Sequential Expression and Redundancy of Pitx2 and Pitx3 Genes During Muscle Development☆

Developmental Biology 307 (2007) 421–433 www.elsevier.com/locate/ydbio

Genomes & Developmental Control Sequential expression and redundancy of Pitx2 and Pitx3 genes during muscle development☆

Aurore L'Honoré a,1, Vincent Coulon b,1, Alexandre Marcil c, Mélanie Lebel d, ⁎ Julien Lafrance-Vanasse a, Philip Gage e, Sally Camper e, Jacques Drouin a,

a Laboratoire de génétique moléculaire, Institut de Recherches Cliniques de Montréal (IRCM), 110, avenue des Pins Ouest, Montréal, QC, Canada H2W 1R7 b CNRS UMR 5535, Institut de génétique moléculaire, 1919 route de Mende, 34293 Montpellier, Cedex 05, France c Centre d'innovation de Génome Québec and de l'Université McGill, Montréal, QC, Canada H3A 1A4 d Hospital for Sick Children, Program in Developmental Biology, Toronto, ON, Canada M5G 1X8 e Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109-0618, USA Received for publication 20 November 2006; revised 26 April 2007; accepted 26 April 2007 Available online 3 May 2007

Abstract

The myogenic program is controlled by different groups of transcription factors acting during muscle development, including bHLH muscle regulatory factors (MRFs), the paired factors Pax3 and Pax7 and the homeobox factors Six1 and Six4. This program is critically dependent on MRFs that target downstream muscle-specific genes. We now report the expression of Pitx2 and Pitx3 transcription factors throughout muscle development. Pitx2 is first expressed in muscle progenitor cells of the dermomyotome and myotome. The onset of myoblast differentiation is concomitant with expression of Pitx3; its expression is maintained in all skeletal muscles while Pitx2 expression decreases thereafter. We have generated Pitx3 mutant mice and this deficiency does not significantly perturb muscle development but it is completely compensated by the maintenance of Pitx2 expression in all skeletal muscles. These experiments suggest that Pitx genes are important for myogenesis and that Pitx2 and Pitx3 may have partly redundant roles. © 2007 Elsevier Inc. All rights reserved.

Keywords: Myogenesis; Transcription factors; Knockout mice; Differentiation

Introduction (MRFs) Myf5, Mrf4 and MyoD (Rudnicki, 2003; Tajbakhsh et al., 1996; Kassar-Duchossoy et al., 2004). These progenitors The skeletal muscles of the body and limbs are derived from give rise to muscle precursors, the myoblasts present in the somites that segment the elongating embryo along the anterior– myotome (Smith et al., 1994; Tajbakhsh, 2003) under the posterior axis. The somites rapidly separate into a ventral influence of myogenin, MyoD and Mrf4 (Tajbakhsh and mesenchymal compartment, the sclerotome, and a dorso-lateral Buckingham, 2000). The myotome is a transient structure and epithelial compartment, the dermomyotome. Cells delaminate at its role appears to be the establishment of the original muscle the dorsal and ventral lips of the dermomyotome to form the masses that form through elongation. Myoblasts from the myotome along its medial surface (Christ and Ordahl, 1995; medial/dorsal myotome will develop into epaxial muscles and Tajbakhsh and Buckingham, 2000). Multipotent muscle intrinsic back muscles whereas cells of lateral/ventral portion of progenitor cells arising in the dermomyotome acquire definitive the dermomyotome and of the lateral myotome will develop identity through the action of the myogenic regulatory factors into hypaxial muscles which include intercostal, abdominal and limb muscles as well as the diaphragm and tongue muscles (Buckingham et al., 2003). At the level of developing limbs, ☆ Pitx2 and Pitx3 in skeletal muscles. ⁎ Corresponding author. Fax: +1 514 987 5575. cells that delaminate from the ventral hypaxial lip of the E-mail address: [email protected] (J. Drouin). dermomyotome migrate towards the developing limb buds and 1 Contributed equally to the work. establish themselves in the early muscle territories of the limbs

