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Keywords:

  • avian embryo;
  • Tbx1;
  • retinoic acid;
  • pharyngeal development

Abstract

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

Both Tbx1 and retinoic acid (RA) are key players in embryonic pharyngeal development; loss of Tbx1 produces DiGeorge syndrome-like phenotypes in mouse models as does disruption of retinoic acid homeostasis. We have demonstrated that perturbation of retinoic acid levels in the avian embryo produces altered Tbx1 expression. In vitamin A-deficient quails, which lack endogenous retinoic acid, Tbx1 expression patterns were disrupted early in development and expression was subsequently lost in all tissues. “Gain-of-function” experiments where RA-soaked beads were grafted into the pharyngeal region produced localized down-regulation of Tbx1 expression. In these embryos, analysis of Shh and Foxa2, upstream control factors for Tbx1, suggested that the effect of RA was independent of this regulatory pathway. Real-time polymerase chain reaction analysis of retinoic acid-treated P19 cells showed a dose-dependent repression of Tbx1 by retinoic acid. Repression of Tbx1 transcript levels was first evident after 8–12 hr in culture in the presence of retinoic acid, and to achieve the highest levels of repression, de novo protein synthesis was required. Developmental Dynamics 232:928–938, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

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

DiGeorge/velocardiofacial syndrome (DGS/VCFS) is characterized by a variable phenotype, which includes abnormalities of the aortic arch and outflow tract, thymic and parathyroid aplasia/hypoplasia, craniofacial defects, and learning and behavioral anomalies. The DGS/VCFS phenotypic spectrum is attributed to the abnormal development of the embryonic pharyngeal arches and pouches, as the tissues affected all arise from this region. The arches have an internal covering of endoderm, forming the pharyngeal pouches and externally are covered in ectoderm. Each arch is composed of neural crest-derived mesenchyme, which migrates in from the neuroectoderm. This surrounds the mesodermal core of cells around the aortic arch arteries. Each of these cell types gives rise to different derivatives of the head and neck, and the aortic arch arteries undergo extensive remodeling to form the aortic arch and other vessels of the head and neck.

The majority of DGS/VCFS patients are hemizygous for a large (typically 3 Mb) region of chromosome 22q11, resulting in haploinsufficiency for the genes contained within the deleted region, including the T-box transcription factor TBX1. Several groups have created mouse models for DGS/VCFS, which implicate Tbx1 in the etiology of DGS/VCFS. Large hemizygous deletions of the region of mouse chromosome 16 syntenic to human 22q11 were produced, similar to human patient deletions. At embryonic day (E) 18.5, these animals displayed characteristic DGS/VCFS-like aortic arch defects, resulting from aplasia or hypoplasia of the fourth aortic arch artery earlier in development. Rescue of the phenotype was accomplished by genetic complementation with PACs or human BACs containing Tbx1 (Lindsay et al., 1999, 2001; Merscher et al., 2001). Null mutations of Tbx1 in the heterozygous state recapitulated the aortic arch anomalies seen in animals with large hemizygous chromosomal deletions. Homozygous null animals at E18.5 displayed defects including the majority of common DGS/VCFS features arising from the earlier loss of caudal pharyngeal structures (Jerome and Papaioannou, 2001; Lindsay et al., 2001; Merscher et al., 2001). Recently, the gene for the van gogh zebrafish mutant, which also has DGS-like defects, has been cloned and identified as the zebrafish homologue of Tbx1 (Kochilas et al., 2003; Piotrowski et al., 2003).

Tbx1 is expressed in the endoderm of the pharyngeal pouches, in the mesodermal core of the pharyngeal arches surrounding the aortic arch arteries, and in the outflow tract. Although neural crest migration is disrupted in Tbx1 −/− embryos, this finding is believed to be secondary to the absence of the pharyngeal pouches and the signals normally emanating from these structures (Kochilas et al., 2002; Vitelli et al., 2002a).

As a result of the mouse model data, Tbx1 currently represents the best candidate gene for DGS/VCFS. However, approximately 10% of DGS/VCFS cases have no detectable deletion of chromosome 22q11 or 10p13 (Gong et al., 2001; Conti et al., 2003). Screens for point mutations of TBX1 in such cases have been largely negative, but three Japanese cases have been described (Yagi et al., 2003). Thus, the mouse chromosome deletion gives Tbx1 haploinsufficiency in the context of hemizygosity of 21 genes and is the model most representative of disease in most patients.

We were interested in investigating the upstream regulation of Tbx1 during development. Shh already has been demonstrated to induce Tbx1 expression (Garg et al., 2001; Yamagishi et al., 2003), but there remain several other potential candidates that may regulate Tbx1. One of these is retinoic acid (RA), well known as a signaling molecule required for normal development.

Disruption of retinoid homeostasis by means of maternal diet or genetic/chemical manipulation can result in embryonic defects similar to those seen in DGS. Human fetuses exposed to retinoids during gestation can phenocopy DGS (Happle et al., 1984; Lammer et al., 1985; Rosa et al., 1986). Exogenous retinoic acid applied to other vertebrate species, including monkeys and rodents results in similar anomalies (Kalter, 1960; Kalter and Warkany, 1961; Kochhar and Johnson, 1965; Shenefelt, 1972; Fantel et al., 1977; Kistler, 1981; Mulder et al., 1998, 2000). Lack of retinoic acid is equally disruptive to the developing embryo; null mutations of the Raldh2 enzyme which generates the majority of embryonic RA, lead to mid-gestational lethality due to severe heart defects (Niederreither et al., 1999, 2000, 2001). Partial rescue by RA supplementation or a targeted mutation to produce a hypomorphic allele produce a closer phenocopy of DGS, with all the characteristic defects attributable to the loss of posterior pharyngeal structures, in particular the endodermal pouches (Niederreither et al., 2003; Vermot et al., 2003). Non-mammalian vertebrate species where production of RA has been suppressed also display a similar phenotype due to loss of posterior branchial structures (Schuh et al., 1993; Begemann et al., 2001; Perz-Edwards et al., 2001; Grandel et al., 2002; Quinlan et al., 2002).

