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Keywords:

  • tbx20;
  • T-box genes;
  • heart development;
  • chick embryo

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The T-box gene family encodes a set of transcription factors that are involved in various developmental processes. We isolated tbx20 gene from chick embryos and examined in detail its expression patterns during heart development. In situ hybridization showed that tbx20 was expressed in the lateral plate mesoderm and subsequently in the primitive heart tube. At stages of looped heart, tbx20 was localized in the outflow tract (OT) and atrioventricular (AV) canal, in which valvuloseptal endocardial cushion develops. At later stages, although tbx20 was expressed predominantly in the nascent right ventricle, transcripts of tbx20 were down-regulated in the left ventricle. These results suggest that tbx20 may play important roles in a variety of developmental processes in cardiogenesis, such as chamber-specification and septation. Developmental Dynamics 230:576–580, 2004. © 2004 Wiley-Liss, Inc.

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

T-box genes, which are transcription factors containing a DNA-binding domain of approximately 200 amino acids (T-box), make up five distinct subfamilies (Tbx1, Tbx2, Tbx6, Tbr1, and T), and they have been isolated in both vertebrates and invertebrates (Papaioannou, 2001). Tbx genes are expressed in distinct spatiotemporal patterns during mouse and chick organogenesis, suggesting that these genes are involved in many developmental processes (Papaioannou, 2001). During chick limb development, Tbx4 is thought to define the identity of the hindlimb, and Tbx5 that of the forelimb (Rodriguez-Esteban et al., 1999; Takeuchi et al., 1999). tbx6-null mice show irregular somite formation in the neck region, while the more posterior paraxial mesoderm does not form somites, but instead differentiates to form a neural tube-like structure (Chapman and Papaioannou, 1998). Genetic analyses have revealed that mutations of TBX genes in several human congenital syndromes: a TBX3 mutation is found in Ulnar–Mammary syndrome (Bamshad et al., 1997), mutations of TBX5 are found in Holt–Oram syndrome (Basson et al., 1997; Li et al., 1997), and TBX1 mutation in del22q11.2 syndrome (Yagi et al., 2003).

In recent years, several T-box genes have been found to be expressed in the developing heart: tbx2, -3, -5, -20 in that of the chick, and tbx1, -4, -5, -12, -18, -20 in that of the mouse (Gibson-Brown et al., 1998; Carson et al., 2000; Yamada et al., 2000; Iio et al., 2001; Kraus et al., 2001a, b; Papaioannou, 2001). Recent experiments showed that tight regulatory interaction between tbx20 and -5 is elucidated to establish chamber-specific gene expression as well as chamber morphology during late heart development (Szeto et al., 2002; Takeuchi et al., 2003). However, little is known about the detailed expression patterns of tbx20 during chick cardiogenesis (Iio et al., 2001; Stennard et al., 2003; Takeuchi et al., 2003). In the present study, we cloned tbx20 gene from chick embryo, examined its spatiotemporal expression patterns with special reference to cardiogenesis, and compared them with those of other Tbx genes expressed in the heart.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We performed detailed observations of spatiotemporal expression patterns of tbx20 during chick cardiogenesis from stage 5 to stage 33 by using in situ hybridization. At stage 5, tbx20 transcripts were seen in the lateral plate mesoderm, including the precardiac region (Fig. 1A,B). An intense signal for tbx20 transcripts was observed in the precardiac mesoderm at stage 6 (data not shown). At stage 8, tbx20 was expressed in the premyocardium of the splanchnic mesoderm (Figs. 1C,D, 2). No tbx20 transcripts were seen in the endoderm, ectoderm, or somatic mesoderm. At stage 10, the expression of tbx20 gene became restricted to the heart region, and it was present throughout the entire myocardium of the primitive heart tube (Figs. 1E,F, 2). After stage 12, the heart tube forms a curved S-shaped structure and consists of five presumptive segments: the conus segment (outflow tract, OT), primitive right ventricle, left ventricle, atrioventricular (AV) canal, and sinoatrial region (de la Cruz et al., 1989). At this stage, the expression of tbx20 was regionalized in the OT, AV canal, and sinoatrial regions (Fig. 1G). At stage 14, tbx20 transcripts were more restricted in the AV and OT regions (Figs. 1H,I, 2). In the AV region of the stage 14 heart, tbx20 transcripts were detected in the myocardium and endocardium (arrowhead in Fig. 1J). At stage 16–17, endothelial cells in the OT and AV regions begin to transform to mesenchymal phenotype and invade into the cardiac jelly (a process known as epithelial–mesenchymal transformation), resulting in the formation of endocardial cushion tissue, the primordia of the valves and septa (Yamagishi et al., 1999; Nakajima et al., 2000). At stage 18, tbx20 was expressed in the endocardium, mesenchyme, and myocardium in the OT and AV regions (Fig. 1K–M). The expression of tbx20 was also seen in the developing epicardium surrounding the AV groove (not shown). At stage 23, an intense signal for tbx20 transcripts was observed in the OT and AV regions (Figs. 1N–P, 2). In the ventricular region of the stage 23 heart, tbx20 was expressed predominantly in the proximal OT (bulbus cordis, future right ventricle), but transcripts of tbx20 appeared to be down-regulated in the primitive ventricle (future left ventricle; Fig. 1N). Epicardial cells surrounding the AV groove expressed tbx20 gene (Fig. 1P–R). In the distal OT region of the stage 27 heart, tbx20 transcripts were seen in the myocardium, endothelial-derived cushion mesenchyme, and neural crest-derived mesenchymal condensations (Fig. 1S,T). Despite the expression of tbx20 in neural crest cell derivatives (such as the mesenchymal condensations of the OT, paraaortic sympathetic ganglia, and adrenal medulla; Table 1), tbx20 transcripts were not found in migrating neural crest cells (data not shown). Thus, the surrounding tissues to which neural crest cells migrate may regulate the expression of tbx20 in such neural crest derivatives. At stage 30, although an intense signal for tbx20 transcripts was observed in the right ventricle (especially in the proximal OT, including the conus cushion tissue), tbx20 expression was apparently down-regulated in the left ventricle (Figs. 1U,V, 2). At stage 33 (early fetal stage), an intense signal for tbx20 was observed in the valves and septa of the OT and AV regions (Figs. 1W,X, 2). At this stage, the expression of tbx20 in the ventricular myocardium appeared to be down-regulated (Fig. 1W). In addition to the heart, tbx20 was expressed both in the developing nervous system and in visceral mesothelia (Table 1).

