DEVELOPMENTAL DYNAMICS 237:2158–2169, 2008

PATTERNS & PHENOTYPES

Btg1 and Btg2 Expression During Early Chick Development

Andre´s Kamaid* and Fernando Gira´ ldez

Btg/Tob encode for a new family of with antiproliferative functions, which are also able to stimulate cell differentiation. Btg1 and Btg2 are the most closely related members in terms of gene sequence. We analyzed their expression patterns in avian embryos by in situ hybridization, from embryonic day 1 to 3. Btg1 was distinctively expressed in the Hensen’s node, the notochord, the cardiogenic mesoderm, the lens vesicle, and in the apical ectodermal ridge and mesenchyme of the limb buds. On the other hand, Btg2 expression domains included the neural plate border, presomitic mesoderm, trigeminal placode, and mesonephros. Both genes were commonly expressed in the myotome, epibranchial placodes, and dorsal neural tube. The results suggest that Btg1 and Btg2 are involved in multiple developmental processes. Overlapping expression of Btg1 and Btg2 may imply redundant functions, but unique expression patterns suggest also differential regulation and function. Developmental Dynamics 237:2158–2169, 2008. © 2008 Wiley-Liss, Inc.

Key words: anterior neural ridge; neural plate; neural development; ectodermal placodes; infundibulum; heart development; Hensen’s node; left-right asymmetry; extraembryonic membranes; notochord; somites; presomitic mesoderm; limb; myotome; mesonephros; rhombic lip; rhombomere

Accepted 15 May 2008

INTRODUCTION ferent model systems suggest that (“antiproliferative” boxes A and B) lo- they act as antiproliferative genes, cated in the first 120 residues of the During development, cell proliferation they can promote cell differentiation (Guehenneux et al., 1997; and cell differentiation are tightly co- and also regulate apoptosis and cellu- Guardavaccaro et al., 2000). This gene ordinated through signaling pathways lar senescence (Matsuda et al., 2001; family is conserved across phylogeny, that ultimately regulate the expres- Tirone, 2001; Duriez et al., 2004; Lim, with two members identified in inver- sion of control genes. Btg/ 2006). Btg/Tob genes encode for a tebrates: the gene Fog-3 in C. elegans Tob genes are a newly characterized group of structurally related proteins, (Chen et al., 2000) and the mRNA family of cell cycle modulators (Gue- characterized by the presence of two with accession number AF177464 in henneux et al., 1997). Studies in dif- novel and highly conserved domains D. melanogaster. Also, a structurally

ABBREVIATIONS AIP anterior intestinal portal ANR anterior neural ridge Btg B-cell translocation gene CNC cranial neural crest DA dorsal aorta DE dorsal ectoderm DRL dorsal rhombic lip EE epidermal ectoderm EEn extraembryonic endoderm EM extraembryonic mesoderm FB forebrain IM intermediate mesoderm IS isthmus ISH in situ hybridization LP lens placode M myotome MZ mantle zone N notochord NE neural ectoderm NPB neural plate border NT neural tube OFP olfactory placode OFT outflow tract OPV optic vesicle OV otic vesicle PSM presomitic mesoderm PSM presomitic mesoderm RV right heart ventricle SN spinal nerve TB tail bud Tob transducer of Erb TP trigeminal placode VE ventral ectoderm VZ ventricular zone

Developmental Biology Group, DCEXS, Universitat Pompeu Fabra, Barcelona, Spain Grant sponsor: Spanish MEC; Grant number: BMC2002-00355. *Correspondence to: Andre´s Kamaid Toth, Developmental Biology, Department of Experimental and Health Sciences, Uni- versitat Pompeu Fabra (UPF), Parc de Rec¸erca Biome`dica de Barcelona (PRBB), Dr. Aiguader 88, Barcelona 08003, Spain. E-mail: [email protected] DOI 10.1002/dvdy.21616 Published online 23 July 2008 in Wiley InterScience (www.interscience.wiley.com).

© 2008 Wiley-Liss, Inc. BTG1 AND BTG2 IN CHICK 2159 related gene has been characterized in al., 2002). Overexpression of Btg2 dur- time RT-PCR as early as stages amphioxus, the AmphiTOB gene (Hol- ing mouse embryonic development in HH3/4, using specific primers (de- land et al., 1997). In vertebrates, the vivo regulates cell cycle exit and differ- scribed in Experimental Procedures whole family is composed of at least entiation of mouse cerebellar progeni- section, data not shown). Results for six members that have been named tors (Canzoniere et al., 2004). Further- each gene are described separately, differently in different species: Btg1, more, Btg2 is expressed at the onset of emphasizing their specific domains of Btg2 (Tis21 in mouse and PC3 in rat), neurogenesis in neuroepithelial cells, expression, and then compared and Btg3 (also named ANA for Abundant concomitantly with the switch from pro- summarized at the end of the report. liferative to neurogenic divisions (Ia- in Neuroepithelium Area), Btg4 Btg2 Expression During (PC3B), Tob (Tob1), and Tob2 (Brad- copetti et al., 1994, 1999; Hammerle et bury et al., 1991; Fletcher et al., 1991; al., 2002). Knock-out mice for Btg1 are Gastrulation and Matsuda et al., 1996; Rouault et al., not yet available and Btg2Ϫ/Ϫ mice Neurulation 1996; Guehenneux et al., 1997; Yo- have minor defects in vertebral pattern- Expression of Btg2 was detected at shida et al., 1998; Ikematsu et al., ing (Park et al., 2004). Morpholino in- HH4 in the posterior epiblast and 1999). It has been proposed to name jection of Btg genes in Xenopus embryos primitive streak (Fig. 1A). Btg2 disrupts some aspects of anterior neu- the family as APRO (AntiPROlifera- showed a distinct and dynamic ex- ral development and notochord differ- tive) (Matsuda et al., 2001), but most pression pattern during neurulation entiation (Sugimoto et al., 2005, 2007; studies and genetic databases con- (Fig. 1B–K). By stage HH6, high lev- tinue using the BTG/Tob nomencla- Wessely et al., 2005). It has been sug- els of Btg2 transcripts were detected ture, which will be adopted in this re- gested that some redundant function in a restricted population of the ecto- port. may exist between different members of derm, at the neural plate border (Fig. In spite of having similar N-termi- the family (Park et al., 2004; Wessely et 1B, NPB), while low levels of expres- nal sequences and activities, BTG pro- al., 2005). sion were detected in the caudal prim- teins are smaller than Tob proteins Information about developmental itive streak (Fig. 1B, PS). Up to stage and both groups are generally consid- expression of Btg genes is still frag- HH8, Btg2 expression was concen- ered as subfamilies (Matsuda et al., mentary and limited to individual trated in this border population, all 2001; Tirone, 2001; Jia and Meng, genes in specific developmental pro- along the anteroposterior axis (Fig. 2007). The Btg2 gene was the first cesses in amphioxus, xenopus, ze- 1C, and see also sections in Fig. 1G member to be identified, as an imme- brafish, and mouse (Iacopetti et al., and H). This domain is the source of diate response gene in mouse and rat, 1994, 1999; Holland et al., 1997; ectodermic placodes and neural crest in two different cellular contexts. The Buanne et al., 2000; Chen et al., 2000; cells in the head, and neural crest mouse gene was isolated in TPA stim- Saka et al., 2000; Sakaguchi et al., ulated NIH 3T3 fibroblasts and was 2001; Thisse et al., 2001; Hammerle et cells in the trunk, and other genes are named Tis21 (for TPA-induced se- al., 2002; Canzoniere et al., 2004; expressed with a similar pattern in quence) (Fletcher et al., 1991), Park et al., 2004; Thisse and Thisse, this region, e.g., Bmp4 (Chapman et whereas the rat homologue was iso- 2004; Sugimoto et al., 2005, 2007; al., 2002), Msx-1 (Streit and Stern, lated as an immediate response gene Wessely et al., 2005; Feng et al., 1999), and Id1 (Kee and Bronner- to NGF stimulation in neural crest– 2007), and there is no detailed de- Fraser, 2001). However, Btg2 expres- derived PC12 cells (Bradbury et al., scription of the expression pattern of sion is rather unique because it is re- 1991). Shortly after, the human Btg1 any member of the family in the chick stricted to the transition zone between (B-cell-translocation gene 1) was embryo. To gain insights on the func- neural ectoderm (NE) and epidermal cloned from a chromosomal transloca- tion of these genes during develop- ectoderm (EE) (Moury and Schoen- tion observed in a lymphoid malig- ment, and since Btg1 and Btg2 are the wolf. 1995) along the entire antero- nancy (Rouault et al., 1992). Human more closely related genes in the fam- posterior axis (Fig. 1G,H). By stage BTG1 and BTG2 proteins share 66% ily, we sought to analyse and compare HH9, Btg2 expression shifted to the sequence identity, and the only signif- their expression profile during early overlying ectoderm, following the ros- icant difference between the two pro- stages of chick development. The re- tro-caudal sequence of neural tube clo- teins is a 10–amino acid insertion in sults showed a dynamic and differen- sure (see asterisks in Fig. 1D,E). In the Carboxy-terminal part of BTG1 tial expression of Btg1 and Btg2, sug- the caudal region, where the neural (Duriez et al., 2004). gesting multiple and specific functions tube was still open, Btg2 was main- Overexpression of Btg/Tob members during early development. tained at the transition zone between in different cellular models have as- the neuroepithelium and the epider- signed them antriproliferative and/or mal ectoderm (Fig. 1K). RESULTS pro-differentiative functions (reviewed Between stages HH9 and HH12, the in (Tirone, 2001; Duriez et al., 2004). The experiments that follow show the expression of Btg2 in the cephalic re- Btg1 has been shown to modulate avian expression patterns of Btg1 and Btg2 gion became concentrated in the ecto- myoblast differentiation in vitro (Mar- in the chick from gastrula stages to derm of the midline, covering the en- chal et al., 1995; Rodier et al., 1999), day 3 of development, carried out by tire anterior neural ridge (Fig. 1D–F, and Btg2 can promote differentiation whole-mount mRNA in situ hybridiza- ANR; see also sections in Fig. 1I,J). and survival of cultured-neural cells tion on chick embryos. Both Btg1 and Interestingly, some Btg2-expressing (Corrente et al., 2002; el-Ghissassi et Btg2 transcripts were detected by real cells were also detected in the infun- 2160 KAMAID AND GIRA´ LDEZ dibulum, the structure from where the trigeminal placode (Fig. 2A, see also a or absent expression of Btg2 up to neural lobe of the hypophysis develops high-magnification view in Fig. 2C). HH22 (not shown). (Fig. 1J). At these stages, the neural This expression was very transient In the epibranchial placodes, the tube was already closed in the mid- and disappeared after stage HH13. Btg2 expression domain was reminis- brain and hindbrain regions, and cra- Transient expression of Btg2 was also cent of that of Sox3 or Neurogenin1 nial neural crest cells started their mi- seen in cells of the early otic placode, (Abu-Elmagd et al., 2001) and is illus- gration, as detected with HNK-1 which was identified by co-immunola- trated by a lateral view of the HH22 antibody (Fig. 2A,A’). Sections at this beling with an anti-PAX2 antibody in embryo shown in Figure 2K. Expres- level showed that Btg2 expression was the same section (Fig. 2B,B’). sion in epibranchial placodes was first absent in the migrating neural crest Btg2 was distinctively expressed detected at HH12 and was maintained cells, but it was high in the ectoderm throughout olfactory placode develop- up to embryonic day 5 (not shown). adjacent to the cephalic neural tube ment (Fig. 2E–J). High levels of Btg2 Btg2 was dynamically expressed in (Fig. 2A,A’,C). In the caudal region of mRNA were detected by stage HH9 at the neural tube. At embryonic day 3, the trunk, Btg2 expression was main- the anterior neural ridge (Fig. 2E). when active neurogenesis takes place, tained in cells of the dorsal neural This domain corresponds to that of ol- Btg2 mRNA was highly expressed in tube, corresponding to the premigra- factory precursors as located by fate the ventricular zone of the CNS, in- tory neural crest, and in the non-neu- map analysis (Bhattacharyya et al., cluding the brain, all along the antero- ral ectoderm (Fig. 2 D). 2004). This expression was main- posterior and dorso-ventral axes (see tained upon closure of the anterior below). Btg2 expression was high in Btg2 Expression in neuropore (Fig. 2F,G), remained high the hindbrain, including the dorsal Ectodermal Placodes and in the olfactory placode at stages rhombic lip, where cerebellar precur- HH13–15 (Fig. 2H,D), and in the ol- sors are located (Wingate and Hatten, Neural Tube factory pit at stages HH19 and HH22 1999) (Fig. 2K,L, DRL), and in the As described above, Btg2 was ex- (Fig. 2J,K). Btg2 expression was not inter-rombomeric boundaries (aster- pressed in the cephalic ectoderm. By detected in the lens placode (see LP in isks in Fig. 2L). stage HH10, Btg2 expression was in- Fig. 2F,H), and histological sections Expression of Btg2 in the neuroepi- tense in the presumptive region of the through the lens confirmed the limited thelium was not detected before the

Fig. 1. Btg2 expression during gastrulation and neurulation. A–F: Dorsal views of embryos labelled by whole-mount in situ hybridization for Btg2 mRNA detection. Anterior is to the top and posterior to the bottom. G–K: Transversal sections (20 ␮m) at the approximate levels indicated in the whole-mount images. Stages are indicated at the bottom of each picture. A: Btg2 expression in the caudal area pellucida epiblast, including the primitive streak. B: Btg2 expression in the neural plate border (NPB) and low levels in the primitive streak (PS). C: High Btg2 expression in the neural folds (NF), concentrated in the border between neural and non-neural ectoderm, and low levels in the regressing primitive streak. G,H: This is also illustrated in sections at the levels indicated. Note that Btg2 expression was also present in the segmental plate mesoderm (SPM), and proamnion (PA). D–F: Btg2 expression shifted to the epidermal ectoderm as the neural tube closes following its rostro-caudal sequence (asterisks mark the caudal limits of neural tube closure). Note that Btg2 expression was concentrated in the anterior neural ridge (ANR), caudal neural folds (NF), and trigeminal placode (TP). Dark blue in the brain vesicles (F) is due to trapping and does not reflect specific signal, as illustrated by section in J. I,J: Sections through the cephalic region at the levels indicated in E and F. Dorsal is to the top, ventral to the bottom. Images show Btg2 expression in the midline ectoderm. Note that at stage HH12, Btg2-positive cells were detected in the infundibulum (IF) (J). K: High-magnification view of a transversal section at the level indicated in E, showing the restricted expression of Btg2 between neural ectoderm (NE) and epidermal ectoderm (EE). Dashed lines indicate the epithelial limits. HN, Hensen’s node; DE, dorsal ectoderm; N, notochord; OPV, optic vesicle; PSM, presomitic mesoderm; TB, tail bud; VE, ventral ectoderm.

Fig. 2. Btg2 expression in ectodermal placodes and neural tube. A–D: Transversal sections (20 ␮m) of an HH12 embryo processed for Btg2 in situ hybridization. The same sections were processed for fluorescent immunohistochemical detection with HNK-1 (green) and anti-Pax2 (red) antibodies (A’,B’). A,A’: Section at the level of the isthmus (IS), as revealed by Pax2 co-labeling in the neuroepithelium (A’). Expression of Btg2 is shown in the ectoderm where trigeminal placode (TP) develops, and in some cells of the somatopleure (SP) (A), but is absent from migrating neural crest cells (MNC) labelled by HNK1 antibody (A’). B,B’: Section at the level of the otic placode (OP) marked by Pax2 immunostaining (B’) shows the expression of Btg2 in some cells of the otic placode (B). C: High-magnification view of the section in A. D: Section through a more caudal region of the same embryo, showing expression of Btg2 in cells of the dorsal neural tube (red arrow), in the presomitic mesoderm (PSM), and in the non-neural epidermal ectoderm (EE). E–J: Embryos processed for whole-mount in situ hybridization showing expression of Btg2 during olfactory placode development. E,F: Dorsal views of the cephalic region of HH9 and HH10 embryos show expression of Btg2 in the ectoderm surrounding the closing neural tube and trigeminal placode (TP). Strong signal was concentrated in the ectoderm of the anterior neural ridge (ANR). G: High-magnification view of the ANR region indicated in F by a dotted rectangle. H: Frontal view of an HH13 embryo shows Btg2 expression in the anterior ectoderm of the olfactory placode (OFP). Dorsal is to the top, ventral to the bottom. I: Lateral view of the head of an embryo at HH15 shows strong Btg2 expression in the olfactory placode. J: Lateral view of the head region at stage HH19, at a higher magnification focusing on the olfactory pit (OFPIT). K,L: HH22 embryo processed for whole-mount in situ hybridization showing Btg2 expression in the hindbrain and epibranchial placodes. K: Lateral view of the cephalic region and anterior part of the trunk. High Btg2 expression was detected in the epibranchial placodes (VII, IX, and X) and in the dorsal rhombic lip (DRL). Note also the expression of Btg2 in the myotome (M) and olfactory pit (OFP). L: Dorsal view of the same embryo shown in K illustrating high Btg2 expression in the dorsal rhombic lip and in the inter-rhombomeric boundaries (asterisks). Anterior is to the top, posterior to the bottom. M,N: Transversal sections (20 ␮m) through the spinal cord at the indicated stages. Dorsal is to the top and ventral to the bottom. M: Section at the level of the second somite in an HH12 embryo shows Btg2 expression in few cells of the neural tube (arrowheads), in the epidermal ectoderm (EE), and in the endoderm (EN). N: Section through the trunk of an HH22 embryo double stained by ISH for Btg2 mRNA (blue) and IHC with TUJ1 antibody (red), labeling the post-mitotic cells in the mantle zone (MZ), and spinal nerve leaving the neural tube (SN). In situ hybridization signal shows high Btg2 mRNA expression exclusively in neuroepithelial progenitors of the ventricular zone (VZ) along the dorso-ventral axis. NE, neuroepithelium; N, notochord; VS, sinus venosus; FB, forebrain; H, heart; LP, lens placode; OPV, optic vesicle; OV, otic vesicle. BTG1 AND BTG2 GENE EXPRESSION IN CHICK 2161

Figure 1.

Figure 2. 2162 KAMAID AND GIRA´ LDEZ onset of neurogenesis and the number 1A,B,H). At stage HH11, Btg2 was ex- was apparent in the myotome (Fig. of neuroepithelial cells expressing pressed in the lateral plate mesoderm, 3H; see also whole-mount image in Btg2 increased in parallel with neuro- but not in the intermediate mesoderm Fig. 2K) and in the mesonephros, genesis (Hollyday and Hamburger, that is fated to become pronephros, as where it was concentrated in the de- 1977; Ericson et al., 1992). This is il- illustrated by the excluding expres- veloping mesonephric tubules (Fig. lustrated by transversal sections sion domains of Btg2 and Pax-2 3H). Btg2 was not detected in the through the spinal cord at stages 12 (Dressler et al., 1990) (Fig. 3A,B). early limb buds up to stages HH18– and 22 (Fig. 2M,N). Note that Btg2 Btg2 was also expressed in the pre- 22, but it was expressed at day 5 mRNA was not detected in the post- somitic mesoderm (Fig. 3A,C, and see (HH27) in the interdigital mesen- mitotic cells, marked by the expres- also Fig. 1F), as reported for its homo- chyme of the limb primordium (not sion of a ␤-tubulin subtype (Lee et al., logues: Xenopus, XBtg2 (Sugimoto et shown). 1990) (Fig. 2N, shown in red by co- al., 2005), zebrafish Btg-b (Sakaguchi Other sites of Btg2 distinctive ex- immunostaining with TUJ1 anti- et al., 2001), and mouse Btg2 (Park et pression include the roof of the phar- body). These results are in agreement al., 2004). In addition, Btg2 mRNA ynx and the initial stages of the respi- with previous reports showing that was also detected in the posterior re- ratory diverticulum (Fig. 3I). Btg2 (Tis21) mRNA expression in the gion of the newly formed somites at ventricular zone starts at the onset of HH11 (Fig. 3C). Btg1 Expression During neurogenesis, is confined to a sub- Btg2 was strongly expressed in the Gastrulation and Heart population of neuroepithelial cells myocardium, the outflow tract, and at that increases with the progression of lower levels in the endocardium and Formation neurogenesis, and is not detected in splanchnic mesoderm of the heart ru- During gastrulation, Btg1 was mark- newborn (Iacopetti et al., diment of the HH11 embryo (Fig. edly expressed in the Hensen’s node 1994, 1999; Hammerle et al., 2002). 3D,E). This previously unknown ex- (Fig. 4A,B). This expression was pression of Btg2 was transient, and it present in the ectodermal and meso- Btg2 Expression in was absent from the heart primor- dermal layers of the node, and from Mesoderm Derivatives and dium at earlier stages of cardiac de- HH4 to HH5 it extended to the axial velopment (see below Fig4, Btg1 ex- mesoderm into the head process (Fig. Other Tissues pression for comparison). In addition, 4B, HP). Noticeably, Btg1 expression Initial expression of Btg2 in the meso- Btg2 was also detected in extraembry- in the Hensen’s node showed an asym- derm was found at the primitive onic membranes (Fig. 3E,F), and in metrical distribution from left to right streak level and in the caudal segmen- the tail bud (Fig. 3G). (Fig. 4AЈ,BЈ,AЉ,BЉ). tal plate mesoderm (see above; Fig. By stage HH22, strong Btg2 signal At gastrula stages (HH4–5), low

Fig. 3. Btg2 expression in other tissues. A,B: Dorsal view of a whole embryo at HH11ϩ triple stained by ISH for Btg2 mRNA (A) and fluorescent immunohistochemical detection with mouse monoclonal HNK1 (green) and rabbit polyclonal anti-Pax2 (red) antibodies (B). At this stage, HNK-1 stained the cephalic migratory neural crest cells (CNC) in the head and cells of the developing heart (HP). Pax2 marked the neural tube at the isthmus level (IS), the pre-otic ectoderm (OP), and the intermediate mesoderm (IM). Note that Btg2 was excluded from the Pax2-positive intermediate mesoderm (arrows IM). C: High-magnification dorsal view of an HH11 embryo at the level of the last formed somite shows Btg2 expression in the presomitic mesoderm (PSM) and the posterior region of newly formed somites (dashed line and S). Ectodermal expression of Btg2 at this level is out of the focal plane. D,G: Transversal sections (20 ␮m) at the levels indicated in A. D: Btg2 expression in the right heart ventricle (RV), the outflow tract (OFT), and low levels in the endocardium (arrowhead). E: Section at the level of the otic placode (OP), showing expression of Btg2 also in extraembryonic mesoderm (EM) and lower levels of expression in the splachnic mesoderm (SM). F: Section at a caudal level where the neural tube is still open. Btg2 was expressed in the neural folds (NF), non-neural ectoderm (EE), presomitic mesoderm (PSM), and a restricted population of extraembryonic endoderm (EEn). G: Btg2 expression in the tail bud (TB). H,I: Transversal sections (20 ␮m) of an HH22 embryo. H: Section at a mid-caudal level showing expression in the myotome (MYOT) and mesonephros (MN). I: Section at the level of the otic vesicle (OV) shows Btg2 expression in the floor of the pharynx (PhX) (arrowhead), and the respiratory diverticulum (RD). Note that sections are not completely transversal. Anterior intestinal portal (AIP), dorsal aorta (DA), neural tube (NT), and notochord (N).

Fig. 4. Btg1 expression during gastrulation and heart formation. A–F: Embryos processed for Btg1 whole-mount ISH at the indicated stages. Anterior is to the top, posterior to the bottom. AЉ,BЉ, G–J: Transversal sections (20 ␮m) at the approximate levels indicated in the whole-mount images. A,B: Dorsal view of gastrulating embryos at HH4 and HH5 showing strong expression of Btg1 in the Hensen’s node (asterisk) and in cells of the midline towards the developing head process (HP). Note that low levels of Btg1 expression can be detected in the pre-cardiac mesoderm (CM). A’,B’: High magnification of the same embryos in A,B, showing asymmetric distribution of Btg1 transcripts in the Hensen’s node area. A؆,B؆: Transversal sections (25 ␮m) at the approximate levels indicated in A’,B’, show that Btg1 is expressed in the ectodermal and mesodermal layers of the node (HN), and at lower levels in the lateral cardiogenic mesoderm (CM). C,D: Ventral views of embryos at HH8–9 showing Btg1 expression in the heart primordium, adjacent to the anterior intestinal portal (AIP). E: Ventral view of an HH10 embryo shows strong Btg1 expression in the sinoatrial region (SA) of the developing heart. Also, lower expression was detected in scattered cells of the extraembryonic membranes (EEM). F: Ventral view of an HH11 embryo shows high Btg1 expression in the developing heart and in the notochord. G,H: Transversal section at the levels indicated in C shows expression of Btg1 in the heart primordium (HP), extraembryonic ectoderm, and in the splanchnic mesoderm (SM) surrounding the anterior intestinal portal (AIP). Arrowhead in G indicates low Btg1 expression in the dorsal aspect of the neuroepithelium of the neural folds. I,J: Transversal sections at the levels indicated in E show strong expression of Btg1 in the myocardium (MC) and in the splanchnic mesoderm (SM) surrounding the anterior intestinal portal (AIP). Low expression of Btg1 was also detected in the pre-placodal ectoderm of the otic region (PPE, J). Arrowhead in I indicates lower levels of Btg1 in the dorsal aspect of the neural tube. BTG1 AND BTG2 GENE EXPRESSION IN CHICK 2163

Figure 3.

Figure 4. 2164 KAMAID AND GIRA´ LDEZ levels of Btg1 mRNA were detected in above). Conversely, Btg1 expression derm, where Btg2 mRNA is concen- bilateral regions corresponding to the was high in the lens vesicle (Fig. trated, has been shown to play primary heart fields (Fig. 4B, CM, and 5A,B), where Btg2 was not expressed. important roles in neural fold forma- see sections in Fig. 4AЉ,BЉ). Btg1 ex- Btg1 expression in the lens was down- tion (Moury and Schoenwolf, 1995; pression persisted in the cardiogenic regulated by stage HH18 (Fig. 5F). Hackett et al., 1997). Interestingly, mesoderm throughout the first stages Also at this stage, Btg1 was broadly regulation of cell cycle and orientation of heart development (Fig. 4C–J). At expressed in the prospective epi- of cell divisions are likely to play key stage HH8, Btg1 expression was high branchial placode domain and in the roles in this process (Smith and in the splanchnic mesoderm sur- otic epithelium (Fig. 5A,C). Later on, Schoenwolf, 1987, 1988), and Btg2 has rounding the anterior intestinal por- expression of Btg1 in the ectoderm of been linked to both cellular processes tal (Fig. 4C, and sections G,H). Later, the branchial arches became re- in other model systems (Malatesta et at stages HH9 and HH12, expression stricted to the neurogenic region of the al., 2000). Moreover, current models in the developing heart was located in epibrancial placodes, and by stage propose that the establishment of the the myocardium of the sinoatrial re- HH21 it displayed a pattern that was neural plate border is regulated by in- gion and ventricle (Fig. 4E,F and sec- similar to that of Btg2 (Fig. 5F, com- teraction between BMP and Wnt sig- tions in I,J). The early expression do- pare with Fig. 2K). At this stage, low nals (Streit and Stern, 1999). Btg2 has mains of Btg1 are similar to that of levels of Btg1 were detected in the been shown to modulate Smad-medi- other cardiac expressed genes, includ- olfactory pit (Fig. 5H). ated BMP signaling (Park et al., ing cardiac troponin T (Antin et al., A remarkable feature of the Btg1 2004), and the related member x-Btgx 2002), the LEK-related factor CMF1 expression pattern was its presence in can modulate Wnt canonical pathway (Pabon-Pena et al., 2000), smooth the myotome (Fig. 5D,E, and section by modulating ␤-Catenin activity muscle ␣-actin (Colas et al., 2000), in I), a pattern that partially overlaps (Wessely et al., 2005). Therefore, it is myocardin’s DNA-binding partner, with that of Btg2. In this context, it is tempting to suggest that Btg2 could SRF (Croissant et al., 2000), and myo- worth mentioning that in vitro studies act as a modulator of the integration cardin (Warkman et al., 2008). in a quail-derived myoblast cell line of both signaling pathways in the neu- During neurulation, Btg1 was not have shown that Btg1 strongly pro- ral plate border, thereby conferring expressed in the neural plate border motes myoblast differentiation (Mar- specific properties of cell proliferation, (Fig. 4G, compare to Btg2 expression chal et al., 1995; Rodier et al., 1999; division, and survival to this cell pop- in Fig. 1G). However, very low levels Busson et al., 2005). ulation. Interestingly, different mem- of Btg1 mRNA were detected in the Finally, Btg1 was expressed in the bers of the Btg family are expressed in dorsal aspect of the neural tube (Fig. developing limb bud, including the similar domains in other animal spe- 4G,H). This weak expression in the apical ectodermal ridge (Fig. 5E). cies, as zebrafish zBtg2 (Sakaguchi et dorsal neural tube slightly increased Transversal sections through the me- al., 2001), Xenopus XBtg2 (Sugimoto during the stages studied (see below), dial region of the limb bud at stage et al., 2007), and XBtg-x (Wessely et and reached maximum levels at em- HH21 showed limited expression in al., 2005), suggesting an evolutionary bryonic day 5 (not shown). Btg1 was the mesoderm, but positive cells in the conserved function of Btg genes dur- also expressed in the extraembryonic ventral ectoderm and apical ectoder- ing neurulation. Furthermore, Wes- ectoderm (Fig. 4H), at low levels in the mal ridge (Fig. 5G). Btg1 expression sely et al. (2005) have shown that loss epidermal ectoderm of the somato- was observed in mesenchymal cells of of function of XBtg-x in Xenopus pro- pleure (Fig. 4H,I), and in the prospec- the limb bud only in caudal sections duced alterations in the spatial distri- tive otic placode (Fig. 4J). (Fig. 6I). bution of neural crest markers, sug- In contrast to Btg2, Btg1 expression gesting a defect in convergent- was observed in the very early stages extension movements. Finally, also in DISCUSSION of notochord formation at HH4–5 Xenopus, a recent report describing (Fig. 4A,B). It decreased in stages This report is an account of the ex- the loss of function of XBtg2 suggests HH6–9 (Fig. 4C,D), and was clearly pression patterns of Btg2 and Btg1 in that this gene is required for differen- detected again at HH11 (Fig. 4F). the early chick embryo. The results tiation but not for cell fate specifica- Btg1 was still expressed in the noto- show that these two genes display tion or patterning of the anterior neu- chord at HH14, but it was down-regu- restricted and dynamic expression ral plate (Sugimoto et al., 2007). lated by HH21 (see below; Fig. 5). patterns throughout development, re- Experiments in vitro and in vivo us- vealing a wide number of developmen- ing different animal species have Btg1 Expression in Placodes, tal processes in which they may be shown that Btg2 is expressed in dif- involved. ferentiating neural precursors that Myotome, and Limb Bud The formation of the neural folds is switch from proliferative to - The pattern of expression of Btg1 in a complex process underlying neuru- generating division (Iacopetti et al., the placodes showed some differences lation (Smith and Schoenwolf, 1997). 1994, 1999), and that Btg2 overex- from that of Btg2. Btg1 was neither Btg2, but not Btg1, is expressed in the pression results in negative regula- expressed in the trigeminal placode neural plate border, including the re- tion of the cell cycle and induction of nor in the anterior neural ridge and gion that originates neural crest and neuronal differentiation (Malatesta et olfactory placode (Fig. 4D,E), both placodal cells. This transition zone be- al., 2000; Corrente et al., 2002; el- sites of high Btg2 expression (see tween the neural and epidermal ecto- Ghissassi et al., 2002; Canzoniere et BTG1 AND BTG2 GENE EXPRESSION IN CHICK 2165

Fig. 5. Btg1 expression in ectodermal placodes myotome and limb bud. A: Ventro-lateral view of an HH14 embryo shows Btg1 pattern in the ectoderm of the branchial arches, otic vesicle (OV), and lens vesicle (LV). B: Transversal section (20 ␮m) at the level indicated in A shows expression of Btg1 in the lens vesicle (LV). C: Transversal section (20 ␮m) at the level indicated in A, shows Btg1 expression in the notochord (N), otic vesicle (OV), and epibranchial placodes (EPP). D: Transversal section of the trunk of the embryo shown in A shows high Btg1 expression in the myotome (MYOT), notochord (N), and lower levels of expression in the dorsal neural tube (NT) and gut endoderm (EN). E: HH21 embryo labelled by whole-mount in situ hybridization with Btg1 antisense probe. Sections revealed that the intense dark blue in the head region is due to trapping. Btg1 expression can be observed in somites and limb buds, including the apical ectodermal ridge (AER). F: Lateral view of the area indicated in E shows Btg1 expression in the epibranchial placodes that originate the VII, IX, and X cranial ganglia. G–I: Transversal sections (20 ␮m) at the levels indicated in E illustrating sites of Btg1 expression. G: Apical ectodermal ridge of the forelimb (AER). H: Low level expression in the olfactory pit (OFP). I: Myotome (Myo), limb mesenchyme (M), and ventral ectoderm of the limb bud (ECT). Note that the notochord is devoid of Btg1 signal. al., 2004). Our results about Btg2 ex- activity, stimulating differentiation of residing in those boundaries has dis- pression in the neural tube are consis- cerebellar progenitors located in the tinct properties and exhibit reduced tent with those reported in mice. In- hindbrain rhombic lip (Canzoniere et rates of cell proliferation (Guthrie et terestingly, in vivo overexpression of al., 2004). Our results in the chick al., 1991). The fact that Btg2 protein Btg2 in mouse neuroepithelial cells af- show that Btg2 is also highly ex- has antiploriferative functions sug- fected cerebellar development by a pressed in the rhombic lip area, sug- gests that it may be one of the mole- mechanism that involves a double ac- gesting again a conserved function. cules controlling the rate of cell prolif- tion of Btg2: (1) down-regulation of Additionally, Btg2 was expressed in eration in these cells. promoting cell cycle exit, the inter-rhombomeric boundaries. The expression of Btg2 and Btg1 in and (2) stimulation of Math1 promoter The population of neuroepithelial cells the neurogenic placodes was not doc- 2166 KAMAID AND GIRA´ LDEZ umented before. Our results show a It is interesting to note that the role of such as MyoD, all-trans retinoic acid distinct expression of Btg2, but not Btg/Tob proteins in muscle develop- receptors, and the T3 receptor TR␣1 Btg1, in the trigeminal and olfactory ment seems to be evolutionary con- (Busson et al., 2005). placodes from early stages of their served, as suggested by experiments In addition, both Btg1 and Btg2 pro- development. Interestingly, another in Xenopus (Saka et al., 2000), and by teins have been shown to interact with member of the family, BTG4 (PC3B), the expression pattern of members of and stimulate the activity of protein ar- is distinctly expressed in the develop- the family in zebrafish (Sakaguchi et ginine methyltransferase-1 (PRMT1), ing olfactory epithelium in the rat al., 2001; Thisse et al., 2001), mouse the major arginine methyltransferase (Buanne et al., 2000). This could re- (Park et al., 2004), and anphioxus in mammals (Lin et al., 1996; Berthet et flect the fact that similar functions (Holland et al., 1997). In particular, al., 2002). Protein arginine methylation can be carried out by different mem- the expression of Btg1 and Btg2 in the is an emergent posttranslational modi- bers of the same gene family, depend- heart primordia suggests a novel role fication involved in a growing number ing on the animal species. This may be for Btg genes in cardiomyogenesis, of cellular processes, including tran- also the case for the expression of Btg and it is noteworthy that Btg1 expres- scriptional regulation, cell signaling, genes in the notochord. In Xenopus, sion precedes that of Btg2, which, in DNA repair, and RNA processing (Rob- Btg2 is expressed in the notochord turn, is very restricted and transiently in-Lespinasse et al., 2007). Further- and translational blockade of this expressed. more, Btg1 and Btg2 can also interact, gene results in alterations of the noto- Another interesting finding is the with the mammalian CCR4-NOT com- chord development (Sugimoto et al., mutually exclusive expression of Btg2 plex, through its subunit CAF1 2005). However, we did not detect and Pax-2 in the intermediate meso- (Rouault et al., 1998). CCR4-NOT is a Btg2 expression in the chick noto- derm that originates the pronephros multi-subunit complex involved in gen- chord. Instead, we found a dynamic (Fig. 3A,B). Induction and patterning eral transcription regulation and repre- expression of Btg1 during notochord of this region has been successfully sents the major cytoplasmic mRNA development. The same occurs in ze- approached using the chick as a model deadenylase in various species brafish, where Btg1 is highly ex- system (James and Schultheiss, (Daugeron et al., 2001; Tucker et al., pressed in the notochord (Thisse et al., 2003), and this kind of study offers a 2001; Viswanathan et al., 2004; Bi- 2001) but Btg2 is not expressed (Sak- possible avenue for future research in anchin et al., 2005). These interactions aguchi et al., 2001). order to elucidate the interactions be- suggest that Btg proteins may contrib- Studies on the chick embryo have tween Pax-2 and Btg2 in the interme- ute to the crosstalk between processes greatly contributed to identify some of diate mesoderm. In this regard, it is such as transcription and mRNA me- the key mechanisms and molecules in- worth mentioning that Btg2 and tabolism (Robin-Lespinasse et al., volved in vertebrate left-right asym- Pax-2 are concomitantly expressed in 2007). In agreement with this, it has metry (reviewed in Mercola and cells of the otic placode (Fig. 2B), sug- been recently shown that Btg2 act as a Levin. 2001; Stern, 2005; Levin and gesting that their interaction depends general activator of mRNA decay Palmer, 2007). The asymmetric ex- on tissue-specific properties. (Mauxion et al., 2008). Thus, it has been pression of Btg1 in the chick Hensen’s Little is known about the molecular proposed that general activation of node has not been reported before in mechanism of action of Btg genes in deadenylation may constitute a com- other species, and resembles that of vivo, and most of the information mon function for BTG family members, other genes involved in establishing comes from studies done in isolated which could allow rapid resetting of pro- the polarity of the node (Mercola and cells in culture. It has been suggested tein production, representing a new Levin, 2001). Thus, the chick embryo that Btg proteins exert cellular func- mode of global regulation of gene ex- may provide a feasible model system tions by interacting with transcrip- pression during development (Mauxion to investigate the function of Btg1 in tional coactivators or corepressors, et al., 2008). left-right patterning. hence modulating their transcrip- Regulation of Btg genes is also com- The role of Btg1 gene in myoblast tional activities (Matsuda et al., 2001; plex. It is known that BTG2 is induced differentiation is well documented by Tirone, 2001; Duriez et al., 2004; Lim, by NGF, FGF, IL-6, TPA, serum, EGF, in vitro studies, and seems to rely on 2006). For example, it has been found and cAMP (Bradbury et al., 1991; its ability to interact with and stimu- that Btg2 impairs G1-S cell cycle- Fletcher et al., 1991; Montagnoli et late the activity of several myogenic phase progression by inhibiting cyclin al., 1996) indicating that a number of factors, including the bHLH tran- D1 transcription (Guardavaccaro et stimuli triggering different transduc- scription factor MyoD (Marchal et al., al., 2000), that it can associate with tion pathways can activate the tran- 1995; Rodier et al., 1999; Busson et HoxB9 (Prevot et al., 2000), and BMP- scription of this gene. Also Btg1 has al., 2005). Our results reveal a high regulated Smad1 and 8 (Park et al., been shown to be a direct target of expression of Btg1 in the myotome, 2004) to promote their transcriptional different molecules, including T3 further suggesting its involvement in activity, and that it can enhance hormone (Rodier et al., 1999), Insu- the regulation of myoblast differenti- Math1 promoter activity (Canzoniere line-like Growth Factor 1 (IGF-1) ation in vivo. Besides, we show that et al., 2004). Similarly, Btg1 has been (Vadgama et al., 2006), or FoxO3A both Btg1 and Btg2 are concomitantly shown to interact with and stimulate (Bakker et al., 2004). These data, to- expressed at high levels in the early the activity of transcription factors gether with the wide distribution and myotome, maybe reflecting some re- that positively regulate myogenic pro- cell types where these genes are ex- dundant function during myogenesis. cesses, in particular myogenic factors pressed during development, suggest BTG1 AND BTG2 GENE EXPRESSION IN CHICK 2167 that their regulation is likely to be used were 1:2 dilution of mouse mono- REFERENCES dependent on cell type and cellular clonal antibody anti CD57, clone context. Given this, and the multiple HNK-1 (B-D), and 1:200 dilution of Abu-Elmagd M, Ishii Y, Cheung M, Rex M, Le Rouedec D, Scotting PJ. 2001. cSox3 and reiterative expression domains of rabbit polyclonal anti-Pax2 (Zymed). expression and neurogenesis in the epi- Btg genes throughout early develop- Embryos were incubated for 48 hr at branchial placodes. Dev Biol 237:258– ment, their in vivo study during em- 4°C in a solution containing the anti- 269. bryogenesis needs to be done at spe- bodies in PBS-T (0.1% Tween) plus Antin PB, Bales MA, Zhang W, Garriock RJ, Yatskievych TA, Bates MA. 2002. cific locations and time points. The use 10% Goat serum. After that, 8 washes Precocious expression of cardiac tropo- of chick as a model organism has with PBS-T were done, and incubated nin T in early chick embryos is indepen- proven to be useful for such studies, for another 48 hr at 4°C with a 1:400 dent of bone morphogenetic protein sig- since it allows transplantation, gain dilution of Alexa-conjugated second- naling. 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