Experimental manipulation of retinoid receptor signaling also produces phenocopies of DGS; compound inactivation of RA receptors RARα and RARβ (Lohnes et al., 1994; Mendelsohn et al., 1994; Ghyselinck et al., 1998; Dupe et al., 1999), retinoid X receptor RXRα and RARs α, β, and γ (Kastner et al., 1997), and chemical alteration with a either a pan-RAR antagonist (Wendling et al., 2000) or an RARβ agonist (Matt et al., 2003) all display phenotypes where development of the pharyngeal region is disrupted.

In this study, we have investigated the possibility that disruption of RA homeostasis can alter the expression of Tbx1, therefore, potentially disrupting caudal pharyngeal development. We present evidence that excess or reduced RA can alter Tbx1 expression in vivo in the avian embryo. In vitro studies in postnatal day (P) 19 cells to quantify the effect of RA upon the level of Tbx1 expression using real-time quantitative-PCR (RTQ-PCR) are also presented.

RESULTS

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

Vitamin A-Deficient Embryos Have Altered cTbx1 Expression

To investigate the effect of retinoic acid deficiency upon cTbx1 expression, fertilized vitamin A-deficient (VAD) quail eggs were incubated to stages 10–14 and in situ hybridization for cTbx1 performed. Expression of cTbx1 was still present at stages 10–12 but clearly differed from expression in normal controls. Notably, at both stages, we observed posterior extension of cTbx1 in the pharyngeal endoderm. In stage 10 embryos, expression was seen bilaterally in the pharyngeal and splanchnic endoderm at the level of the hindbrain from rhombomere 1 terminating subjacent to rhombomere 7, just above the normal position of somite 1 (Fig. 1a,h). Expression at lower levels was also seen in the head mesenchyme and in somatic and splanchnic mesoderm in a small number of cells (Fig. 1a,h). In VAD quails at the same stage, expression was attenuated, with the endodermal expression diminishing significantly in width and extending much farther caudally past the end of the hindbrain. Endodermal expression was apparent adjacent to the spinal cord down to the level of somite 4/5(Fig. 1b). Given the slight anterior shift of somites in VAD embryos, this position is equivalent to the level of normal somite 3, a considerable posterior extension of expression compared with controls. Sections through these embryos confirmed that Tbx1 transcripts were seen at more caudal levels in the pharyngeal endoderm of VAD embryos than in normal embryos. Additionally, more Tbx1-positive cells were seen in the mesoderm above and adjacent to the expanded caudal expression domain of the pharyngeal endoderm in VAD embryos (Fig. 1h,i).

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Figure 1. In situ hybridization for Tbx1 expression in normal and vitamin A-deficient (VAD) embryos. a,b: Dorsal views of a stage 10 normal (a) and VAD embryo (b). Transcripts in pharyngeal and splanchnic endoderm showed posterior extension and lateral reduction in the VAD embryo. c,d: Dorsal views of a stage 12 normal (c) and VAD embryo (d). Transcripts were normally localized to the otic vesicle and surrounding mesoderm/endoderm, and there was some more anterior, lighter expression. In VAD embryos, expression extended caudally past the otic vesicle, down to the level of somite 4/5. Some nonspecific background staining was also seen in the head in d. a–d: White arrows indicate anterior limits of expression and black arrows the posterior limit of expression. Red arrows point to the caudal end of the hindbrain (r7 in normal embryos). r1, rhombomere 1; ov, otic vesicle. e–g: Lateral views of stage 14 normal (e) and VAD embryos (f,g). Strong expression in pharyngeal arch mesodermal cores and pouch endoderm was present in normal embryos. This expression is greatly reduced in VAD embryos and pharyngeal structures were malformed/lost. Arrowheads indicate small patches of remaining expression. pa, pharyngeal arch. h,i: Transverse sections taken just above somite 1, subjacent to the end of the hindbrain in a normal stage 10 embryo (h), and at a similar level in a VAD stage 10 embryo (i). Tbx1 transcripts were not evident in the normal pharyngeal endoderm (pe) at this level but were present in the same tissue in the VAD embryo(arrowheads). Expanded expression was also evident in the mesoderm (m). j,k: Coronal sections through the pharynx and gut in stage 13 normal (j) and VAD (k) embryos. Arrowheads indicate the increased posterior limit of Tbx1 expression in the pharyngeal endoderm in normal vs. VAD embryos. l,m: Transverse sections through stage 14 normal (l) and VAD embryos (m). Normal expression in the pharyngeal arch mesodermal core and pouch endoderm was greatly reduced in the VAD embryo and most of pharyngeal arch 2 is lost. acv, anterior cardinal vein; da, dorsal aorta; p, pharynx.

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In normal controls at stage 12/13, the most posterior expression was seen at the level of the forming otic vesicle (r4) and presumptive pharyngeal arch 2 in mesoderm and endoderm but transcripts were not seen in tissues caudal to the level of r4. Faint expression was also apparent in more anterior head mesenchyme (Fig. 1c,j). In VAD embryos, mesodermal and endodermal expression extended from the level of the otic vesicle down past the caudal end of the rhombencephalon/anterior spinal cord, to the level of somite 4/5 (equivalent to normal somite 3 as explained above). The most intense expression was present in and below the otic vesicle, adjacent to the caudal hindbrain/anterior spinal cord (Fig. 1d,k).

In normal embryos strong expression was present in the mesodermal core of the pharyngeal arches and in the endoderm of the pharyngeal pouches by stage 14 (Fig. 1e,l). In VAD embryos, this expression was either lost or greatly reduced (Fig. 1f,g,m). Where any expression remained, it was generally in the area of the first two pharyngeal arches and head mesenchyme. However, although the pharyngeal endoderm is still present in VAD embryos, caudal pharyngeal arches and pouches fail to form (Quinlan et al., 2002), and the lack of Tbx1 expression at stage 14 may reflect these morphological defects.

The lack of retinoic acid in VAD embryos seemed to affect Tbx1 expression in different tissues in a similar manner. In earlier embryos, Tbx1 patterning was affected, with extension of the caudal expression limit in both pharyngeal mesoderm and endoderm. Head mesenchyme expression was relatively unaffected and otic vesicle expression was maintained. Later in development, expression was lost in both the pharyngeal endoderm and mesoderm and the otic epithelium but was still present in patches of head mesenchyme.

Exogenous Retinoic Acid Down-Regulates cTbx1 Expression In Vivo

To exclude the possibility of global toxic effects all-trans retinoic acid (at-RA) was applied by grafting RA acid-soaked beads into the embryo, where the RA was slowly released from the bead into the surrounding tissues. Beads were soaked in either 2 or 5 mM of at-RA in dimethyl sulfoxide (DMSO) or DMSO alone and implanted into stage 12–14 embryos in contact with tissues known to express cTbx1, including the pharyngeal pouch endoderm and mesoderm (Fig. 2a–f) and otic vesicle (Fig. 2g–i). Figures show results with the lower 2 mM concentration of RA. After a further 18–24 hr of culture embryos were processed for cTbx1 mRNA expression. No difference was seen in the results between embryos cultured for 18 hr after grafting and those cultured for longer.

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Figure 2. In situ hybridization for Tbx1 expression in embryos at stage 20/21 after culture for 24 hr with grafts of 2 mM retinoic acid-soaked beads. Images from bead-grafted sides have been inverted for the sake of comparison. The position of the grafted bead has been outlined by dashed lines where appropriate. a,c: When RA-soaked beads were grafted into presumptive pharyngeal arch, normal Tbx1 expression in pharyngeal pouch endoderm (white arrowheads) and pharyngeal arch mesoderm (black arrowheads) was lost. b,d: Expression remained strong in the ungrafted contralateral side of the embryos. pp; pharyngeal pouch. e,f: Coronal sections through grafted tissues confirmed that expression in pharyngeal endoderm (arrowhead) and core mesoderm (star) present in ungrafted pharyngeal arch 3 (pa3) was lost in grafted pharyngeal arch 4 (pa4) both in the direct vicinity of the bead (e) and in sections 10–30 μm away (f). g,h:Tbx1 expression in the caudal part of the otic vesicle (ov) was also down-regulated by grafting an RA-bead (g) but remained high in the ungrafted contralateral side (h). i: Coronal sections showed that Tbx1 transcripts present in the ungrafted otic epithelium (arrowhead) were absent from the RA-bead grafted otic vesicle. j–m: Analysis of Shh (j,k) and Foxa2 (l,m) genes, which are part of a Tbx1 regulatory pathway, showed that they were unaffected by RA as expression levels in pharyngeal pouch (pp) endoderm for both these genes remained the same on the grafted (j,l) and contralateral control (k,m) sides of embryos.

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Local expression of cTbx1 was down-regulated with both RA concentrations at all implant locations in 64% of embryos (n = 36), compared with both nongrafted contralateral side of the same embryo and embryos grafted with DMSO-control beads (n = 24).

Tbx1 down-regulation was observed in all tissues grafted with a bead, including the pharyngeal endoderm and mesoderm (Fig. 2a–f) and the otic epithelium (Fig. 2g–i). Decrease of transcripts was seen at fairly similar levels in all grafted tissues, and this reflected results obtained with global embryonic RA treatment (data not shown).

Section data suggested that otic epithelium Tbx1 mRNA was possibly the most efficiently down-regulated, but this finding was most likely a reflection of the particular properties of the otic vesicle as a graft site; the bead was in a more secure location and was less likely to change position due to embryonic morphogenetic movements compared with those beads grafted into pharyngeal arches/pouches. Additionally, the target tissue completely surrounded the bead, whereas in pharyngeal grafts, only a small portion of Tbx1-expressing tissue is exposed to the bead.

Exogenous Retinoic Acid Does Not Alter Pharyngeal Shh or Foxa2 Expression In Vivo

Experiments by Yamagishi et al. (2003) have shown that Tbx1 can be up-regulated by Shh acting by means of Fox-binding sites in the Tbx1 promoter sequences. We therefore examined the expression of these two genes in embryos grafted with RA beads to establish if the repressive effect of RA was achieved by a decline in either Shh or Foxa2 expression. RA beads were grafted into pharyngeal locations known to express these genes, cultured overnight, and processed for in situ hybridization with either Shh or Foxa2. No convincing reduction of expression was seen for either probe (Shh; n = 19; Foxa2; n = 15; Fig. 2j–m).

Retinoic Acid Reduces Tbx1 RNA Expression in P19 Cells

P19 cells grown as a monolayer have been shown to express Tbx1 under normal culture conditions and are known to be responsive to retinoic acid. We used P19 monolayer cultures treated with differing concentrations of at-RA as a model system in which to quantitate the apparent repression of Tbx1 by at-RA, using RTQ-PCR. Cells were grown in monolayers in the presence of at-RA or DMSO alone for 24 hr. Untreated cells were also grown to exclude any effect of DMSO alone upon Tbx1 levels. Additional controls showed that cell morphology appeared normal for undifferentiated P19 cultures just before harvesting and no changes in markers for differentiated (neurofilament 68) vs. undifferentiated (vimentin) P19 cells were apparent (data not shown). After RNA extraction and generation of cDNA from each experimental treatment, RTQ-PCR for Tbx1 and Gapdh was performed. Using standard curves for Tbx1 and Gapdh, normalized Tbx1 copy numbers were calculated from Ct values. Mean Tbx1 copy numbers for each experimental treatment are displayed in Table 1 below.