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Figure 1. Expression of tbx20 during heart development. A:tbx20 was expressed in lateral plate mesoderm at stage (st) 5. B: Cross-section approximately at the level indicated by the line in A. tbx20 transcripts were detected in the anterior lateral plate mesoderm (meso) but not in the ectoderm (ecto) or endoderm (endo). C: Whole-mount in situ hybridization of stage 8 embryo showed an intense expression of tbx20 in the lateral plate mesoderm. D: Cross-section through the precardiac region indicated by the line in C. tbx20 was localized in the splanchnic mesoderm (sm) but not in the somatic mesoderm (arrow). E: Whole-mount in situ hybridization of stage 10 embryo. tbx20 transcripts were observed in the primitive heart tube. F: Frontal section of the stage 10 heart. tbx20 transcripts were detected in the myocardium (my) but not in the endocardium (ec). G: Frontal section of the stage 12 heart. The signal for the tbx20 gene was seen in the myocardium (my) of the OT (arrow) and sinoatrial region (arrowhead). H: Whole-mount in situ hybridization of stage 14 embryo. tbx20 was observed in the OT, AV, and atrium. I: Sagittal section of stage 14 heart. tbx20 was expressed in the OT, AV canal, and atrium. J: High magnification of boxed area in I. tbx20 transcripts were detected in the myocardium (my) and endocardium (ec, arrowhead). K: Frontal section of stage 18 heart showing tbx20 in the OT, AV, and atrium. L,M: High magnification of the OT and AV regions shown in K (boxes). tbx20 signal was seen in the myocardium (my), endocardium (ec), and mesenchymal cells (mes). N: Frontal section of the stage 23 heart. tbx20 transcripts were detected in the OT, AV canal, and atrium (a). A weak signal was seen in the primitive ventricle (v). O,P: High magnification of the boxed regions shown in N. An intense signal for tbx20 was seen in the proximal OT and AV myocardium (my), endocardium (ec), and mesenchymal cells (mes). Epicardium (epc) expressed tbx20 in the AV region. Q: Horizontal section of the stage 27 AV region. tbx20 was expressed in the right ventricle (rv), atria (ra and la) and cushion tissue (ct). R: High magnification of boxed area in Q. tbx20 was expressed in the epicardium (epc), myocardium (my), and cushion mesenchyme (mes). S: Horizontal section of the stage 27 distal OT region. tbx20 transcripts were detected in the OT and atria (ra and la). T: High magnification of boxed area in S. tbx20 transcripts were seen in the myocardium (my) and mesenchymal condensation (mc; arrowheads). U: Horizontal section of the stage 30 heart. tbx20 transcripts were detected in the right ventricle (rv) and conus cushion tissue (ct). A weak signal for tbx20 was seen in the left ventricle (lv). V: High magnification of boxed area in U. tbx20 was expressed in the endocardium (ec) and mesenchymal cells (mes). W: Frontal section of the stage 33 heart. tbx20 was localized in the AV canal, including AV septal cushion tissue (ct) and valve mesenchyme. X: High magnification of the boxed area in W. tbx20 was expressed in the endocardium (ec) and mesenchymal cells (mes). Myocardial expression of tbx20 signal was down-regulated.

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Figure 2. Expression patterns of Tbx genes during chick cardiogenesis.