(Scaal et al., 1999; Dietrich, 1999; Christ and Brand-Saberi, subset of midbrain dopaminergic neurons (Smidt et al., 1997; 2002); these territories appear to be defined independently of van den Munckhof et al., 2003, 2006). muscle progenitor cells (Kardon et al., 2003). These migratory We now report the detailed investigation on the expression of cells require Pax3 for proper migration and establishment of Pitx2 and Pitx3 during myogenesis. Pitx2 is expressed the limb muscle masses (Bober et al., 1994; Daston et al., 1996; earliest in muscle progenitor cells. This expression is followed Tremblay et al., 1998b). Indeed, a migration defect was ascribed and partly overlapping with the appearance of Pitx3 in to the failure to express the receptor tyrosine kinase c-met in the differentiated muscle cells. Pitx3 then appears to be widely absence of Pax3 (Bladt et al., 1995; Epstein et al., 1996; expressed in all skeletal muscles of the body and limbs. The Birchmeier and Brohmann, 2000). Migrating progenitor cells investigation of Pitx3−/− mice indicates that Pitx3 on its own is also express Mox2 and Lbx-1 which are required for migration not required for myogenesis. However, Pitx2 is strongly and under the control of Pax3 (Mankoo et al., 1999; Mennerich upregulated in the absence of Pitx3 and appears to fully et al., 1998; Schafer and Braun, 1999; Gross et al., 2000). compensate during muscle formation. Taken collectively, these Pax7,aPax3 paralogue, has also been implicated in the data suggest that Pitx genes are important for myogenesis and specification of cells that enter the myogenic program. In the that their activities may be partly redundant. absence of both Pax3 and Pax7, there is a major deficit in skeletal muscle development with the arrest of the myogenic program occurring during late embryonic and fetal develop- Materials and methods ment (Relaix et al., 2005). Indeed in this mutant, cells which should have expressed these two genes die or change cell Construction of Pitx3 gene targeting vector fate. Interestingly, Pax7 is not expressed in early embryonic Pitx3 genomic DNA was cloned from a 129sv genomic library. Genomic muscle progenitors present at the edges of the dermomyo- fragments corresponding to the Pitx3 locus were cloned into pBluescript KS+ tome. Rather, Pax3/Pax7-positive cells are derived from the (for construction of a targeting vector, Fig. 5). Homologous recombination with central part of the dermomyotome (Ben Yair and Kalcheim, this vector will lead to insertion of a fragment containing the neomycin 2005; Gros et al., 2005). These cells can either activate the resistance gene flanked by FRT sites and to insertion of LoxP sites upstream of the neo cassette and downstream of exon 3. Expression of Cre recombinase myogenic program through the action of Myf5 and MyoD should lead to excision of this whole fragment, including Pitx3 exon 3 that codes or remain as a proliferating progenitor population that for the homeodomain responsible for the DNA binding activity of Pitx3. reside within the muscle mass (Gros et al., 2005; Kassar- Duchossoy et al., 2005; Relaix et al., 2005). In late-stage ES cell screening and chimeric mouse production fetal muscle, these cells adopt a satellite cell position, suggesting that they can become the pool of adult satellite cells Linearized targeting DNA was electroporated (250 V, 500 μF) into μ responsible for postnatal skeletal muscle growth and integrity embryonic stem (ES) cells. ES cells were selected with 250 g/ml of G418 48 h after electroporation. The DNA of ≈600 resistant clones was analyzed by (Gros et al., 2005; Kassar-Duchossoy et al., 2005; Relaix et al., Southern blot after digestion. A 3′ genomic fragment was used as external probe. 2005). Three independent homologous recombinant clones were identified and Two homeobox genes related to drosophila sine oculis (Six1 microinjected into C57BL6 blastocysts for implantation into pseudopregnant and Six4) are also expressed in muscle cells starting in mice. Chimeric males were obtained for the 3 clones and tested for germline transmission. Two chimeric males were chosen and heterozygous progenies dermomyotome and myotome (Ozaki et al., 2001; Laclef et − − were generated by backcrosses to C57BL6 and 129/SvJ females. Pitx2 / and al., 2003; Grifone et al., 2005). Inactivation of both genes CMV-Cre transgenic mice were described previously (Gage et al., 1999). appears to prevent myogenic precursor delamination and migration, in part through deficient Pax3 expression and in Mouse genotyping part through deficient MRF expression (Grifone et al., 2005). These different studies clearly illustrate that the pathway for Genotyping was carried out by PCR using DNA isolated from the control of myogenesis is not linear and includes many inputs umbilical cord/amniotic membrane of the embryos or adult tail sections. Separate reactions were carried out for Pitx3 and Cre genotyping. Three whose interrelations are not well defined. specific primers were used for Pitx3 alleles: one forward primer in intron 2: 5′- Prior work has shown expression of the homeobox GGGAGCGAGAGTGATAACCT-3′, and two reverse primers located in intron transcription factor Pitx2 in what appeared to be myotomes 3: 5′-GGAACAGCTCGGGATCAAAG-3′ and in the neomycin gene 5′-TAC- and putative migrating myoblasts (Logan et al., 1998; Kioussi et CGGTGGATGTGGAATGT-3′. Two primers were used for Cre detection: ′ ′ ′ al., 2002). The related Pitx3 is also expressed in somites (Zhao forward primer 5 -ATCCGAAAAGAAAACGTTGA-3 and reverse primer 5 - ATCCAGGTTACGGATATAGT-3′. For embryo studies, noon of the day on et al., 2004) and we have recently identified a muscle-specific which a vaginal plug was detected was considered as e0.5. Embryos were staged promoter responsible for its expression in myotome and more precisely by counting the number of somites posterior to the forelimb bud muscles (Coulon et al., submitted for publication). Otherwise, and scoring the first one counted as somite 13 (Lewandoski et al., 2000). Pitx2 has been implicated in left–right asymmetry (Yoshioka et al., 1998; Logan et al., 1998; Piedra et al., 1998; Ryan et al., Preparation and characterization of affinity-purified anti-Pitx2 and 1998), in cranio-facial development and in pituitary function as anti-Pitx3 antibodies indicated by the name of these factors (Lin et al., 1999; Lu et al., Antibodies were raised in rabbits using 100 μg MBP-Pitx2 or MBP-Pitx3 for 1999; Gage et al., 1999). Pitx3 is required for ocular the primary injection and three subsequent boosts. Affinity purification was development (Semina et al., 1997; Semina et al., 1998) and performed as previously described (Lanctôt et al., 1999b) except that a GST- for the development (Smidt et al., 2004) and maintenance of a Pitx2 or Pitx3 column was used. Two different antisera were produced against A. L’Honoré et al. / Developmental Biology 307 (2007) 421–433 423

MBP fusion antigens containing amino acids 1–38 of mouse Pitx2 protein and Whole-mount in situ hybridization amino acids 121–180 of mouse Pitx3. Sections and whole-mount in situ hybridization was performed as described Histology, immunohistochemistry and immunofluorescence in the protocols from Dr. Janet Rossant's laboratory using Pitx2 and Pitx3 probes. These two protocols used can be found at http://www.sickkids.ca/ Histology and section immunohistochemistry was performed as described rossant/custom/protocols.asp. (Lanctôt et al., 1999a) using rabbit anti-Pitx2 (1/250), rabbit anti-Pitx3 (1/ 250) and a previously described rabbit Pitx1 antibody (Tremblay et al., 1998a). Other primary antibodies were purchased from Pharmingen (anti- Results mouse MyoD 1/100 and myogenin 1/100) or from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA Expression of Pitx2 and Pitx3 in muscle cells 52242 (anti-mouse α-actin, MF20 1/20, Pax3 1/100 and Pax7 1/10). Biotinylated anti-mouse and anti-rabbit (Vector Labs, 1/150) were used as The embryonic pattern of Pitx gene expression was revealed secondary antibodies and revealed using streptavidin–HRP (NEL 750, NEN, by whole-mount in situ hybridization. For both Pitx2 and Pitx3, 1/1000) and DAB for immunohistochemistry. Slides were then counter- expression was detected in myotome and in forming muscles stained with Methyl Green. For immunofluorescence, biotinylated anti-rabbit, – anti-mouse-Alexa 488 or 546, and streptavidin-Alexa 488 or 546 (Vector masses of limb buds (Figs. 1A H). This expression appears Labs, 1/150) were used as secondary or tertiary antibodies. Slides were later than the Pitx2 expression in oral ectoderm and lateral plate mounted using Vecta-Shield (Vector Labs). For Pitx3 and Pax7 co-staining, mesoderm that are already evident at e9.5 (Fig. 1A). Examina- rabbit anti-Pitx3 (1/250) and anti-rabbit alkaline phosphatase (Vector Labs, 1/ tion of multiple embryos suggested that myotome expression of 300) were used together with mouse anti-Pax7 (1/10), biotinylated anti-mouse Pitx2 and Pitx3 starts around e10.5 and that both are well (Vector Labs, 1/150) and streptavidin–HRP (NEL 750, NEN, 1/1000). The reactions were revealed using NBT/BCIP (Roche Diagnostics) and NovaRED established by e11, with Pitx2 signal appearing stronger than (Vector Labs) substrates. No counter-staining was performed and slides were that of Pitx3 (Figs. 1B and F). By e11.5, both genes appear to mounted using aqueous medium (9:1 glycerol/PBS). have similar patterns in developing muscles (Figs. 1C and G)

Fig. 1. Early expression of Pitx2 and Pitx3 genes in muscle cells. Whole-mount in situ hybridization of wild-type embryos reveals the pattern of expression for Pitx2 (A–D) and Pitx3 (E–H). At e9.5 (A, E), only Pitx2 is expressed in oral ectoderm and lateral plate mesoderm (arrowheads). (B, F) Both Pitx2 and Pitx3 are expressed in myotomes by e11 at the same time as Pitx3 expression in eye lens (F) and Pitx2 in ocular muscles (B). By e11.5 (C, G) (lateral views) and e12.5 (D, H) (dorso-posterior views), both genes are expressed in all developing muscles (myotome and limbs muscles). Immunohistochemistry experiments performed on serial transverse sections of e10.5 embryos reveal early Pitx2 and Pitx3 expression in myotome at the forelimb (I–M) but not at hindlimb (N–R) level. At the FL level, Pitx2 (L) and Pitx3 (M) are both detected in the ventral hypaxial part of the myotome (m) delineated by myogenin (I) and MF20 (J) staining while Pitx2 (but not Pitx3) is also expressed in ventro-lateral lip (vll) of dermomyotome (dm) that stains for Pax3 (K). 424 A. L’Honoré et al. / Developmental Biology 307 (2007) 421–433 and this is illustrated for hindlimb (HL) muscles at e12.5 (Figs. performed co-staining of Pitx2 with Pax3 (Figs. 3A–C): 1D and H). indeed, many of these vll cells co-express Pitx2 and Pax3 In order to correlate the expression of Pitx2 and Pitx3 with (Fig. 3C2). In the central myotome, most Pitx2-positive cells myotome and/or dermomyotome development, sequential are also Pax3-positive (Fig. 3C1). We then checked Pitx2 and transverse sections from e10.5 embryos taken at the level of Pitx3 expression at limb level in migrating myoblast forelimb (FL) and HL buds were analyzed by immunohisto- precursors by co-labeling with Pax3; we did not find any chemistry for Pitx factors and for markers of myogenesis (Figs. co-labeling in migrating cells in agreement with Figs. 1K, L 1I–R). At the FL level, expression of Pitx2 is first detected in and P, Q. Interestingly, all Pax3-positive cells that have the hypaxial part of the myotome (Fig. 1L), defined by completed migration at the proximal limb bud also express myogenin (Fig. 1I) and MF20 staining (Fig. 1J). It is also Pitx2 (Figs. 3D–I). However, all Pitx2-positive cells are not expressed in the ventro-lateral lip (vll) of the dermomyotome positive for Pax3: these may represent myoblast cells that (Fig. 1L), delineated by Pax3 staining (Fig. 1K). No expression have entered differentiation. In parallel co-labeling experi- is detected at this stage at the HL level (Fig. 1Q). Serial sections ments, Pitx3 was never co-expressed with Pax3 (data not stained for Pitx3 reveal expression in the hypaxial part of rostral shown). myotomes but not in dermomyotome (Fig. 1M), in contrast to Pax7 is expressed with Pax3 in a large subset of muscle Pitx2. Interestingly, neither Pitx gene is expressed in less progenitors (Relaix et al., 2005). In agreement with the developed myotomes at HL level (Figs. 1Q, R), whereas Pax3 interpretation that muscle progenitors express Pitx2, we found and myogenin are already detected (Figs. 1N, P), suggesting extensive co-labeling of myotome cells with Pitx2 and Pax 3 that Pitx2 and Pitx3 switch on after myogenic commitment in (Fig. 3C1), and with Pax7 (Figs. 3J–L). In order to assess myotome. In addition, migratory cells (arrowhead) and HL whether Pitx2-positive cells are indeed proliferating progeni- precursors expressing Pax3 do not express Pitx genes (Fig. 1P). tors, we performed BrdU incorporation in e11.5 embryos At later developmental stages (data not shown), caudal (Figs. 3M–O, Y). Analysis of developing FL muscle masses myotomes exhibit Pitx expression patterns similar to those of indicated that a large number of Pitx2-positive cells e10.5 rostral myotomes. incorporated BrdU (Figs. 3O, Y). In contrast, co-staining for Pitx3 revealed fewer double-positive cells with BrdU Sequential expression of Pitx2 and Pitx3 (Figs. 3V–X, Z). Phospho-histone H3 is a marker of early mitosis and in agreement with the idea that Pitx2-positive In order to better define expression of Pitx2 and Pitx3 genes progenitors are in proliferation, we detected cells positive for during myotome development, we investigated their expression both Pitx2 and phospho-H3 in interlimb myotomes of e11.5 by co-localization with various markers in e11.5 myotomes that embryos (Figs. 3P–R, Y). In contrast, double-positive cells are progressively more advanced in development along the for phospho-H3 and Pitx3 could only rarely (about 1.5%) be anterior to posterior axis (Fig. 2). In these embryos, the least observed (Fig. 3Z), suggesting that the few Pitx3- and BrdU- developed myotomes at the caudal end show a significant positive cells (Figs. 3X, Z) correspond to cells that recently number of nuclei positive for Pitx2 (Figs. 2D and J), but very exited the cell cycle. In order to test the idea that Pitx3 few positive for Pitx3 (Figs. 2P and V). Co-labeling with switches on at the onset of muscle differentiation, we did co- myogenin and MF20 indicates that many cells co-express these labeling of myotomal and limb muscle masses with Pitx3 and differentiation markers with Pitx2 (Figs. 2F, L1 and 3Y). It is the MRFs MyoD and myogenin (Fig. 3Z). Whereas in interesting to note that Pitx2-positive and MF20-negative cells myotome, about 80% of Pitx3-positive cells are also are present at the ventral lip of the dermomyotome (Fig. 2L2). myogenin-positive, we observed fewer double-positive cells Taken together, these data suggest that Pitx2 is expressed before in limb muscles (about 40%); it is however noteworthy that Pitx3 in myotome; in agreement with its expression in vll (Figs. all limb bud myogenin-positive cells are also Pitx3-positive 1L and 2L2), Pitx2 may be associated with cell progression (Figs. 3S–U). It is also interesting to note different pro- from dermomyotome to myotome. portions of double MRF- and Pitx2-positive cells in myotome The same co-labeling experiments were performed in more compared to limb muscle masses (Fig. 3Y). These data may advanced rostral somites and showed a majority of Pitx3- suggest that Pitx3 expression precedes MRFs in limb bud positive cells co-expressing myogenin (Figs. 2M–O, 3Z) and muscles, in contrast to myotomes. MF20 (Figs. 2S–U). In contrast, only a fraction of Pitx2- positive cells express these markers (Figs. 2A–C, G–I, 3Y). Maintenance of Pitx3 and decreasing Pitx2 expression These results suggest that Pitx3 is expressed in differentiated myotome cells, and that Pitx2 may be expressed earlier in Whole-mount in situ hybridization revealed that both Pitx2 muscle progenitors and precursors. and Pitx3 genes have similar patterns of expression in embryonic developing muscles (Fig. 1). Since Pitx3 early expression Pitx2 is expressed in muscle precursors while Pitx3 appears at suggests widespread presence in all skeletal muscles, we verified differentiation its expression in muscles of e15.5 embryos by in situ hybridization. This analysis (Figs. 4A–C) confirmed Pitx3 In order to determine if the Pitx2-positive MF20-negative mRNA in HL muscles as well as in abdominal and back muscles cells of the myotome and vll are muscle progenitor cells, we that derive from hypaxial and epaxial myotome, respectively. A. L’Honoré et al. / Developmental Biology 307 (2007) 421–433 425

Fig. 2. Pitx2 and Pitx3 are expressed sequentially. Double staining experiments for Pitx2 (A, D, G, J) or Pitx3 (M, P, S, V) and myogenin (B, E, N, Q) or MF20 (H, K, T, W) were performed on transverse sections of e11.5 embryos at rostral (A–C, G–I), thoracic (M–O, S–U) and caudal (D–F, J–L, P–R, V–X) levels. Pitx3 expression in caudal myotome is restricted to a few cells (P–R, V–X), whereas a significant number of cells are positive for Pitx2 (D–F, J–L). A majority of these cells co-express both myogenin (D–F) and MF20 (J–L, L1), but interestingly, a population of Pitx2-positive cells located in the ventro-lateral part of dermomyotome (L2) do not express MF20. The same co-staining performed at thoracic and rostral levels show a large number of Pitx3-positive cells that mostly co-express myogenin (M–O) and MF20 (S–U), whereas only a fraction of Pitx2-expressing cells are positive for these markers (A–C, G–I).

In order to assess the relative expression of Pitx2 and Pitx3 and α-actin showed a close correlation between Pitx3, but in developing muscles, we followed their expression during less so for Pitx2, and α-actin expression (data not shown), in fetal development in a variety of muscles. While Pitx2 agreement with the conclusion that Pitx3 is expressed in expression is already well established at e11.5 in shoulder differentiated muscle cells. This is further supported in e17.5 girdle muscle (Fig. 4D) together with MyoD and myogenin muscles that all show strong Pitx3 expression (Fig. 4Q) but (Figs. 4F, G), fewer Pitx3-positive cells are detected in these very weak Pitx2 (Fig. 4P). In order to directly assess Pitx3 muscles (Fig. 4E). In contrast, by e13.5 the same muscles expression in satellite cells, we did co-labeling of e18.5 exhibit a strong Pitx3 signal (Fig. 4I), but greatly decreased hindlimb muscles with Pitx3 and Pax7 (Fig. 4R). This Pitx2 (Fig. 4H). This switch in relative expression of Pitx2 analysis revealed that most Pax7-positive nuclei are also and Pitx3 is also evident for many muscles on a low Pitx3-positive, in addition to a population of Pitx3 single- magnification view of e13.5 FL (Figs. 4L, M). It is interesting positive nuclei. These data indicate expression of Pitx3 in to note that staining of serial limb sections for Pitx2, Pitx3 both muscle fiber and satellite cells. 426 A. L’Honoré et al. / Developmental Biology 307 (2007) 421–433

Fig. 3. Pitx2 is expressed in proliferative progenitors while Pitx3 is induced at the onset of differentiation. Co-staining experiments for Pitx2, Pitx3, Pax3, Pax7, BrdU, phospho-histone H3 or myogenin were performed on transverse sections of e11.5 embryos at caudal myotome (A–C), HL bud (D–I), FL (M–O, S–U), interlimb (P–R, V–X) and rostral myotome (J–L) level. Pitx2 is co-expressed with Pax3 (A–C) in cells of the myotome (C1), of the ventro-lateral edge of the dermomyotome (C2) and in limb bud progenitor cells (circled in panels D–F, enlarged in panels G–I), while no Pitx3 and Pax3 co-staining can be detected in consecutive sections (not shown). Pitx2-positive cells of the myotome are also mostly positive for Pax7 (J–L). A large number of Pitx2-expressing cells incorporated BrdU in FL muscle masses (M–O) indicating that they were in proliferation whereas only a few Pitx3-positive cells stained for BrdU at thoracic level (V–X). Proliferation of Pitx2- positive cells was confirmed by phospho-histone H3 (pH3) co-staining (P–R) but co-staining could not be observed with Pitx3 (not shown), suggesting that the few BrdU-positive Pitx3-positive cells observed in panels V–X were just completing their last cell cycle. A subset of Pitx3-positive cells of FL muscle masses express myogenin (S–U), suggesting that Pitx3 switches on in early differentiating cells. (Y, Z) Quantitation of cells double-positive with Pitx2 (Y) or with Pitx3 (Z). Data are means±SEM for triplicate counts of at least 100 cells. A. L’Honoré et al. / Developmental Biology 307 (2007) 421–433 427

Fig. 4. Maintenance of Pitx3 and decreasing Pitx2 expression. In situ hybridization performed with MyoD (A) and Pitx3 (B) probes on thoracic transverse sections of e15.5 embryos reveal that both are co-expressed in all back (b), abdominal (ab) and HL muscles. Bl, bladder; tl, tail. Immunohistochemical localization of Pitx2, Pitx3, MyoD and myogenin in sagittal FL (D–O) and HL (P, Q) sections of e11.5 (D–G), e13.5 (H–O) and e17.5 (P–Q) embryos. At e11.5, shoulder girdle muscles already express Pitx2 (D) as well as MyoD (F) and myogenin (G), but not Pitx3 (E). The same muscles examined in e13.5 embryos (H–K) show a switch of relative expression between Pitx2 and Pitx3: a strong expression of Pitx3 is detected (I), while Pitx2 expression is decreasing (H). Similar distributions were observed in e13.5 FL muscles (L–O) and e17.5 HL muscles (P, Q). (R) At e18.5, co-immunostaining was performed for Pitx3 (blue staining) and Pax7 (red staining). Pitx3 was detected in myofiber nuclei (Pax7-negative nuclei marked by asterisk) and in satellite cells (Pax7-positive nuclei marked by arrows). 428 A. L’Honoré et al. / Developmental Biology 307 (2007) 421–433

Normal myogenesis in Pitx3 mutant embryos the targeted sequences (Fig. 5A) identified 7 correctly targeted clones among ≈600 tested (Fig. 5B). Of three ES Since Pitx3 is the most widely expressed Pitx factor in clones that were selected and used for blastocyst injection, fetal muscles (Fig. 4), we undertook to knockout this gene. two transmitted the floxed allele to the germline and were For this purpose, the naturally occurring Pitx3 mutant, the interbred with C57BL6 and 129SV females. Pitx3+/flox mice aphakia mouse, is not useful since this mouse retains obtained in both genetic backgrounds were mated with mice muscle expression of Pitx3 (Coulon et al., submitted for expressing Cre recombinase under the control of the CMV publication). For Pitx3 gene inactivation, we targeted the promoter. Deletion of the third exon of the Pitx3 gene was third exon of the gene that encodes the homeodomain (Fig. confirmed by PCR analysis (Fig. 5B). Pitx3+/− mice are 5A). LoxP sites were introduced upstream and downstream viable, fertile and did not present any obvious phenotype. of exon 3 in a vector that also contains a PGKneo cassette To analyze the effect of Pitx3 loss-of-function, Pitx3+/− flanked by FRT sites. These FRT sites will permit in vivo mice were intercrossed. Pitx3−/− mice in both genetic back- deletion of the neo cassette if required (Dymecki, 1996). grounds are viable, fertile, and have a life expectancy The Pitx3flox-neo allele was generated through homologous similar to wild-type siblings despite the absence of muscle recombination in isogenic 129 mouse embryonic stem (ES) Pitx3 protein (Figs. 7I–L). Further investigation of Pitx3−/− cells. Southern blotting using a 3′ probe located outside of animals showed eye dysmorphogenesis (Fig. 5C) and

Fig. 5. Targeted disruption of the mouse Pitx3 gene. (A) Schematic representation of the Pitx3 gene with its exons shown in black boxes and of the downstream cig30 gene with its exons in open boxes. Upon successful homologous recombination, the Pitx3flox allele was obtained as shown for the targeted gene in which neo represents the PGKneo gene and the open arrows indicate the position the LoxP sites. Upon deletion by cre recombinase, the Pitx3null (−/−) allele was obtained. (B) Southern blot analysis (left) of tail DNA digested with XbaI and hybridized with the probe indicated in panel A reveal mice harboring the three Pitx3 alleles. PCR analysis (right) of genomic DNA from embryos used for genotyping as described in Materials and methods. (C) Eye phenotype of a newborn Pitx3−/− mouse (bottom) compared to wild- type littermate (top). A. L’Honoré et al. / Developmental Biology 307 (2007) 421–433 429 selective loss of midbrain dopaminergic neurons, similar Compensation of Pitx3 deficiency by Pitx2 and not worse than aphakia mice (data not shown), but did not reveal any gross defect in musculature. Since Pitx2 is expressed early during myogenic differ- Despite the absence of an apparent muscle phenotype, we entiation, we tested whether its expression might be altered in investigated Pitx3−/− embryos for expression of muscle the absence of Pitx3. All muscles of Pitx3−/− embryos markers that may reveal partial defects in myogenesis. No examined showed no immunoreactivity for Pitx3 (Figs. 7I–L) defects could be observed in e11.5 myotome (Figs. 6A–H), in whereas their wild-type counterparts showed expression in developing e11.5 intercostal muscles (Figs. 6I–N), or in e13.5 almost all nuclei (Figs. 7A–D); this included paravertebral FL muscle masses (Figs. 6O–R) or in e17.5 HL muscles (A, E, I, M), intercostal (B, F, J, N), lower back (C, G, K, O) (Figs. 6S–V). Analysis of these different muscles at different as well as hip muscles (D, H, L, P) of e15.5 embryos. As times in their development indicated no differences in the shown previously (Fig. 4), these muscles express very low patterns of expression for Pax3 (Fig. 6E), MyoD (Figs. 6F, L, levels of Pitx2 in wild-type embryos (Figs. 7E–H). In striking R, V), myogenin (Figs. 6G, M), MF20 (Figs. 6H, N) or Pax7 contrast, the same muscles of Pitx3−/− embryos exhibit robust (Figs. 6Q, U) compared to heterozygous siblings (Figs. 6A– Pitx2 immunoreactivity (Figs. 7M–P), indicating that Pitx2 D, I–K, O–P, S–T). expression is compensating for the absence of Pitx3 in these

Fig. 6. Embryonic and fetal myogenesis occur normally in Pitx3−/− mice. Immunohistochemical analysis of muscle markers on transverse (A–H) and sagittal (I–V) sections of e11.5 (A–N), e13.5 (O–R) and e17.5 (S–V) embryos did not show any difference between Pitx3−/− and Pitx3+/− littermates. (A–H) Absence of Pitx3 does not impair early somitic differentiation: e11.5 caudal myotomes show similar immunostaining for Pax3 (A, E), MyoD (B, F), myogenin (C–G) and MF20 (D–H). (I–V) Absence of Pitx3 does not impair expression of MyoD (I, L), myogenin (J, M) or MF20 (K, N) in e11.5 inter-costal muscle masses, or expression of Pax7 (O, Q, S, U) and MyoD (P, R, T, V) in e13.5 FL muscles (O–R) as well as in e17.5 HL muscles (S–V). 430 A. L’Honoré et al. / Developmental Biology 307 (2007) 421–433

Fig. 7. Pitx2 compensates the loss of Pitx3 in muscles of Pitx3−/− mice. Expression of Pitx2 (E–H, M–P) and Pitx3 (A–D, I–L) revealed by immunohistochemistry on sagittal sections of e15.5 Pitx3−/− (I–P) and Pitx3+/+ (A–H) embryos. In wild-type embryos (+/+), Pitx3 is detected in most nuclei of para-vertebral, inter-costal, lower back and hip muscles (A–D) whereas Pitx2 expression is relatively weak (E–H). In contrast, Pitx2 is strongly expressed in most nuclei of the same muscles of Pitx3−/− embryos (M–P) that no longer express Pitx3 (I–L). mutant animals. These data suggest that a feedback mechan- large number of these are positive for BrdU (Figs. 3M–O, Y) ism exists to maintain a level of Pitx transcription factors in and for phospho-H3 (Figs. 3P–R, Y) indicating that they are muscle cells and that this mechanism can feedback onto the in proliferation. However, Pitx2 expression is not restricted to Pitx2 gene in absence of Pitx3 resulting in complete undifferentiated proliferating cells since the myotome and compensation (Fig. 8C). forming muscles contain a large number of cells that co-stain for Pitx2 and myogenin (Figs. 2A–F, Y) and for MF20 (Figs. Discussion 2G–L). The expression of Pitx2 is strongest in early muscle cells from about e10.5 to e12.5. Thereafter, Pitx2 expression The present work identifies the Pitx transcription factors as decreases in all muscle masses to be apparently replaced by markers of skeletal muscle development (Fig. 8). All skeletal Pitx3 (Figs. 4 and 8A). In all developed muscles at e17.5, muscles of the body and of the limbs express Pitx3 and this expression of Pitx2 is very discrete with a few nuclei often expression correlates closely with the expression of muscle observed in clusters or within the same fiber (Figs. 7E–H). markers and with the onset of myogenic differentiation. Pitx3 The nature of those cells/fibers remains undefined. The co- expression is preceded by Pitx2 in myotome and in limb muscle expression of Pitx2 with Pax3 in proliferating BrdU-positive progenitors (Fig. 8A). The knockout of Pitx3 expression does cells may be suggestive of a role in proliferation of pro- not impair muscle development in any significant way and it genitors (Kioussi et al., 2002; Baek et al., 2003; Martinez- appears that Pitx2 completely compensates through enhanced Fernandez et al., 2006). If so, its replacement by Pitx3 in and prolonged expression for the loss of Pitx3 (Figs. 6, 7). differentiated post-mitotic muscle cells may be a way to maintain a Pitx-dependent myogenic program of gene Pitx2 in muscle progenitor cells expression independently of a Pitx2 proliferative activity that may not be shared by Pitx3. Should Pitx2 have such role The earliest expression of Pitx2 appears to be in in muscle progenitor proliferation, it is clear that it is not the undifferentiated muscle progenitors of the myotome and sole regulator of proliferation as its maintenance in Pitx3−/− ventro-lateral lip of the dermomyotome: these cells co-express muscles did not lead to bigger or dysmorphic muscles (data Pax3 (Figs. 3A–C) and Pax7 (Figs. 3J–L) but do not express not shown). MF20 (Fig. 2L). Progenitors of developing limb muscle A recent study of lacZ expression from a Pitx2 gene masses also co-express Pax3 and Pitx2 (Fig. 3D–F) and a insertion showed similar Pitx2 expression patterns as we A. L’Honoré et al. / Developmental Biology 307 (2007) 421–433 431

Fig. 8. Schematic representation of the expression of Pitx2 and Pitx3 in myogenic lineages. (A) The least differentiated cells that express Pitx2 are in the ventro-lateral lip (vll) of the dermomyotome (DM) and the progenitor cells that populate the limb muscle territories; these cells do not express Pitx3. The onset of myogenic differentiation in both myotome (M) and limbs is accompanied by Pitx3 expression that eventually becomes predominant as myoblasts differentiate into myocytes and myotubes. (B) Expression of cell markers during myogenic differentiation. Pitx2 is expressed in both Pax3 and Pax3+Pax7 progenitors. The myoblast precursors switch on Pitx3 expression that replaces Pitx2 almost completely in differentiated muscle cells. (C) In Pitx3−/− embryos, Pitx2 expression is not switched down and compensates for Pitx3 deficiency in all muscles. report here (Shih et al., 2007). However, there appears to be 4). These differences may be due to the stability of the lacZ significant discrepancies: for example, Shih et al. report lacZ protein and/or to the greater sensitivity of lacZ relative to expression in migrating myoblast and in late stage and adult immunoreactive Pitx2 detection. The lacZ expression may be muscles. We did not find significant Pitx2 immunoreactivity indicative of weak Pitx2 promoter activity but very low in these tissues compared to e10.5–e12.5 embryos (Figs. 2– protein accumulation: a similar situation has been proposed 432 A. L’Honoré et al. / Developmental Biology 307 (2007) 421–433 for Myf5 (Smith et al., 1994; Cornelison and Wold, 1997; preparation and sections. A.L. was supported by the Cooper et al., 1999; Beauchamp et al., 2000). Similarly, the Fondation pour la recherche médicale (FRM). V. Coulon adult Pitx3−/− muscles did not show more Pitx2 expression was supported by fellowships from INSERM and the French than wild-type animals (data not shown). Ministère des Affaires étrangères. This work was funded by grants from the Canadian Institutes of Health (CIHR). Pitx3 is expressed at the onset of muscle cell differentiation and is maintained in all skeletal muscles References In contrast to Pitx2, Pitx3 is rarely associated with phospho- Baek, S.H., Kioussi, C., Briata, P., Wang, D., Nguyen, H.D., Ohgi, K.A., Glass, H3-positive cells (Figs. 3Y, Z) and is less present in BrdU- C.K., Wynshaw-Boris, A., Rose, D.W., Rosenfeld, M.G., 2003. Regulated positive cells (Figs. 3V–Z). In myotome, Pitx3 is detected in subset of G1 growth-control genes in response to derepression by the Wnt cells that have entered myogenic differentiation as they are, for pathway. Proc. Natl. Acad. Sci. U. S. A. 100, 3245–3250. the most part, already positive for myogenin (Figs. 2M–R, 3Z), Beauchamp, J.R., Heslop, L., Yu, D.S., Tajbakhsh, S., Kelly, R.G., Wernig, A., for MF20 (Figs. 2S–X) and for MyoD (Fig. 3Z). 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