Table 1. RTQ-PCR Analysis of RA-mediated Tbx1 Repressiona
P19 cell treatmentMean Tbx1 copy no.Ratio of Tbx1 expression relative to controlsP value
  • a

    Normalized results from RTQ-PCR analysis of Tbx1 expression levels in P19 cells cultured in the presence of different all-trans retinoic acid concentrations compared to control cells cultured with DMSO alone. Copy number is given as molecules/μl of cDNA. Change in Tbx1 expression levels in the presence of RA compared to controls is expressed as a ratio to 1. ‘n’ indicates the number of duplicate RTQ-PCR reactions performed for each sample. An unpaired t-test shows that copy number after RA treatment at all concentrations is significantly different than in control treated cells (P < 0.001 in all cases). RTQ-PCR, real-time quantitative polymerase chain reaction; at-RA, all-trans retinoic acid; DMSO, dimethyl sulfoxide.

3.0 × 10−6M at-RA2.06 × 104 (n = 4)0.14< 0.001
3.0 × 10−7M at-RA2.37 × 104 (n = 9)0.16< 0.001
1.5 × 10−7M at-RA2.49 × 104 (n = 4)0.17< 0.001
1.5 × 10−8M at-RA6.47 × 104 (n = 5)0.45< 0.001
DMSO1.44 × 105 (n = 22)1.0N/A

Addition of at-RA to P19 cells at physiological levels down-regulates expression of Tbx1 after 24 hr compared with levels in control cells treated with DMSO alone. A graph of RA concentration vs. Tbx1 copy number shows dose-dependent significant repression of Tbx1 expression with increasing at-RA concentration (P < 0.001 for all RA concentrations, Table 1) and demonstrates that the repressive response begins to plateau between 1.5–3.0 × 10−7 M at-RA (Fig. 3a). The lowest concentration of added at-RA, 1.5 × 10−8 M, reduced Tbx1 copy number just over twofold from 1.44 × 105 molecules/μl of cDNA in the DMSO control cells to 6.47 × 104 molecules/μl of cDNA. Increasing the concentration of at-RA 10-fold to 1.5 × 10−7 M repressed Tbx1 levels further to 2.49 × 104 molecules/μl of cDNA, a sixfold decrease compared with DMSO controls. Concentrations of 3 × 10−7 M at-RA and 3 × 10−6 M at-RA repressed Tbx1 levels further, producing up to sevenfold repression vs. DMSO controls. Copy numbers for untreated cells were also calculated and showed that DMSO treatment alone had no effect upon gene expression (data not shown).

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Figure 3. Results of real-time quantitative polymerase chain reaction for Tbx1 upon postnatal day (P) 19 cells after different experimental treatments. Error bars represent standard error of the mean. Tbx1 copy number is plotted against experimental treatment. a: Effect of culture with increasing concentrations of retinoic acid (RA) for 24 hr, showing dose-dependent repression of Tbx1. From left to right, bars represent dimethyl sulfoxide (DMSO) control, 1.5 × 10−8 M RA, 1.5 × 10−7 M RA, 3.0 × 10−7 M and 3.0 × 10−6 M RA, respectively, for increasing RA concentrations. b: Time course for the effect of 3 × 10−7 M RA upon Tbx1 copy no. i. After 8 hr of RA treatment. ii. After 12 hr of RA treatment. iii. After 16 hr of RA treatment. c: Effect of cycloheximide (CHX) blockade of protein translation on RA-mediated Tbx1 repression. Cells were grown in the absence/presence of 1.5 × 10−7 M RA retinoic acid, either with no CHX, 2 μg/ml CHX, or 5 μg/ml CHX.

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RA Repression of Tbx1 Becomes Apparent Between 8 and 12 hr Culture

A time course for the effect of at-RA upon P19 cells was performed to assess whether the at-RA repressive effect was likely to be direct, acting upon Tbx1 at the transcriptional level, or an indirect mechanism by means of another protein(s). The results of RTQ-PCR for Tbx1 expression levels in P19 cultures treated with either 3 × 10−7 M at-RA or DMSO and harvested after 8, 12, and 16 hr are presented in Table 2 and Figure 3b.

Table 2. Time-course of RTQ-PCR Analysis of RA-mediated Tbx1 Repressiona
P19 cell treatmentMean Tbx1 copy no.Ratio of Tbx1 expression relative to controlsMean Gapdh copy no.P value for Tbx1 repression
  • a

    Normalized results from RTQ-PCR analysis for Tbx1 expression levels in P19 cells at different time-points after treatment with 3 × 10−7 M all-trans retinoic acid or DMSO alone. Copy number is given as molecules/μl of cDNA. Change in Tbx1 expression levels in the presence of RA compared to control for each time point is expressed as a ratio to 1. ‘n’ indicates the number of duplicate RTQ-PCR reactions performed for each sample. An unpaired t-test shows that copy number after RA treatment for 12 and 16 hours was significantly different than in control treated cells (P < 0.001 in both cases). For abbreviations, see Table 1.

+RA 8h culture9.94 × 103 (n = 6)1.044.76 × 105 (n = 5)> 0.1
+DMSO 8h culture9.51 × 103 (n = 4)1.005.28 × 105 (n = 5) 
+RA 12h culture1.42 × 104 (n = 6)0.261.04 × 106 (n = 8)< 0.001
+DMSO 12h culture5.43 × 104 (n = 8)1.001.07 × 106 (n = 6) 
+RA 16h culture1.71 × 104 (n = 6)0.208.12 × 105 (n = 4)< 0.001
+DMSO 16h culture8.52 × 104 (n = 5)1.008.94 × 105 (n = 6) 

At 8 hr after treatment with retinoic acid, no significant difference was seen between copy number for Tbx1 between P19 cells treated with at-RA and controls treated with DMSO alone (P > 0.1, Table 2). However, after 12 hr of culture at-RA–treated cells began to display down-regulation of Tbx1 copy number compared with control cultures. Tbx1 was repressed nearly fourfold in RA-treated cells with copy numbers dropping from 5.43 × 104 to 1.42 × 104 molecules/μl of cDNA between DMSO controls and RA-treated samples (P < 0.001). By 16 hr of culture in the presence of RA, repression of Tbx1 had reached similar levels to those seen in previous experiments, with copy number dropping in retinoid-treated cells to 1.71 × 104 molecules/μl of cDNA, a sixfold decrease compared with the copy number of 8.52 × 104 molecules/μl of cDNA in control cells (P < 0.001).

Tbx1 transcription is up-regulated in normal control cells with time after plating. This finding is probably partly due to increase in cell number, as confluence of each plate increased with time, and Gapdh levels increased twofold between the 8- and 12-hr time points. Similar levels of confluency were seen in cultures with and without RA at each time point and Gapdh copy number did not vary greatly within each pair.

However, Tbx1 transcription in normal controls rises more than twofold between these time points. This finding is not an effect of DMSO because, as mentioned previously, no difference was seen between Tbx1 or Gapdh copy numbers for untreated and DMSO-control P19 cells (data not shown). The presence of RA represses the Tbx1 transcription seen in controls, raising the possibility that RA acts as an active repressor or prevents an activator of Tbx1 transcription from acting.

Full RA-Mediated Repression of Tbx1 Requires De Novo Protein Synthesis

The results from the time course suggested that the repression of Tbx1 was not an immediate early response to RA, as the difference in levels of transcription between controls and RA-treated cells might be expected to be detected sooner than 12 hr if this were the case. However, it was still unclear if the repressive effect of RA was acting directly at the level of Tbx1 transcription, or indirectly, mediated by another protein(s). For example, a possible indirect mechanism might involve retinoic acid acting to alter the levels of other transcription factor/s which are up-stream regulators of Tbx1. This process would require de novo protein synthesis; to test this possibility we compared Tbx1 copy numbers derived from real-time PCR for cells cultured for 24 hr with 1.5 × 10−7 M at-RA/DMSO and DMSO alone, in the presence or absence of cycloheximide (CHX), a known inhibitor of protein synthesis (Table 3; Fig. 3c).

Table 3. RTQ-PCR Analysis of Cycloheximide Treatment of RA-mediated Tbx1a
P19 cell treatmentTbx1 copy no.Ratio of Tbx1 expression relative to controlsP value
  • a

    Normalized results from real time PCR analysis for Tbx1 expression levels in P19 cells after treatment with 1.5 × 10−7 M all-trans retinoic acid or DMSO alone in the presence or absence of different concentrations of the protein synthesis inhibitor cycloheximide (CHX). Copy number is given as molecules/μl of cDNA. Change in Tbx1 expression levels in the presence of RA compared to control for each concentration of cycloheximide is expressed as a ratio to 1. ‘n’ indicates the number of duplicate RTQ-PCR reactions performed for each sample. An unpaired t- test shows that, although CHX partially rescued Tbx1 from repression by RA, the copy number after RA treatment both with and without cycloheximide remained significantly different than in control treated cells (P < 0.001 in all cases). For abbreviations, see Table 1.

+ RA1.26 × 104 (n = 5)0.18< 0.001
+ DMSO6.98 × 104 (n = 5)1.00 
+ RA + 2μg/ml2.80 × 104 (n = 9)0.43 
CHX  < 0.001
+ DMSO + 2μg/ml6.23 × 104 (n = 4)1.00 
CHX   
+ RA + 5μg/ml1.80 × 104 (n = 7)0.49 
CHX  < 0.001
+ DMSO + 5μg/ml3.65 × 104 (n = 7)1.00 
CHX   

In the absence of CHX, RA repressed Tbx1 copy number over fivefold compared with control levels, from 6.98 × 104 molecules/μl of cDNA down to 1.26 × 104 molecules/μl of cDNA. However, in the presence of 2 μg/ml CHX, the repression factor was reduced to 2.2-fold, from 6.23 × 104 molecules/μl of cDNA in DMSO+CHX controls to 2.8 × 104 molecules/μl of cDNA in cells treated with at-RA. Increasing the concentration of CHX further to 5 μg/ml did not greatly increase the rescue of Tbx1 RA-mediated repression as the repression factor between DMSO+CHX (3.65 × 104 molecules/μl of cDNA) and RA+CHX (1.8 × 104 molecules/μl of cDNA) -treated cells remained at 2.03-fold. Tbx1 copy numbers for all treatments remained significantly different to control-treated cells (P < 0.001 in all cases, Table 3), showing that RA-mediated repression does not require de novo protein synthesis. However, statistical tests (t-test) showed the partial rescue of Tbx1 copy number in the presence of CHX to be significant (P < 0.001), suggesting that, although significant repression of Tbx1 does not require de novo protein synthesis, this process may be necessary to achieve the highest levels of repression.

DISCUSSION

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

We have presented evidence that homeostasis of the signaling molecule retinoic acid is important for the normal expression of Tbx1, a transcription factor required for the proper development of the pharyngeal apparatus. Lack of retinoic acid during development resulted in early aberrant Tbx1 expression followed by loss of Tbx1 later on, when pharyngeal tissues should be forming.

Addition of exogenous retinoic acid clearly repressed expression of Tbx1; local grafting of retinoic acid soaked beads showed a specific reduction of Tbx1 in the vicinity of the grafted bead in pharyngeal endoderm and mesoderm and the otic vesicle. This effect was seen with beads soaked in 2mM RA (0.6 mg/ml), roughly half of the concentration used to produce posterior duplications in the chick limb (Riddle et al., 1993). Analysis of the effect of different RA concentrations upon Tbx1 expression in P19 cells showed negative regulation of Tbx1 in a dose-dependent manner where the larger the dose of RA, the greater the repression of Tbx1.

A time course for the induction of mRNA repression in P19 cells suggested this effect of RA upon Tbx1 might be partially due to an indirect mechanism, as down-regulation of Tbx1 became apparent between 8 and 12 hr of culture in the presence of RA. This finding was further investigated by experiments where Tbx1 repression by RA could be moderated by culture with the protein synthesis inhibitor CHX, suggesting that de novo protein synthesis may be required to achieve maximum RA-mediated repression of Tbx1. However, increasing cycloheximide concentrations did not fully rescue the level of Tbx1 copy number, and levels of repression continued to be statistically significant even in the presence of CHX, suggesting that at least part of the action of RA on Tbx1 does not require de novo protein synthesis. This mechanism could potentially be a direct RAR-mediated effect upon Tbx1 control sequences or act through direct effects upon other transcriptional regulators for Tbx1 (see below).

Since Tbx1 is not expressed in neuronal lineages and P19 cells can differentiate into neurons after a specific RA treatment protocol, we assessed the possibility that our results are due to the differentiation of the P19 cultures into neurons. This is highly unlikely, because the cells were cultured as a monolayer for only 24 hr. Normally, a monolayer culture will only produce neurons at very low efficiency 4 to 5 days after RA treatment (McPherson and McBurney, 1995). No cells of neuronal phenotype were seen and analysis of RA-treated cells and control cells with markers for undifferentiated P19 cells (vimentin) and neurons (neurofilament 68) also showed that all P19 cells were still undifferentiated.

A growing body of work has demonstrated that the correct levels of retinoic acid are required for normal pharyngeal development. Disruption of RA homeostasis has a profound affect upon the development of these tissues and their derivatives, and results in DGS-like phenotypes. Interestingly, overexpression of Tbx1 can cause a similar spectrum of DGS-like defects to those observed in the haploinsufficiency models (Merscher et al., 2001). Previously, the effects of hyper- and hypovitaminosis A were attributed to defects within the neural crest, but a role in pharyngeal endodermal patterning now seems most likely (Lammer et al., 1985; Mulder et al., 1998, 2000; Wendling et al., 2000; Quinlan et al., 2002; Niederreither et al., 2003; Vermot et al., 2003), particularly because retinoic acid treatment of amphioxus embryos, which do not contain neural crest cells, results in loss of pharyngeal arches/pouches by means of action upon the pharyngeal endoderm (Escriva et al., 2002).

RA has been shown to induce alterations in the expression of other transcription factors involved in the organogenesis of structures affected in DGS/VCFS. Using a single large dose of RA administered maternally to E9.0 embryos, an increase in the expression of Hoxa3 and an alteration in the expression of Pax1 were noted (Mulder et al., 1998). Both of these genes are expressed in the pharyngeal endoderm and are both are required for normal thymic and parathyroid development (Manley and Capecchi, 1995; Wallin et al., 1996). In RA-treated embryos, Hoxa3 pharyngeal endoderm expression was up-regulated although the anterior expression limit remained unchanged. Pax1 expression was maintained at normal levels in RA-treated embryos, but was distorted, reflecting a loss of the third pouch or its fusion with the second pouch. Other genes expressed abnormally in pharyngeal regions where retinoic acid homeostasis has been disrupted by maternal vitamin A-deprived diet include Hoxa1 and b1 (pharyngeal endoderm and mesoderm), Pax9 (endoderm), and Fgf8 (endoderm and ectoderm; Quinlan et al., 2002). Our data from the avian embryo and P19 cells suggest that Tbx1 can be added to this list of molecules important for pharyngeal development and regulated by retinoic acid.

Retinoic acid homeostasis appears to be vital for normal Tbx1 expression as both addition and inhibition of retinoic acid leads to aberrant expression of Tbx1. At early stages (stage 10) in VAD embryos, the Tbx1 endodermal domain is extended caudally along the anteroposterior axis. This posterior extension is also evident for Fgf8 in VAD embryos (Quinlan et al., 2002), which is interesting because work in the mouse has shown that Fgf8 and Tbx1 are genetic interactors and endodermal Fgf8 is postulated to be a direct downstream target of Tbx1 (Vitelli et al., 2002b). Later in development (stage 14), Tbx1 is greatly reduced across the entire pharyngeal region in VAD embryos, and this finding is again mimicked by Fgf8 expression, which is lost in the endoderm and reduced in the ectodermal domain (Kochilas et al., 2002; Quinlan et al., 2002; Vitelli et al., 2002b). Studies of VAD embryos (Quinlan et al., 2002) have showed that the first pharyngeal pouch is generally formed, with a possible attempt at a second pouch sometimes being evident, but more caudal pouches do not arise. This loss may be the reason for the failure of caudal pharyngeal Tbx1 expression. However, in the more anterior pharyngeal structures, which are partially formed, down-regulation of Tbx1 is still seen, suggesting that, at later stages, reduced expression is not entirely due to tissue loss.

Because Shh and Foxa2 have been shown to be required for tissue specific Tbx1 expression in the pharyngeal endoderm and head mesenchyme (Yamagishi et al., 2003), we examined their expression after the implantation of RA-beads. Neither Shh nor Foxa2 was down-regulated in the pharyngeal endoderm or head mesenchyme, and two further pieces of evidence suggest that it is unlikely that the RA repression of Tbx1 in pharyngeal endoderm is mediated by a decrease in expression of Shh or Foxa2. Firstly, axial expression of Shh exerts a long-range signaling effect upon the otic vesicle, but in Shh null mice, Tbx1 expression in the otic epithelium is maintained (Riccomagno et al., 2002), whereas in RA bead experiments otic Tbx1 is completely lost. Secondly, pharyngeal expression of Tbx1 is abnormal in the VAD quail at all stages, but expression of Shh is normal at stage 11 in the presumptive second pharyngeal pouch (Quinlan et al., 2002). If Shh expression is unaffected in the presence of exogenous RA and in the absence of endogenous RA in avian embryos, whereas Tbx1 expression is affected in both cases, then it seems doubtful that RA acts by means of Shh to alter Tbx1 expression.

Previous work has suggested that Tbx1 expression in mouse embryos either homozygous for a hypomorphic allele of raldh2 or nulls partially rescued by administration of maternal RA is only a mildly disrupted. Investigators have proposed that this finding may mean that either retinoic acid acts downstream of/in combination with Tbx1 to regulate the expression of signaling molecules such as Fgf8 (Niederreither et al., 2003) or that it does not regulate Tbx1 (Vitelli and Baldini, 2003). The results from both the avian embryo experiments (as discussed above) and the P19 cell RTQ-PCR suggest that RA does regulate Tbx1 in vivo and in vitro. The explanation for this discrepancy between the mouse and avian data may be that, in the VAD embryos, there is no dietary vitamin A. Thus, all the embryonic RA production pathways, including those mediated by retinol dehydrogenases, e.g., adh1 and adh4 (Rossant et al., 1991; Vonesch et al., 1994; Haselbeck and Duester, 1998), and retinaldehyde dehydrogenases such as Raldh1, 2 and 3 (Blentic et al., 2003; Fan et al., 2003) are blocked. In the hypomorphic raldh2 mouse mutants, only one RA-producing enzymatic pathway is partially blocked, perhaps resulting in a less severe effect upon Tbx1 expression. Alternatively, the genetic manipulation of raldh2 might result in compensation/up-regulation of the other genes involved in RA-production, which again might mitigate the effect upon Tbx1. Additionally, several Cyp26 genes, which metabolize RA to an inactive form during embryogenesis, have been shown to require the presence of RA for normal expression (Fujii et al., 1997; Reijntjes et al., 2003, 2004), suggesting further disruption of normal retinoic acid homeostasis in VAD embryos.

The biological actions of RA are mediated by specific ligand-activated receptors that bind to RA response elements (RAREs) within target gene promoters. Typically, targets are up-regulated by RA not down-regulated as described here for Tbx1, although down-regulation of endothelin-1, a Tbx1 target gene, (Piotrowski et al., 2003), has been described in endothelial cells 8 hr after the addition of RA (Yokota et al., 2001). As some of the repression we observed was dependent upon de novo protein synthesis, it is possible that an intermediary transcriptional repressor is involved. An example of such RA-mediated repression is the ligand-dependent binding of RXRα to Fog2, which enhances Fog2s' repression of GATA-4 activation of ANF (Clabby et al., 2003). RA also represses AP-1–mediated gene activation, partly by disrupting c-fos/c-jun interaction, and this action may account for much indirect ligand-induced repressive activity (Zhou et al., 1999). In some cases, RA repression may be mediated directly; RA significantly represses the activity of zebrafish raldh2-promoter within 2 hr in luciferase reporter constructs and in the embryo (Dobbs-McAuliffe et al., 2004). RA also represses the expression of Bmp-4 in both a cell line model and through bead delivery to the chick otocyst—experiments reminiscent of those conducted here. This finding was a direct effect mediated by RARs, not requiring de novo protein synthesis, through a second promoter in the second intron. No classic RAREs were to be found in this Bmp-4 promoter, but they are present in the upstream (1A) promoter, where they mediate RA activation of Bmp-4 expression in osteoblasts (Thompson et al., 2003). It has been reported that transcriptional repression of ovine FSH occurs by means of a standard RARE (Xing and Sairam, 2002). A search for RAREs in the Tbx1 promoter was carried out using MatInspector within the Genomatix suite (http://www.genomatix.de/software_services/software/ElDorado/Dorado.html), as some of the Tbx1 repression observed here could be direct. The program predicted two RAREs in a region 600 bp upstream of Tbx1 in human and rat, and one in mouse. Whether these sites are of biological significance remains to be seen, because previous studies have demonstrated that some Tbx1 control elements and evolutionary conserved sequences are located many kilobases from the coding sequence (Yamagishi et al., 2003; Brown et al., 2004). Thus, dissection of the sites responsible for RA-mediated repression of Tbx1 could be quite difficult but might be achieved through manipulation of BACs (recombineering) (Testa et al., 2003) to produce the many reporter constructs likely to be required. We recently have obtained reporter gene expression in P19 cells after integration of an internal ribosome entry site–green fluorescent protein element into Tbx1 using these techniques, demonstrating such an approach is feasible.

EXPERIMENTAL PROCEDURES

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

Embryos

Fertilized chicken eggs (White Leghorn, from Henry Stewart and Co. Ltd., UK) and quail eggs (Brian C. Potter, UK) were incubated at 38°C humidified until the required stage of development. Fertilized vitamin A-deficient quail eggs obtained from Dr. Malcolm Maden (King's College, London) were treated likewise.

Whole-Mount In Situ Hybridization

A 373-bp fragment of chick Tbx1(cTbx1) containing the conserved T-box domain was amplified by reverse transcriptase-PCR from stage 9–12 chick embryo total RNA using the primers and conditions described by Garg et al. (2001) and cloned into pGEMT. After linearization with Pst1 and Nco1, digoxigenin sense and antisense probes were transcribed by using T7 and SP6 polymerases, respectively. In situ hybridization was performed at 68°C by using standard protocols as described previously (Streit et al., 1998; Wilkinson, 1992). cTbx1 probes hybridized to both quail and chick embryos with the same expression pattern.

Bead Implantation

AG1-X2 beads (Bio-Rad) were incubated in 2 mM or 5 mM at-RA in DMSO or DMSO alone for 20 min at room temperature (4 beads/100 μl). The beads were then rinsed briefly twice in DMEM/10% fetal bovine serum (FBS). Two further 20-min washes in DMEM/10% FBS were performed, the first at 37°C and the second at room temperature.

After the removal of 5 ml of albumin, a window was cut into the egg shell and beads were inserted into various cTbx1-expressing positions in the pharyngeal region of stage 12–14 chick embryos, including the otic vesicle, pharyngeal pouches, and pharyngeal mesoderm. Eggs were sealed with tape and incubated for a further 20–24 hr, and embryos were dissected and fixed in 4% paraformaldehyde/phosphate buffered saline and then processed for in situ hybridization with cTbx1.

Cell Culture

P19 cells were cultured in 100-mm dishes in Glutamax αMEM (Gibco), 7.5% bovine calf serum and 2.5% FBS, 1× nonessential amino acids until nearly confluent, then trypsinized, spun down, and the medium removed. Cells were resuspended in 20 ml of medium and split 50:50 between two fresh 100-mm dishes. A total of 2 mM at-RA in DMSO was diluted in medium to produce a stock concentration of 3 × 10−5 M at-RA. The same dilution of DMSO alone was made as a control stock concentration. Volumes of 100, 50, and 5 μl of the at-RA stock were added to 10-ml cell cultures, to produce a final concentration of 3 × 10−7 M, 1.5 × 10−7 M and 1.5 × 10−8 M RA, respectively. Final concentrations of 3 × 10−6 M at-RA was made by diluting 2 mM at-RA/DMSO to give a 3 × 10−4 M stock solution and adding 100 μl of this to 10-ml cultures. Furthermore, 10-ml plates of cells received no treatment or 100 μl of the DMSO/medium stock as negative controls (final concentration in culture 0.015% DMSO). All cells were then cultured for a further 24 hr, when they were harvested. For the time course, separate plates of P19 cultures were set up as above and grown in the presence of 3 × 10−7 M at-RA/DMSO or DMSO alone for 8, 12, and 16 hr before harvesting.

The culture of P19 cells with at-RA was also performed in the presence of protein synthesis inhibitor cycloheximide. Plates of cells were grown as above and on splitting cultured in the presence of one of the following; 1.5 × 10−7 M at-RA/DMSO and 2–5 μg/ml CHX (the lower concentration of at-RA was used because culture with 3 × 10−7 M at-RA and CHX was found to be cytotoxic), or an equal volume of DMSO plus 2–5 μg/ml CHX. Cells were cultured 24 hr and then harvested. Total RNA was Trizol-extracted (Invitrogen) from cells, and 1 μg of RNA for each experimental treatment was reverse-transcribed by using random hexamer primers to make cDNA.

RTQ-PCR

RTQ-PCR for mTbx1 and mGapdh was performed, in at least quadruplicate for each sample, using the Qiagen SmartCycler PCR machine and SYBRGreen dye mix. Mouse Tbx1 primers were forward, 5′-CGACAAGCTGAACTGACCA-3′ and reverse, 5′-CAATCTTAAGCTGCGTGATCC-3′. Those for mGapdh PCR were forward, 5′-TTCACCACCATGGAGAAGGC-3′ and reverse, 5′-GGCATGGACTGTGGTCATGA-3′. RQT-PCR conditions comprised 95°C for 15 min, then 30 sec at 95°C, 57°C, and 72°C for 40 cycles.

Standard curve cDNA templates were made by Pfx PCR from mouse Tbx1 cDNA using forward primer 5′-CGCACAGTGGATGAAACAGA-3′ and reverse, 5′CAATCCCGGAAGCCTTTG-3′ and from mouse Gapdh cDNA with forward primer 5′-GCTGAGTATGTCGTGGAGTC-3′ and reverse primer, 5′-CATCCACAGTCTTCTGGGTG-3′. The resulting PCR products were gel purified, sequence confirmed using MegaBACE sequencing, and the concentration measured by spectrophotometer. Copy number in molecules per microliter was calculated using the equation (nanograms DNA × 10−9)/(length in base-pairs × 660) × (6.022 × 1023). Copy number was diluted to 1 × 1011 molecules/μl, and serial dilutions down to 1 × 103 were prepared. Mouse Tbx1 and Gapdh PCR was performed as above, in duplicate on at least five serial dilutions for the mTbx and mGapdh standard template, respectively, from 1 × 107 to 103 molecules/μl. Standard curves of the logarithm of copy number vs. the Ct value (cycle number at which the fluorescent signal reaches the threshold separating significant increase of the PCR product fluorescence above background) were generated using the SmartCycler software. Tbx1 Ct values for each cell sample were plotted on the standard curve to calculate copy number and normalized to adjust for variation in the initial amount of cDNA using the copy numbers generated from the mGapdh standard curve and Ct values.

Acknowledgements

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

We thank Drs. Malcolm Maden and Emily Gale for providing vitamin A-deficient quail embryos, Dr. Martin Koltzenburg for the use of the SmartCycler PCR machine, and Dr. Paris Ataliotis for critical reading and helpful comments on the manuscript.

REFERENCES

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  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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