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Table 1. Localization of tbx20 Transcripts During Chick Embryogenesis (Stage 5–33)
Tissuetbx20 (stage at which signal was detected)
  • *

    basal plate of neural tube;

  • **

    paraaortic sympathetic ganglion and adrenal grand;

  • ***

    visceral peritoneum;

  • ****

    visceral pericardium. st, stage.

Neural tissue 
 Central nervous system+ (st14–33)*
 Peripheral nervous system+ (st18–33)**
Optic vesicle
Otic vesicle
Body wall
Notochord
Somite 
 Myotome
 Dermatome
 Sclerotome
Urogenital mesoderm 
 Nephrotome
Mesothelia 
 Pleura
 Peritoneum+ (st18–23)***
 Pericardium+ (st18–33)****
Heart 
 Myocardium+ (st10–33)
 Cushion mesenchyme+ (st16–33)
 Endocardium+ (st14–33)
 Epicardium+ (st18–33)****
Great vessels
Liver 
 Endothelial cells+ (st18–23)
 Hepatic cells
Lung 
 Epithelium
 Mesenchyme
Other digestive organs
Pharyngeal region 
 Pouch epithelium
 Arch mesenchyme
 Pharynx epithelium
Allantois 
 Mesodermal lining+ (st18–23)
 Endodermal lining
Limb

During chick cardiogenesis, at least four Tbx genes (tbx2, -3, -5, and -20) are expressed (Bruneau et al., 2000; Liberatore et al., 2000; Yamada et al., 2000; this study and our unpublished observations); therefore, it is important to compare the expression patterns among these genes. As shown in Figure 2, before the formation of primitive heart tube (stage 8 in Fig. 2), these four genes are expressed homogeneously in the precardiac region. At later stages, each Tbx gene is expressed in a manner with a distinct spatiotemporal pattern. Furthermore, each heart segment appears to have regional specific transcripts of Tbx gene(s). During zebrafish development, although hrT (an ortholog of tbx20) is expressed in the precardiac region, inhibition of hrT function does not affect early heart development (Szeto et al., 2002). As shown in Figure 2, several Tbx genes are expressed in the precardiac region during chick cardiogenesis. Therefore, functional redundancy between the Tbx genes expressed in the early zebrafish heart may compensate for the reduced hrT function in the hrT-knockdown embryos. Recent tbx5 misexpression experiment showed that Tbx5 specifies the identity of left ventricle through interactions among heart-specific factors (Takeuchi et al., 2003). Accordingly, it is suggested that other Tbx gene(s), including tbx20, may contribute to the specification of the heart segment during cardiogenesis. In the present study, we showed detailed observations of spatiotemporal expression patterns of tbx20 during cardiogenesis and compared them with those of other Tbx genes expressed in the heart. Taking our results into account with those of others may suggest that Tbx20 may play important roles in a variety of developmental processes in cardiogenesis, such as chamber specification and septation.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Fertilized chicken eggs (Gallus domesticus) were incubated at 37.8°C until embryos reached appropriate stages within the range from 24 hr to 8 days. Embryos were collected in ice-cooled phosphate-buffered saline (PBS), then staged according to Hamburger and Hamilton (1951). Staged embryos were subjected to cloning of Tbx genes and to in situ hybridization for examination of the tissue distribution of their mRNAs, as described below.

Two sets of degenerate polymerase chain reaction (PCR) primers were designed against conserved regions within the T-box. First-strand cDNA synthesized using total RNA from 4-day chick embryos was used as the template to amplify the T-box regions. PCR products were subcloned into pCRscript (Stratagene, La Jolla, CA) and sequenced. These cDNAs corresponded to Tbx2 and Tbx5. The cloned T-box region of Tbx2 was then used to screen the cDNA library of chick whole embryo (stage 22). A total of 95 positive clones were isolated, and these were classified into four cDNAs encoding Tbx2, -3, -5, and -20.

Digoxigenin (DIG) -labeled single-strand RNA probes were prepared by using a DIG RNA labeling kit (Roche Diagnostics, Tokyo, Japan) according to the manufacturer's instructions. To produce an antisense probes, tbx20 (2.1 kb) subcloned into pGem7Zf(+) was linearized by using EcoRI and transcribed by using SP6 RNA polymerase. RNA probes for tbx20 were hydrolyzed to an average size of 0.6 kb. Embryos at appropriate stages were fixed in 4% paraformaldehyde in PBS, then embedded in paraffin. Sections were deparaffinized, hydrated, digested with proteinase K (3 μg/ml), refixed with 4% paraformaldehyde, and then acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). The sections were dehydrated, air-dried, and hybridized at 60°C. After the hybridization step, sections were rinsed with 5× standard saline citrate (SSC) followed by 50% formamide at 60°C, then 2× SSC at 60°C, and 0.2× SSC at 60°C (twice). Hybridization was detected by using alkaline phosphatase-conjugated anti-DIG antibody and nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). Whole-mount in situ hybridization was performed essentially as described by Nieto et al. (1996).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

T.Y. was funded by a Grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES