells,
59 MRFs are regulated differently in the head and trunk. For example,
60 mice lacking Myf5 and Pax3 do not develop skeletal muscle in the
61 trunk and limb, whereas they do develop normal head muscles
62
(Tajbakhsh et al., 1997). Taking another example, Lbx1/Pax7/Paraxis
63
in chick are necessary for trunk myogenesis, but they are not
64
necessary for head myogenesis (Mootoosamy and Dietrich, 2002). In
65
fact, the Wnt signals, which promote trunk myogenesis, have been
66
proven to block head myogenesis in chick (Tzahor et al., 2003). In
67
zebrafish, we have clearly defined the distinct functions of Myf5 and
68
Myod that regulate head muscle development, and we have
demon-69
strated that they exhibit their own regulatory pathways (Lin et al., 70 2006). Although myogenic progression is similar in all developing
71
muscle groups, it seems, therefore, that the specification of cells just
72
before myoblast differs significantly between head and trunk (Rawls 73 and Olson, 1997; Mootoosamy and Dietrich, 2002).
74
Only a few factors have been reported to play roles in head
75
myogenesis, and we enumerate them here. Mice lacking Capsulin and
76
myoR fail to express myf5 in the first arch and lose a subset of
77
mandibular arch-derived muscle (Lu et al., 2002). It has been found
78
that tbx1, which is expressed in the premyoblast mesoderm in thefirst
79
and second branch arch, is required for the development of some head
80
muscles (Kelly et al., 2004; Dastjerdi et al., 2007). Although bmp4
81
promotes cardiac differentiation, it also inhibits head skeletal muscle
82
differentiation (Tirosh-Finkel et al., 2006). Similarly, fgf8 is shown to
83
promote branchiomeric muscle development, but it inhibits
extrao-84
cular muscle development (von Scheven et al., 2006). Finally, pitx2 is a
85
paired-related homeobox gene, which is required for the expression of
86
the premyoblast specification markers tbx1, tcf21 (Capsulin), and msc
87
(MyoR) to set up the premyoblast in thefirst branch arch (Dong et al.;
Developmental Biology xxx (2009) xxx–xxx
⁎ Corresponding author. Fax: +886 2 2363 8483. E-mail address:hjtsai@ntu.edu.tw(H.-J. Tsai).
YDBIO-04296; No. of pages: 15; 4C: 4, 5, 6, 7, 8, 9, 10, 11
0012-1606/$– see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.ydbio.2009.04.029
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Developmental Biology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / d e v e l o p m e n t a l b i o l o g y
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88 2006; Shih et al., 2007; L'Honore et al., 2007). However, it remains89 unclear what additional factors may be involved in head myogenesis.
90 The six gene is a vertebrate homolog of the Drosophila homeobox
91 gene sine oculis (so) and plays important roles in morphogenesis,
92 organogenesis, and cell differentiation (Kawakami et al., 2000). Six
93 protein is a transcription factor that contains two conserved domains,
94 the Six domain (SD) and the homeodomain (HD). Both domains are
95 required for specification through binding DNA and cooperative
96 interaction with cofactors (Kawakami et al., 1996; Chen et al., 1997; 97 Pignoni et al., 1997). For example, Drosophila so is required for eye
98 formation through binding the synergistic regulatory network, such as
99 eyeless (Pax), eyes absent (Eya) and dachshund (Dach) (Chen et al., 100 1997; Pignoni et al., 1997).
101 In vertebrates, Six protein displays a similar regulatory network
102 during myogenesis and development of the metanephric kidney and
103 inner ear (Heanue et al., 1999; Xu et al., 1999; Xu et al., 2003; Li et al., 104 2003). Moreover, Six protein is reported to directly control the
105 expressions of myf5 and myogenin through binding at the MEF3 in
106 their promoters (Spitz et al., 1998; Giordani et al., 2007). There are six
107 Six genes (Six1 to Six6) in mouse and human genomes (Kawakami 108 et al., 2000). Six1 is expressed from E8 stage and throughout skeletal
109 muscle development in mouse embryos (Grifone et al., 2004). Six1
110 and Six4 are expressed as overlapping in muscle territories, such as
111 dermomyotome, myotome, limb bud and migrating muscle precursors
112 (Ozaki et al., 2001; Laclef et al., 2003). Six1-knockout fetuses suffer
113 from muscle hypoplasia (Laclef et al., 2003), whereas Six1 and Six4
114 double knockout embryos appear to have more severe muscle defects,
115 especially in leg muscles (Grifone et al., 2005), suggesting that Six4
116 shares a common function with Six1 during myogenesis. Furthermore,
117 in both Six1−/−Six4−/−and Eya1−/−Eya2−/−double mutants, pax3
118 fails to express in the hypaxial dermomyotome, which then causes cell
119 death and reduces muscle progenitor cells in the limbs (Grifone et al., 120 2005; Grifone et al., 2007). In zebrafish, six members of the six gene
121 have been defined: six1a–1b, six2–2.1, six3a–3b, six4.1–4.3, six7 and
122 six9 (Kobayashi et al., 1998, 2000, 2001; Drivenes et al., 2000; 123 Wargelius et al., 2003; Bessarab et al., 2004, 2008). Both six1a and
124 six4.2 are expressed in the presomitic mesoderm, somites and pectoral
125 fin bud. Moreover, Bessarab et al. (2004) reported that six1a
126 expression is regulated by the Notch pathway during trunk muscle
127 differentiation. Knockdown of six1a causes myogenin expression to be
128 reduced in somites, resulting in abnormal differentiation of trunk fast
129 muscles (Bessarab et al., 2008). They also demonstrated that the six1a
130 transcript is expressed in craniofacial muscle. More importantly, it has
131 been reported that the six1a gene is involved in branchio-oto-renal
132 syndrome (Ruf et al., 2004). Therefore, detailed knowledge about the
133 mechanisms controlling molecular interaction among genes involved
134 in head muscle development should not only give insight into
135 craniofacial morphogenesis but also help in the development of
136 therapies designed to treat clinical syndromes affecting head and facial
137 development. However, the function that Six1a plays in head muscle
138 development is still unknown.
139 In this study, we focus on the role of Six1a in head myogenesis.
140 When Six1a is absent by injection of six1a-specific morpholino (MO),
141 we show that myf5 fails to express in the cranial muscles that
142 originate from trunk paraxial mesoderm, whereas myf5 continues to
143 be normally expressed in cranial muscles that originate from head
144 mesoderm. In contrast, myod is lost in the cranial muscles that
145 originate both from trunk and head mesoderm. We also demonstrate
146 that injection of myod mRNA can rescue the six1a-MO-induced defect,
147 but that injection of myf5 mRNA could only rescue the muscle defects
148 that originate from trunk paraxial mesoderm. We prove that the
149 function of Six1a is equivalent to Pax3 and that Six1a is not involved in
150 the Tbx1 pathway. Furthermore, we propose four putative regulatory
151 pathways to demonstrate that six1a interacts separately with either
152 myf5 or myod to modulate the development of craniofacial muscles
153 in zebrafish.
154
Materials and methods
155
Fish embryos
156
The wild-type AB strain (University of Oregon, Eugene, OR) and the
157
transgenic line Tg(α-actin:RFP) (Lin et al., 2006) of zebrafish (Danio
158
rerio) were used. The culture condition, embryo stage, egg production
159
and collection were described previously (Lin et al., 2006).
Fluor-160
escent signal in embryos was observed under afluorescent
stereo-161
microscope (MZ FLIII, Leica) equipped with 583 nm (emission)filters.
162
Whole-mount in situ hybridization
163
Whole-mount in situ hybridization has been described previously
164
(Lee et al., 2006), except that the following genes were used as probes:
165
six1a (Bessarab et al., 2004); myf5, myod, myogenin, et1 (Miller et al., 166 2000); fgf3, dlx2 (Akimenko et al., 1994); tbx1 (Piotrowski et al.,
167 2003); pax3 (Seo et al., 1998) and eya1 (Sahly et al., 1999) cDNAs
168
(GenBank Accession Nos. NM207095, NM131576, NM131262,
169
NM131006, AF281858, NM131291, U03875, NM183339, AF014366,
170
and BC154187, respectively).
171
MOs for blocking translation and mRNAs for rescue experiments
172
MOs designed specifically as translational inhibitors of six1a were
173
(1) six1a-MO (Nica et al., 2006), 5
′-CGAAAGAAGGCAACATTGACAT-174
GAC-3′, which is complementary to nucleotides (nt) 142–166 of
175
zebrafish six1a cDNA (GenBank Accession No. NM207095) and was
176
injected at the concentration of 8, 6, 4, 2, or 1 ng per embryo; (2)
UM-177
MO (Bessarab et al., 2008), 5′-TCTCCTCTGGATGCTA-CGAAGGAAG-3′,
178
which is complementary to nt 93–117 of zebrafish six1a cDNA
179
(GenBank Accession No. NM207095) and was injected at 8 ng per
180
embryo; and (3) SM-MO (Bessarab et al., 2008), 5
′-CGCTTAAT-181
TACCTTTCTTTCGCCTC-3′, which is complementary to nt 87073–87097
182
(intron sequence is underlined) of the clone DKEY-225H23 (GenBank
183
Accession No. BX649231), binding the splice donor site of six1a
pre-184
mRNA, and was injected at 8 ng per embryo. Regarding MOs that were
185
designed specifically as translational inhibitors of MRFs, they were (1)
186
myf5-MO, 5′-TCTGGGATGTGGAGAATACGTCCAT-3′, which is
comple-187
mentary to nt 44–68 of zebrafish myf5 cDNA (GenBank Accession No.
188
NM131576) and was injected at 4 ng per embryo; and (2) myod-MO
189
(Lin et al., 2006), 5′-ATATCCGAC-AACTCCATCTTTTTTG-3′, which is
190
complementary to nt 172–196 of zebrafish myod cDNA (GenBank
191
Accession No. NM131262) and was injected at 4 ng per embryo.
192
Regarding MOs that were designed specifically as translational
193
inhibitors of tbx1, pax3 and eya1, they were (1) tbx1-MO, 5
′-194
GGGCTTGATATTGCTGAAA-TCATTC-3′, which is complementary to nt
195
359–383 of zebrafish tbx1 cDNA (GenBank Accession No. NM183339)
196
and was injected at 10 ng per embryo; (2) pax3-MO (Lee et al., 2006),
197
5′-ACGAAAAAAGGATGCACGAAGCACT-3′, which is complementary to
198
nt 241–265 of zebrafish pax3 cDNA (GenBank Accession No.
199
AF014366) and was injected at 3 ng per embryo; and (3) eya1-MO
200
(Bricaud and Collazo, 2006), 5′-AGCTAGATCCTGCATTTCCATAGAC-3′,
201
which is complementary to nt 274–298 of zebrafish eya1 cDNA
202
(GenBank Accession No. AF014366) and was injected at 10 ng per
203
embryo. All MOs were prepared at a stock concentration of 1 mM and
204
were diluted to the desired concentrations for microinjection.
205
In order to further prove the specific effectiveness of six1a-,
myod-206
and myf5-MO, we designed the following synthetic mRNA: (1)
six1a-207
egfp mRNA, in which the six1a cDNA, including six1a-MO target
208
sequence, is fused in frame with egfp cDNA; (2) myod-egfp mRNA, in
209
which the myod cDNA, including myod-MO target sequence, is fused in
210
frame with egfp cDNA; and (3) myf5-MO-target-egfp mRNA, in which
211
the myf5-MO target sequence is fused in frame with egfp cDNA.
212
Regarding that the introduced six1a mRNA is not bound by six1a-MO
213
during the rescue experiment, we designed (1) a wobble six1a mRNA,
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214 in which we changed the nt 145–166 of zebrafish six1a cDNA (GenBank
215 Accession NO. NM207095) from 5′-ATGTCAATGTTGCCTTCTTTCG-3′ to
216 5′-ATGAGTATGCTCCCGAGCTTCG-3′, but without altering the amino
217 acid residues; and (2) a wobble six1a-egfp mRNA, in which the wobble
218 six1a cDNA was fused in frame with egfp cDNA. Capped mRNA of
219 wobble six1a was synthesized according to the protocols of the
220 manufacturer (Epicentre). The mRNAs of myf5 and myod were also
221 synthesized. The generated mRNAs were diluted with distilled water to
222 110 ng/μl and 66 ng/μl for six1a mRNA, to 44 ng/μl and 22 ng/μl for
223 myf5 mRNA and to 22 ng/μl for myod mRNA (Lin et al., 2006). Each
224 time, approximately 2.3 nl of solution was injected into the one-cell
225 stage of zebrafish embryos.
226 Western blot analysis
227 The Western blot was performed after the total proteins were
228 analyzed on a 12% SDS-PAGE by following the procedures described
229 previously (Lee et al., 2007), except that the yolk was removed, and
230 the antibodies of anti-Six1a (abcan, ab22072) and anti-Glyceraldehyde
231 3-phosphate dehydrogenase (ABBIOTEC, 250504) were used at the
232 dilution of 1:1000.
233 Results
234 Expression patterns of six1a, myf5 and myod in zebrafish head
235 muscle development
236 To study the roles of Six1a during cranial muscle development,
237 wefirst analyzed the spatiotemporal expression of six1 from 24 to
238 72 hpf and made a comparison with the expression patterns of
239 some MRFs, particularly myf5 and myod. At 24 hpf, six1a was
240 detected in the olfactory placode (olp), otic vesicle (ov), anterior
241 lateral line and vestibular ganglia (allg) (Fig. 1A), which was
242 consistent with what was reported byBessarab et al. (2004). We
243 also noted that six1a was only expressed in the neural ectoderm,
244 but not in the cranial mesoderm, during 24 hpf. However, myf5, but
245 not myod, were detected in the cranial muscle precursors during
246 24 hpf (Figs. 1B, C). At 32 hpf, six1a initiated expression in
247 branchial arch and in extraocular muscle primordial of the medial
248 rectus (mr) and inferior rectus (ir) (Fig. 1D), while myf5 started to
249 gradually reduce its expression in thefirst branchial arch, but began
250 to express in the extraocular muscle primordial inferior oblique (io)
251 and superior oblique (so) (Fig. 1E). At the same time, myod
252 transcripts were initially detected in the head muscle primordia of
253 the mr, ir, lateral rectus (lr), andfirst branchial arch mesoderm core
254 (Fig. 1F), which was similar to the expression pattern of six1a. At
255 36 hpf, six1a was strongly expressed in the branchial arch (Fig. 1G),
256 while the myf5 transcripts were gradually decreased in the arch
257 region (Fig. 1H). However, myod was now detected in head muscles
258 derived from the first (masticatory plate, MP; intermandibularis,
259 IM) and the second arch mesoderm cores (constrictor hyoideus
260 dorsalis, CHD; constrictor hyoideus ventralis, CHV) (Fig. 1I). This
261 result was consistent with the report of Schilling and Kimmel 262 (1994). At 72 hpf, all cranial muscles were six1a- (Figs. 1J, L) and
263 myod-positive (Figs. 1K, M).
264 Comparing the expression patterns of six1a, myf5 and myod during
265 the head muscle development of zebrafish, we concluded that six1a
266 was expressed in all cranial muscles. The expression stage of six1a in
267 hyoid (1st), mandibular (2nd), and branchial arch (3rd) was later than
268 that of myf5, but obviously earlier than that of myod. In extraocular
269 muscle, six1a started to express in mr and ir primordia and sustained
270 its expression to the later stage of 72 hpf, which was similar to myod,
271 but different from myf5, which was expressed in io and so primordia.
272 Thus, we can further conclude that the expression of myf5 is earlier
273 than that of six1a in the cranial mesoderm, whereas the expression of
274 six1a is earlier than that of myod in the arches.
275
Six1a is involved in zebrafish cranial muscle development
276
To understand whether six1a plays roles in craniofacial muscle
277
development, we used a transgenic line, Tg(α-actin:RFP), in which the
278
RFP reporter is labeled in all craniofacial muscles (Figs. 2A, B), as
279
previously reported (Lin et al., 2006). When the embryos derived from
280
this line were injected with six1a-MO, we observed that the muscle
281
primordia of extraocular muscles mr and ir, all arch muscles and sh
282
were missing at 72 hpf (Figs. 2C, D), whereas the muscle primordial of
283
so, io, sr, and lr, and some remnants of arch muscle cells, were all
284
normal and presented as RFP-positive (Figs. 2A, B). Furthermore,
285
besides six1a-MO, we also designed two other types of morpholinos,
286
UM-MO and SM-MO, to specifically knockdown the translation of
287
six1a mRNA. The defective phenotype induced by injection of either
288
UM-MO or SM-MO was similar to that of injection of six1a-MO
289
(Supplemental Fig. S1). Western blot analysis proved that the Six1a
290
expression level was greatly reduced in the six1a-MO-injected
291
embryos (Supplemental Fig. S2). In addition, we co-injected a wobble
292
six1a-egfp mRNA with a six1a-MO, and the Six1a-GFP fusion protein
293
was detected in embryos (Supplemental Fig. S3), indicating that the
294
injected six1a-MO cannot inhibit the translation of the introduced
295
wobble six1a-egfp mRNA. Furthermore, co-injection of six1a-MO with
296
wobble six1a-mRNA, but not egfp mRNA, enabled embryos to rescue
297
the defective phenotypes induced by six1a-MO and resulted in the
298
normal development of all head muscles (Figs. 2E, F; andTable 2). We
299
also noticed that the degree of defective phenotype induced by
six1a-300
MO was dose-dependent (Table 1). Overall, evidence indicates that
301
the defects induced by six1a-MO are specific and we therefore
302
concluded that six1a is necessary for the development of extraocular
303
muscles mr and ir, all arch muscles and sh, which migrate from trunk
304
and contribute to head muscle.
305
Six1a functions with Myf5 and Myod in cranial muscle development, but
306
in different modulations
307 Lin et al. (2006)categorized all zebrafish cranial muscles into three
308
groups and defined three regulatory pathways involved in cranial
309
muscle development. Among them, the extraocular muscles so and io,
310
the dorsal pharyngeal arch muscles lap, do, am, ah and ao, and the
311
trunk migratory head muscle sh, are categorized as Group I, whose
312
primordial cells require Myf5 to activate their downstream MRFs, such
313
as myod and myogenin. In this study, we found that myf5 was normally
314
expressed in pharyngeal arch muscle precursors of the
six1a-MO-315
injected embryos during 36–48 hpf (Figs. 2I vs. M and J vs. N). In
316
addition, compared to the wild-type embryos, the expressions of
317
myf5, myod and myogenin remained unchanged in the extraocular
318
muscles so and io of six1a morphants (Figs. 2J vs. N, K vs. O, and L vs. P).
319
However, the expressions of myod and myogenin were greatly reduced
320
in the pharyngeal arch muscle precursors, lap, do, am, ah, and ao, of
321
six1a-MO-injected embryos at 48 hpf (Figs. 2K vs. O and L vs. P),
322
suggesting that Six1a is required for the normal expressions of myod
323
and myogenin in the precursors of dorsal pharyngeal arch muscles. On
324
the other hand, the primordial muscle sh, which originates from trunk,
325
lost both myf5 and myod expression in the six1a morphants (Figs. 5B
326
vs. E and C vs. F), suggesting that Six1a is required for the expressions
327
of myf5 and myod in sh primordial muscle.
328
The extraocular muscle lr and the ventral pharyngeal arch muscles
329
ima, imp, ih and hh are categorized as Group II, whose primordial cells
330
are myf5-expressed precursors and require myod to play a major role in
331
myogenesis. Both myf5 and myod are necessary for the development of
332
Group II precursors. Here, we revealed that myf5 was normally expressed
333
in the precursor of pharyngeal arch muscles of six1a morphants at 36–
334
48 hpf, compared to the wild-type embryos. In addition, the myod and
335
myogenin expressions remained unchanged in the extraocular muscle lr
336
of six1a morphants at 48 hpf (Figs. 2K vs. O and L vs. P). However, the
337
myod and myogenin expressions were totally lost in the ventral
3 C.-Y. Lin et al. / Developmental Biology xxx (2009) xxx–xxx
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338 pharyngeal arch muscles ima, imp, ih and hh in the six1a morphants at
339 48 hpf (Figs. 2K vs. O and L vs. P), suggesting that Six1a affects myod
340 expression in the ventral pharyngeal arch muscle of zebrafish.
341 The extraocular muscles sr, mr and ir belong to Group III, whose
342 primordial cells require myod, but not myf5, as a major factor in
343 muscle development. The expressions of myod and myogenin in the
344 extraocular muscle sr of the six1a-MO-injected embryos appeared the
345 same as the extraocular muscle sr of the wild-type embryos (Figs. 2K
346 vs. O and L vs. P). However, the extraocular muscles mr and ir of the
347 six1a-MO-injected embryos were completely lost when observed at
348 48 hpf (Figs. 2K vs. O and L vs. P). This evidence suggests that Six1a
349 modulates myod expression in extraocular muscles mr and ir.
350
Taken together, during craniofacial muscle development of
zebra-351
fish, we conclude that Six1a is required for (1) myf5 expression in
352
trunk migratory head muscle sh and (2) myod expression in the
353
extraocular muscles mr and ir in all pharyngeal arch muscles and the
354
trunk migratory head muscle sh.
355
The defective pharyngeal arch muscles are induced specifically by loss
356
of Six1a
357
Pharyngeal arch is developed from three germ layers: the
358
mesoderm core, the endoderm pharyngeal pouch and the ectoderm
359
neural crest cells (Graham and Smith, 2001). It was necessary to Q1
Fig. 1. The temporal expressions of six1a, myf5 and myod during cranial muscle development of zebrafish. The temporal expressions of six1a (A, D, G, J, L), myf5 (B, E, H) and myod (C, F, I, K, M) transcripts of zebrafish were analyzed by whole-mount in situ hybridization in embryos from lateral view (A–K) and ventral view (L–M). The transcript of six1a in the olp, ov, and allg at 24 hpf (A); in the mr, ir, 1st, 2nd, and 3rd arches at 32 and 36 hpf (D, G); and in all the cranial muscles at 72 hpf (J, L). The myf5 transcript was detected in the craniofacial region at 24 hpf (B, arrow); in the so, io, 2nd and 3rd arches at 32 hpf (E); and in the so, io, and 3rd arches at 36 hpf (H). Although the myod was not expressed in the craniofacial muscles at 24 hpf (C), it was detected in the mr/ir, lr, and 1st arch at 32 hpf (F); in the mr, ir, sr, lr, MP, IM, CHD, and CHV at 36 hpf (I); and in all the cranial muscles at 72 hpf (K, M). The schematic diagram illustrates the expression of six1a in the cranial muscles during 32–72 hpf (N). ah, adductor hyoideus; allg, anterior lateral line and vestibular ganglia; am, adductor mandibulae; ao, adductor operculi; do, dilator operculi; dpw1–5, dorsal pharyngeal wall 1–5; hh, hyohyoideus; ih, interhyoideus; ima, intermandibularis anterior; imp, intermandibularis posterior; io, inferior oblique; ir, inferior rectus; lap, levator arcus palatini; lr, lateral rectus; mr, medial rectus; olp, olfactory placode; ov, otic vesicle; sh, sternohyoideus; so, superior oblique; sr, superior rectus and tv 1–5, transversus ventralis 1–5. CHD: the constrictor hyoideus dorsalis, which differentiates to ah and ao; CHV: the constrictor hyoideus ventralis, which differentiates to ih and hh; IM: the intermandibularis, which differentiates to ima and imp; MP: the masticatory plate, which differentiates to CD and am. CD: the constrictor dorsalis, which differentiates to lap and do.
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360 confirm whether the defects in pharyngeal muscles in the six1a
361 morphants were exclusively the result of Six1a loss or the result of
362 defective mesoderm core, defective endoderm pharyngeal pouch, or
363
neural crest cells. To accomplish this, we detected the expressions of
364
et1, fgf3 and dlx2, which are the gene markers of the ventral mesoderm
365
core (Miller et al., 2000), the endoderm pouch (David et al., 2002) and
Fig. 2. Six1a is required for the development of mr, ir, sh and all pharyngeal muscles. Embryos derived from the transgenic line Tg(α-actin:RFP) (A–H), all of whose skeletal muscles appear as redfluorescent protein (RFP), were injected with 8 ng of six1a-morpholino oligonucleotide (MO) to specifically inhibit six1a mRNA translation. RFP signal was detected only in the so, io, sr, lr, and remnant dorsal branchial arch muscle (white star) primordia in the six1a-MO-injected embryos (A vs. C and B vs. D). When embryos were injected with 8 ng of six1-MO together with 150 pg of six1a mRNA, results showed that the defective muscle primordia induced by six1a-MO were rescued and appeared as RFP-labeled muscles (E, F; the rescued muscles are marked in green typeface). In contrast, the rescue experiment failed when embryos were injected with 8 ng of six1a-MO with 200 pg of gfp mRNA (G, H), suggesting that the defects of six1a morphants were specific. The expressions of myf5 (I, J, M, N), myod (K, O), and myogenin (L, P) were also observed at the stages indicated. When wild-type embryos were injected with six1a-MO, myf5 was expressed normally in the six1a morphants, both at 36- (I vs. M) and at 48-hpf (J vs. N), except sh. On the other hand, the expressions of myod (K vs. O) and myogenin (L vs. P) were decreased in the extraocular io, so, sr and lr in the six1 morphants at 48 hpf. Weak signals of myod and myogenin were also noticed in the remnant dorsal branchial muscles (black stars) of six1a morphants. The schematic diagram illustrates the cranial muscle defects in six1a morphants and compares the expressions of myf5, myod and myogenin between wild-type (upper row, Q) and six1a morphants (lower row, Q). Lateral view: A, C, E, G and I–P; and ventral view: B, D, F and H. ah, adductor hyoideus; am, adductor mandibulae; ao, adductor operculi; do, dilator operculi; hh, hyohyoideus; ih, interhyoideus; ima, intermandibularis anterior; imp, intermandibularis posterior; io, inferior oblique; ir, inferior rectus; lap, levator arcus palatini; lr, lateral rectus; mr, medial rectus; sh, sternohyoideus; so, superior oblique and sr, superior rectus.
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366 neural crest cells (Akimenko et al., 1994), respectively. Results showed367 that both et1 (Figs. 3A vs. B), fgf3 (Figs. 3C vs. D) and dlx2 (Figs. 3E
368 vs. F) were normally expressed in the six1a-MO-injected embryos at
369 36 hpf, indicating that mesoderm core, endoderm pouch and neural
370 crest cells develop normally. The loss of pharyngeal arch muscle in
371 six1a morphants does not arise from the lost structures of mesoderm
372 core, pharyngeal pouch and neural crest cells. Therefore, we proposed
373 that Six1a is directly involved in pharyngeal arch myogenesis.
374 six1a links with either myf5 or myod to modulate the development of
375 craniofacial muscles
376 Based on the expression patterns and the muscle defects which
377 occurred in the six1a morphants, we hypothesized the plausibility of a
378
myf5-six1a-myod regulatory pathway in craniofacial muscle
develop-379
ment. After we confirmed the specific activities of myf5-MO and
380
myod-MO (Supplemental Fig. S3), we microinjected either myf5-MO
381
or myod-MO together with six1a mRNA to determine which embryo
382
would be rescued by six1a mRNA from MO-induced defect. Results
383
showed that both myf5- (Figs. 4E–H) and myod-morphants (Figs. 4I–K)
384
failed to be rescued from their muscle defects through the addition of
385
exogenous six1a mRNA (Wild type control,Figs. 4A, B). Therefore, we
386
next microinjected six1a-MO with either myf5 mRNA or myod mRNA
387
to determine which mRNA enabled rescue of the embryos from the
388
defects induced by six1a-MO (six1a-MO phenotype control,Figs. 4C,
389
D). By co-injection of six1a-MO with myf5 mRNA, results showed that,
390
while the loss of mr, ir and the remnants of pharyngeal arch muscle
391
were observed, muscle sh had been rescued (Figs. 4M, N; andTable 2).
392
This evidence suggests that myf5 may not be an upstream modulator
393
of six1a. Instead, myf5 and six1a may independently regulate the
394
craniofacial muscles derived from the head paraxial mesoderm, mr, ir
395
and the pharyngeal arch muscles. In contrast, co-injection of myod
396
mRNA enabled embryos to rescue all head muscle defects induced by
397
six1a-MO, suggesting that six1a was the upstream regulatory gene of
398
myod (Figs. 4O, P; andTable 2).
399
It is noteworthy that the muscle sh, which originates from the
400
trunk, could be rescued by myf5 mRNA in the six1a morphants. Based
401
on this evidence, we hypothesized that Six1a plays roles in different
402
regulatory pathways between cranial and trunk myogenesis. To
403
demonstrate this hypothesis, we analyzed the development of the
404
sh,fin bud (fb) and posterior hypoaxial muscle (phm) that come from
405
the dermomyotome in the anterior somites. Using whole-mount in
406
situ hybridization, we observed that six1a, myf5 and myod were all
407
expressed in the muscle primordia of wild-type at 36 hpf (Figs. 5B, C;
Table 1 t1:1
Six1 is required for the developments of medial rectus (mr), inferior rectus (ir), sternohyoideus (sh) and all arch muscles.
t1:2
t1:3 six1-MO-injected concentration
Defect (%)
t1:4 Absent-muscle Reduced-muscle Wild-type like t1:5 Uninjected 0 (0/107) 0 (0/107) 100 (107/107) t1:6 1 ng 3.1 (3/98) 91.8 (90/98) 5.1 (5/98) t1:7 2 ng 19.0 (20/105) 76.2 (80/105) 4.8 (5/108) t1:8 4 ng 42.1 (48/114) 55.2 (63/114) 2.7 (3/114) t1:9 6 ng 65.1 (58/89) 34.9 (31/89) 0 (0/89) t1:10 8 ng 81.8 (54/66) 18.2 (12/66) 0 (0/66)
The morphological defects were observed at 72 hpf. Absent-muscle defect indicated that mr, ir, sh and ventral arch muscles were completely lost but remnants of dorsal arch muscles still remained. Reduced-muscle defect indicated that mr, ir, sh and all arch muscles were partially lost. Wild-type like phenotype indicated that the head muscles were not lost.
t1:11
Fig. 3. Loss of Six1a function does not impede the normal development of mesoderm core and pharyngeal pouch. The expression patterns of et1, fgf3 and dlx2 were examined in the wild-type and in the six1a-MO-injected embryos at 36 hpf. Results showed that the transcripts of et1, fgf3 and dlx2 exhibited similarly in ventral mesoderm cores (A vs. B, arrows), pharyngeal pouches (C vs. D, arrows), and neural crest cells (E vs. F, six arches), respectively, between wild-type embryos and six1a morphants.
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408 andSupplemental Fig. S4). However, we also observed that the sh, fb
409 and phm were absent in the six1a-MO-injected embryos derived from
410 the transgenic line Tg (α-actin:RFP) at 72 hpf (Fig. 5D). In addition,
411 myf5 and myod lost their expressions in these muscles at 36 hpf (Figs.
412 5E, F). Thus, we reasoned that Six1a is required for the trunk
413
migratory muscles. When six1a-MO was co-injected with six1a mRNA
414
into embryos, the sh, fb and phm primordia appeared normally at
415
72 hpf (Fig. 5G). Furthermore, both myf5 and myod were detected at
416
48 hpf (Figs. 5H, I). Interestingly, co-injection of either myf5 or myod
417
mRNA could rescue the defective development of sh, fb and phm
418
primordia induced by six1-MO at 72 hpf (Figs. 5J, M). The expressions
419
of myod and myogenin were partially restored in the embryos
co-420
injected with myf5 mRNA and six1a-MO when observed at 48 hpf
421
(Figs. 5K, L). Similarly, the expressions of myf5 and myogenin were
422
also partially rescued in the embryos co-injected with myod mRNA
423
and six1a-MO (Figs. 5N, O). Thus, we concluded that six1a is required
424
for the expressions of myf5 and myod in sh and trunk myogenesis, but
425
six1a activates myf5 and myod through the six1a-myf5 pathway and
426
six1a-myod pathway, respectively.
427
Regulatory pathways that control six1a expression
428
Six1a plays important roles in pharyngeal arch muscle, mr and ir
429
development. It has been reported that mouse T-box gene, tbx1, is an
430
early cranial mesoderm inducer which regulates arch muscle
Fig. 4. Injection of myod mRNA enables embryos to rescue the defective muscle derived from cranial mesoderm in the six1a morphants. Embryos derived from Tg (α-actin:RFP) (A, B as non-treated) were injected with 8 ng of six1a-MO (C, D) and either 4 ng of myf5-MO (E, F) or myod-MO (I, J) to serve as control groups. Embryos injected with 4 ng of myf5-MO and 150 pg of six1a mRNA (G, H), 4 ng of myod-MO and 150 pg of six1a mRNA (K, L), 8 ng of six1a-MO and 100 pg of myf5 mRNA (M, N), and 8 ng of six1a-MO and 50 pg of myod mRNA (O, P) were used to examine the appearance of RFP-labeled muscles. Results showed that only sr and mr/ir muscles exhibited in the myf5-MO-injected embryos and in the myf5-MO-six1a-mRNA-injected embryos (E vs. G and F vs. H). In contrast, only so, io, lap/do, ah, ao, and sh muscles exhibited in the MO-injected embryos and in the myod-MO-six1a-mRNA-injected embryos (I vs. K and J vs. L). Similar to six1a morphants, embryos co-injected with six1a-MO and myf5 mRNA exhibited the so, io, sr, lr and remnant dorsal branchial arch muscles. However, injection of myf5 mRNA enabled embryos to rescue only sh primordia muscle among defects induced by six1a-MO (M, N), while injection of myod mRNA enabled embryos to rescue all the defective muscle primordia induced by six1a-MO (O, P). The rescued muscles are marked in green typeface. Lateral view: A, C, E, G, I, K, M and O; Ventral view: B, D, F, H, J, L, N and P. ah, adductor hyoideus; am, adductor mandibulae; ao, adductor operculi; do, dilator operculi; hh, hyohyoideus; ih, interhyoideus; ima, intermandibularis anterior; imp, intermandibularis posterior; io, inferior oblique; ir, inferior rectus; lap, levator arcus palatini; lr, lateral rectus; mr, medial rectus; sh, sternohyoideus; so, superior oblique and sr, superior rectus.
Table 2 t2:1
The loss of mr, ir and arch muscles, which originate from cranial mesoderm, in the six1-morphants was rescued by myod mRNA, but not myf5 mRNA.
t2:2
t2:3 six1-MO-injected concentration
Defecta
(%)
t2:4 Absent-muscle Reduced-muscle Wild-type like t2:5 8 ng 85.7 (66/77) 13.0 (10/77) 1.3 (1/77) t2:6 8 ng + 150 pg six1 mRNA 20.5 (14/68) 44.1 (30/68) 35.4 (24/68) t2:7 8 ng + 250 pg six1 mRNA 23.4 (11/47) 36.2 (17/47) 40.4 (19/47) t2:8 8 ng + 50 pg myf5 mRNA 86.0 (49/57) 14.0 (8/57) 0 (0/57) t2:9 8 ng + 100 pg myf5 mRNA 84.1 (53/63) 14.3 (9/63) 1.6 (1/63) t2:10 8 ng + 50 pg myod mRNA 21.3 (16/75) 42.7 (32/75) 36.0 (27/75)
The morphological defects were observed at 72 hpf. Absent-muscle defect indicated that mr, ir and ventral arch muscles were completely lost but the remnants of dorsal arch muscles still remained. Reduced-muscle defect indicated that mr, ir and all arch muscles were partially lost. Wild-type like phenotype indicated that the head muscles were not lost.
t2:11 a
The sh muscle was not included because it originates from the trunk. t2:12
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431 development (Grifone and Kelly, 2007;Kelly et al., 2004; Piotrowski 432 et al., 2003). To understand whether tbx1 is upstream of six1a, we
433 detected the expression of tbx1 and found that tbx1 was expressed in
434 zebrafish pharyngeal arch region at 36 hpf (Fig. 6A). In
tbx1-MO-435 injected embryos derived from the transgenic line
Tg(alpha-actin-436 RFP), all the pharyngeal arch muscles were lost when observed at
437 72 hpf underfluorescent microscopy. However, the six extraocular
438 muscles were normally developed (Fig. 6E). We also detected six1a,
439 myf5 and myod expressions in the tbx1-MO-injected embryos.
440 Results showed that the expressions of six1a, myf5 and myod were
441 lost in pharyngeal arch muscles, but not the extraocular muscles
442 (Figs. 6B–D), suggesting that tbx1 is required for the expressions of
443 six1a, myf5 and myod in pharyngeal arch muscle. Interestingly, when
444
six1a-, myf5- or myod mRNA was co-injected with tbx1-MO, we
445
found that myf5-mRNA (Fig. 6G), but not six1a-mRNA or myod
446
mRNA, enabled rescue of embryos from the defects induced by
tbx1-447
MO (Figs. 6F, H). This evidence strongly suggests that tbx1 is an
448
upstream modulator of myf5, regulating the specification of cranial
449
muscle development through myf5; however, the findings also
450
indicate that tbx1 is not a direct upstream regulator of six1a.
451
Next, we studied whether the EYA-DACH-SIX-PAX pathway, which
452
plays a critical function in the trunk muscle development of mouse, is
453
also involved in the head muscle development of zebrafish. When we
454
detected the myf5 and myod expressions in the eya1-knockdown
455
morphants, we found that both myf5 (Figs. 7A vs. C) and myod
456
(Figs. 7Dvs. F) were normally expressed in the eya1-MO-injected
Fig. 5. Injection of six1a-, myf5-and myod mRNA enables embryos to rescue trunk migratory head muscle sternohyoideus (sh) defect in six1a morphants. Dorsal views of embryos derived either from the transgenic line Tg(α-actin:RFP) (A, D, G, J, M) or from the wild-type (B, C, E, F, H, I, K, L). The RFP expression in the embryos derived from the transgenic line at 72 hpf (A) and the detection of myf5 and myod by whole-mount in situ hybridization at 36 hpf (B, C) served as control groups. Injection of embryos with either 8 ng of six1a-MO alone (D–F) or co-injection with 8 ng of six1a-MO and 150 pg of six1a mRNA (G–I), 100 pg of myf5 mRNA (J–L) or 50 pg of myod mRNA (M–O) were examined. RFP, myf5 and myod were not detected in sh, fb, or phm primordia in the six1a morphants (D–F); however, co-injection of six1a mRNA enabled embryos to rescue the defective expressions of RFP, myf5 and myod in sh, fb and phm primordia induced by six1a-MO at 48 hpf (H, I) and at 72 hpf (G). Meanwhile, injection of myf5 mRNA enabled embryos to rescue the defective expressions of RFP, myod and myogenin in sh, fb and phm primordia induced by six1a-MO at 48 hpf (K, L) and at 72 hpf (J). Injection of myod mRNA enabled embryos to rescue the defective expressions of RFP, myf5 and myogenin in sh, fb and phm primordia induced by six1a-MO at 48 hpf (N, O) and at 72 hpf (M). fb,fin bud; phm, posterior hypoaxial muscle; and sh, sternohyoideus. 8 C.-Y. Lin et al. / Developmental Biology xxx (2009) xxx–xxx
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457 embryos. In addition, we injected eya1-MO into the embryos
458 derived from transgenic line Tg(α-actin:RFP) and found that the
459 head muscles were still developed normally in the eya1 morphants
460 (Figs. 7I, L), even though the eya1 morphant suffered from reduced
461 size of the inner ear (Supplemental Fig. S5), a phenotype similar to
462 that of the eya1 mutant described by Kozlowskia et al. (2005). In
463 contrast, when we detected the myf5 and myod expressions in the
464 pax3-knockdown morphants, we found that myf5 was detected in
465 the head muscles that originated from the mesoderm (Figs. 7A vs.
466 B), and myod was detected only in so, io, sr and lr muscle primordia
467 in the pax3-MO-injected embryos (Figs. 7D vs. E), suggesting that
468 pax3 is necessary for the development of mr and ir, all arch muscles
469 and sh. Moreover, when we injected pax3-MO into the Tg(α-actin:
470 RFP) embryos, we found that only the so, io, sr and lr muscles
471 remained unchanged, which was similar to that of six1a-MO
472
morphants (Figs. 7G, H, J, K). When we co-injected six1a mRNA
473
with pax3-MO, it was observed that the head muscle defects
474
induced by pax3-MO could not be rescued (Figs. 7M, P). Similarly,
475
neither myf5 nor myod mRNA was able to rescue the pax3-morphant
476
defects (Figs. 7N, O, Q, R). To better understand the role pax3 plays
477
in craniofacial muscle development, we analyzed the expression
478
pattern of pax3 by whole-mount in situ hybridization during late
479
embryogenesis. Results showed that pax3 was not detected in the
480
cranial muscles, with the exception of sh (Supplemental Fig. S6).
481
However, the absence of Pax3 function caused a severe defect in the
482
head muscles. Based on this evidence, we suggest that the
483
modulation of pax3 on the cranial muscle development from head
484
mesoderm is indirect in manner. It is also clear that six1a and pax3
485
do not function in the same regulatory network during cranial
486
muscle development.
Fig. 6. The function of Six1a in branchial muscle development requires Tbx1 and Myf5 to play a specification role on arch muscle cell fate. Embryos derived from the wild-type strain (A–D) and from the transgenic line Tg(α-actin:RFP) (E–H) were examined at lateral view. Whole-mount in situ hybridization was used to detect the tbx1 expression in arch muscle and cranial mesoderm in wild-type embryos at 36 hpf (A). Compared to the above control embryos, the expressions of six1a (B), myf5 (C) and myod (D) in the 10 ng group of tbx1-MO-injected embryos were lost in arch muscles, but retained in extraocular muscles. We also observed that all the pharyngeal arch muscles were lost, but that 6 extraocular muscles developed normally in the 10 ng group of tbx1-MO-injected embryos derived from Tg(α-actin:RFP) at 72 hpf (E). With co-injection of 10 ng of tbx1-MO and either 150 pg of six1a mRNA (F), 100 pg of myf5 mRNA (G) or 50 pg of myod mRNA (H) in embryos, we found that only myf5 mRNA enabled embryos to rescue the RFP expression in lap, do, ah, ao, am, ima/ imp and ih/hh (marked in green typeface of G). The heart defect induced by injection of tbx1-MO is labeled with a white star. ah, adductor hyoideus; am, adductor mandibulae; ao, adductor operculi; do, dilator operculi; hh, hyohyoideus; ih, interhyoideus; ima, intermandibularis anterior; imp, intermandibularis posterior; io, inferior oblique; ir, inferior rectus; lap, levator arcus palatini; lr, lateral rectus; mr, medial rectus; sh, sternohyoideus; so, superior oblique and sr, superior rectus. Embryos were all lateral views.
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Fig. 7. Similar to Six1a, Pax3 is required for the development of mr, ir and all arch muscles. Embryos derived from the wild-type strain (A–F) and from the transgenic line Tg(α-actin: RFP) (G–R) were examined. Whole-mount in situ hybridization was used to detect the expressions of myf5 and myod at 48 hpf in the wild-type embryos, which served as a control group (A, D). Compared to the control group, embryos injected with 3 ng of pax3-MO exhibited a normal expression of myf5 in head muscle primordia at 48 hpf (B), but these embryos expressed myod only in so/sr, lr, io, and some remnant branchial muscles (E). Embryos injected with 10 ng of eya1-MO expressed myf5 and myod normally in head muscle primordia at 48 hpf (C, F). We also noticed that the RFP expression of embryos injected with six1a-MO was similar to that of control group at 72 hpf (G vs. J). The RFP signal appeared in the so, io, sr, lr and some remnant branchial muscles in pax3-MO-injected embryos, which was similar to that of the six1a morphants at 72 hpf (G vs. H and J vs. K). The RFP signal appeared in all cranial muscles of the eya1-MO-injected embryos at 72 hpf (I and L). By co-injection of 10 ng of pax3-MO with 150 pg of six1a mRNA (M, P), 100 pg of myf5 mRNA (N, Q) or 50 pg of myod mRNA (O, R), we found that the defective expressions of six1a, myf5 and myod could not be rescued in the pax3 morphants.
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487 Discussion
488 Myf5 and Myod play crucial functions in modulating the
expres-489 sion of genes encoding the muscle structural proteins and,finally,
490 permitting the assembly of myofibers (Molkentin and Olson, 1996; 491 Buckingham, 2001). In previous studies, we demonstrated that the
492 role of myogenic regulatory factor myf5 is markedly different from
493 that of myod during craniofacial muscle development in zebrafish
494 through three pathways (Lin et al., 2006). However, it remained
495 unclear whether other factors might be involved in the regulation of
496 myf5 and myod expression in craniofacial muscle development.
497 Here, we study the roles that transcriptional factors six1a, tbx1 and
498 pax3 play in regulating myf5 and myod expressions in craniofacial
499 muscle development. Based on expression patterns and knockdown
500 phenotypes, we, therefore, proposed four putative regulatory
path-501 ways along which these myogenic regulatory factors function with
502 particular focus on Myf5 and Myod (Fig. 8).
503 Regulatory networks of Tbx1, Six1a, Pax3, Myf5 and Myod during
504 cranial myogenesis
505 During zebrafish cranial muscle development, the arch I and II
506 mesoderm cores are subdivided into dorsal and ventral mesoderm
507
cores. The dorsal mesoderm cores are the precursors of lap, do, am, ah,
508
and ao, whereas the ventral mesoderm cores are the precursors of
509
ima, imp, ih, and hh. We previously found that both myf5 and myod
510
are expressed in the dorsal mesoderm cores, but only myod is
511
expressed in the ventral mesoderm (Lin et al., 2006). Nathan et al.
512
(2008)also showed that chick myf5 expresses in the dorsal region of Q2
513
arch I mesoderm core at an early stage and functions synergistically
514
with Isl1 to commit cell fates to be different from those in ventral arch
515
I mesoderm core.
516
Pathway I is involved in dorsal arch muscle development. In this
517
study, we demonstrate that the expressions of myf5, six1a and myod
518
are not detected in the dorsal arch region of embryos treated with
519
tbx1-MO. However, only injection of myf5 mRNA can enable rescue
520
of the embryos from the defects induced by tbx1-MO (Fig. 6). We
521
therefore propose that tbx1 determines cell fates to myogenic lineage
522
through the modulation of myf5. Once myf5 is expressed, myod
523
expression starts to increase. On the other hand, we found that myf5
524
was expressed normally in the embryos injected with either six1a- or
525
pax3-MO (Figs. 2,7), whereas myod (Figs. 2,7) and myogenin (Fig. 2)
526
transcripts were severely reduced in the dorsal arch of six1a-and
527
pax3-morphants. In addition, injection of myod mRNA enables rescue
528
of embryos from the defects induced only by six1a-MO (Figs. 3,7).
529
Taken together, we conclude that six1a and pax3 are not involved in
Fig. 8. Regulatory network model for tbx1, six1a, pax3, myf5 and myod, which are involved in the craniofacial muscle development of zebrafish embryos. Based on the evidence presented in this study, we propose that the development of all cranial muscles of zebrafish is through four regulatory pathways, which is a modification of model presented byLin et al. (2006). To summarize, Pathway I (marked in green): for dorsal arch muscles, lap, do, ah, ao and am. The myogenic regulatory factor tbx1 activates myf5 to initiate myogenesis. As a consequence, the basal level of myf5 triggers myod expression to further myogenic processes. Subsequently, myod, which is directly controlled by six1, but indirectly controlled by pax3, maintains and enhances a high level of myogenesis. Pathway II (marked in yellow): for the precursors of ventral arch muscles, ima, imp, ih and hh. Before subdivision, tbx1 activates myf5 expression to determine the myogenic cell fate. After subdivision, the major role of myod is to trigger the continuation of myogenic processes. Subsequently, myod, which is directly controlled by six1a, but indirectly controlled by pax3, maintains and enhances a high level of myogenesis. Pathway III (marked in red): for extraocular muscles, mr and ir. The myogenic regulatory factor myod initiates myogenesis, and its expression is controlled by six1a directly, and by pax3 indirectly, to maintain and enhance myogenesis. Pathway IV (marked in blue): for trunk migratory head muscle, sh. The MRF six1a directly controls both myf5 and myod in the myogenesis process, but myf5 and myod have redundant function.
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530 myogenesis initiation. Rather, they are required for maintaining a high531 level of myod transcripts so that myogenesis can be processed. We also
532 notice that myod mRNA enables rescue of embryos from the defects
533 induced by six1a-MO, but not by pax3-MO, suggesting that, while the
534 expression of myod in the dorsal arch is directly controlled by six1a, it
535 is only indirectly controlled by pax3, perhaps through the interaction
536 of still unknown regulatory modules.
537 Pathway II is involved in ventral arch muscle development.
538 Ventral mesodermal core primordia originate from the myf5-positive
539 core and require Tbx1 and Myf5 to initiate myogenic lineage. In this
540 study, we found that the expressions of six1a and myod are lost in
541 the ventral arch region of tbx1 morphants, but that this defect can be
542 rescued by overexpression of myf5 mRNA (Fig. 6). Therefore, we
543 propose the involvement of two steps in the development of ventral
544 arch muscle: tbx1 initiates myf5 expression in the beginning, and,
545 subsequently, six1a enhances and maintains myod expression.
546 Furthermore, similar to Pathway I in dorsal arch, we also found
547 that the expressions of myod (Figs. 2,7) and myogenin (Fig. 2) are
548 severely reduced in ventral arch when embryos are injected with
549 both six1a-and pax3-MO. Thus, we conclude that six1a and pax3 are
550 required to maintain myod expression in the ventral mesodermal
551 core cells. Again, as in Pathway I, we found that overexpression of
552 myod mRNA enables rescue of embryos from the defects induced
553 only by six1a-MO, but not pax3-MO, suggesting that the regulation
554 of myod in the ventral arch is directly controlled by six1a, but
555 indirectly controlled by pax3.
556 PathwayШ is involved in the development of extraocular muscles,
557 mr and ir. We found that six1a and myod transcripts are detected, but
558 not myf5 transcript, in mr and ir (Fig. 1). Meanwhile, the expressions
559 of myod (Figs. 2,7>) and myogenin (Fig. 2) were lost in the mr and ir
560 of six1a- and pax3-morphants. Injection of myod mRNA enables
561 rescue of embryos from the defects induced by six1a-MO, but not
562 induced by pax3-MO (Figs. 3,7). Therefore, myod is required for the
563 development of mr and ir, and myod is directly regulated by six1a.
564 Interestingly, although six1a and myod transcripts are expressed in all
565 extraocular muscles, the so, io, sr and lr are still observed in the six1a
566 morphants, suggesting that other factors may be involved in
567 controlling the development of so, io, sr and lr.
568 Pathway IV is involved in the development of sh primordia, which
569 originates from anterior trunk somites. We found that the transcript
570 of sh was lost in the six1a morphants, but injection of either myf5
571 mRNA or myod mRNA enables embryos to be rescued from this defect
572 (Fig. 5). Since sh muscle is derived from anterior trunk somites, the
573 regulatory mechanisms controlling the muscle development between
574 head and trunk paraxial mesoderm are different. This evidence
575 suggests that six1a is governed by a head-specific regulatory cascade
576 in cranial myogenesis, which is fundamentally distinct from that
577 which is governed by a trunk-specific regulatory cascade. Taken
578 together, we conclude that six1a performs its function in head muscle
579 development, but it does so separately and by two distinct pathways,
580 one where muscle development originates from head mesoderm and
581 one where muscle development originates from the trunk.
582 Networks supporting modulation of Sixla and MRFs are dependent on
583 mesoderm origin
584 The branchiomeric muscles originate from cranial paraxial
meso-585 derm. Knockdown of Six1a function results in greatly decreased
586 expressions of myod and myogenin, while myf5 is expressed normally.
587 Because of the existence of myf5 in these muscles, the remaining arch
588 muscles in Six1a knockdown embryos are observed. Nevertheless,
589 myod transcripts are reduced, with the result that these muscles
590 eventually lose their function (Fig. 3). Like branchiomeric muscles, the
591 extraocular muscles originate from cranial paraxial mesoderm. Both
592 six1a and myod transcripts are expressed in mr and sr muscles at
593 32 hpf, whereas myf5 is not expressed (Fig. 2). Thus, when six1a is
594
knocked down, the myod transcripts are lost, with the result that
595
muscle primordia of mr and ir are not developed (Fig. 3). On the other
596
hand, unlike branchiomeric muscles and extraocular muscles, the
597
cranial muscle sh originates from trunk paraxial mesoderm, which is
598
named dermomyotome. During sh muscle development, Six1a is
599
required for both myf5 and myod expressions (Fig. 5). This result is
600
consistent with what we observe in fb and phm which originate from
601
anterior trunk mesoderm in zebrafish. Therefore, both myf5 mRNA
602
and myod mRNA were injected in the attempt to rescue muscle defects
603
of six1a morphants. While injection of myf5 mRNA could rescue only
604
the defective sh muscle in six1a morphants, injection of myod mRNA
605
enabled rescue of embryos from all defective cranial muscles (Fig. 4).
606
Based on thesefindings, it seems clear that the modulatory network
607
between Six1a and these two MRFs in cranial paraxial mesoderm is
608
quite different from that which is observed in trunk (sh) paraxial
609
mesoderm. This conclusion is similar to, and supported by, the
610
findings ofGrifone et al. (2005)in mice. They described how six1 and
611
six4 genes control mrf4 expression and that six1−/−six4−/−embryos
612
display reduced and delayed expressions of myod and myogenin,
613
whereas the early activation of myf5 transcripts in the epaxial somite
614
is unaffected. However, in the limb muscles, the Six1/4 are shown to
615
be involved in myf5 transcription through binding the MEF3 site in the
616
145-bp regulatory sequence located at −57.5 kb of myf5 gene
617
(Giordani et al., 2007).
618
The six genes constitute a large family of genes that are highly
619
conserved within the animal kingdom. In mammals, six members of
620
the Six family have so far been identified, and these can be divided
621
into three subclasses designated as Six1/2, Six3/6 and Six4/5
622
subfamilies (Laclef et al., 2003; Seo et al., 1999). Moreover, it has
623
been subsequently demonstrated that Six1, Six2, Six4 and Six5 have a
624
similar binding specificity to the ARE/MEF3 site possessing a
625
consensus sequence TCAGGTTTC (Ohto et al., 1999; Spitz et al., 626 1998). In mice, the defects of muscle hypoplasia in six1−/−six4−/−
627
embryos are more severe than those seen in six1−/− embryos
628
(Grifone et al., 2005; Laclef et al., 2003). Similarly, the reduced
629
expression of myf5 in the hind limb of six1−/−six4+/−mice is more
630
severe than that observed in six1−/−embryos. These lines of evidence
631
suggest that Six4 in myogenic progenitor cells displays a redundant
632
function with Six1. Meanwhile, in zebrafish, three isoforms, six4.1–4.3,
633
and their expression patterns have been defined, and six4.2 is
634
expressed in the presomitic mesoderm, somites and pectoralfin bud
635
(Kobayashi et al., 2000). In addition,Bessarab et al. (2008)reported
636
that the fast muscles differentiate abnormally in the trunk muscles of
637
six1a morphants in contrast to the slow muscles which develop
638
normally. The expression of myogenin is reduced in all somites in the
639
six1a morphants at the 9-somite stage. However, myogenin increases
640
its expression at the 10-somite stage andfinally reaches its normal
641
expression level at the 13-somite stage. In our case, we also noticed
642
that the cranial muscles in six1a morphants are partially developed.
643
Thus, we speculate that zebrafish six4.2 may have redundant function
644
with six1 during muscle development of zebrafish. The zebrafish
645
Six4.2, like mouse Six4, might partially compensate for the absence of
646
Six1 to activate MRFs in the trunk muscle cells. According to this
647
hypothesis, the selective muscle hypoplasia described in six1
648
morphants could result from either insufficient levels of Six4.2 to
649
compensate for Six1 in the affected myogenic precursor cells or from
650
the existence of specific Six1 target genes.
651
Comparison of Six1a, Pax3 and Eya1 functions in head muscle
652
development between zebrafish and other model animals
653
Genetic studies in Drosophila have identified that the eyeless (pax)
654
is synergistic with DNA binding homeodomain factors, such as sine
655
oculis (so/six), and nuclear cofactors, such as eyes absent (eya) and
656
dachshund (dach) (Cheyette et al., 1994; Bonini et al., 1993; Mardon 657 et al., 1994). Mutation of any gene encoding for these proteins leads
12 C.-Y. Lin et al. / Developmental Biology xxx (2009) xxx–xxx