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RESEARCH REPORT |
1 Department of Orthodontics & Pediatric Dentistry, University of Michigan School of Dentistry 1011 N. University Avenue, Ann Arbor, MI 48109-1078, USA;
2 Kresge Hearing Research Institute, Department of Otolaryngology/Head-Neck Surgery, University of Michigan; and
3 Physiology, Pharmacogenetics and Injury Program, Division of Basic and Translational Sciences (DBTS), National Institute of Dental and Craniofacial Research (NIDCR), NIH, Building 45, Room 4AN-18B, 45 Center Drive, MSC 6402, Bethesda, MD 20892-6402, USA;
* corresponding author, sggong{at}umich.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: primary palate • microarray analysis • RT-qPCR
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Various genes have been implicated in the biological processes involved in growth and fusion of the midfacial processes (reviewed in Francis-West et al., 1998; Young et al., 2000). The analyses and functions of most of the genes during craniofacial development have been obtained primarily from a combination of their expression patterns during growth and development of the midfacial region, analysis of the phenotypes generated through loss-of-function experiments, functional studies, and linkage analyses in human populations. As yet, many of the possible pathways that operate in the patterning and regulation of growth and fusion of this region remain unclear. We took advantage of the Clontech Atlas Arrays to search for genes that may play a critical role in the growth and fusion of the midfacial region to become the primary palate. We identified about 70 genes that were potentially expressed at different levels between TS 6-8 (pre-fusion) and TS 12-14 (fusion) in LNP, MNP, and MxP. We describe here the quantitative and spatial characterization of 4 of these genes, Tbx14/15, Fgf8, Keratin-18 (K18), and Dickkopf-1 (Dkk-1).
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis of Gene Expression by Clontech Atlas Arrays
The mouse array, Mouse 1.2 Array, contains 1176 genes covering "crucial cellular pathways and functions" (Clontech, Palo Alto, CA, USA). Membranes were hybridized in parallel with radiolabeled cDNAs generated from these 2 RNA populations so that we could identify genes that were differentially expressed between these 2 stages of development. Hybridization was carried out according to the vendor’s protocol. Scanned images were analyzed with AtlasImage v1.01 software; signal intensities from both membranes were globally normalized, followed by calculation of the ratio between the 2 developmental stages, as described previously (Lomax et al., 2000). Signal ratios between the 2 developmental stages of > 2 or < 0.5 were considered differentially expressed.
Reverse-transcriptase and Real-time Polymerase Chain-reaction (RT-qPCR)
Real-time PCR was carried out in triplicate per sample for all genes assayed. To compare expression levels across samples, we assayed transcripts of any given gene from all 6 samples (3 independent pools of 2 developmental stages) at once, i.e., in the same PCR plate. One µg total RNA was reverse-transcribed with RNase H–-MMLV (SuperScipt III, Invitrogen, Carlsbad, CA, USA) and oligo (dT)12–18 (400 ng). Aliquots (1 to 2 µL) of the 200-µL diluted first-strand cDNAs were subjected to PCR amplification in a 25-µL reaction and analyzed on an ABI Prism 7000 (Applied Biosystems; Foster City, CA, USA) with gene-specific primers, and, for some genes, in conjunction with TaqMan probes (sequences in Appendix Table IIIa). Thermocycling conditions were: 50°C for 2 min to degrade any cDNA contamination from previously amplified products, 95°C for 10 min to activate Taq polymerase, followed by 40 cycles of amplification at 95°C for 15 sec alternating with 60°C for 1 min. Expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was determined as an internal standard for normalization, since its level was relatively unchanged between these 2 developmental stages. The relative difference in expression of genes of interest was determined with the use of threshold cycle CT, defined as the PCR cycle number at which intensity of fluorescence labeling crosses a threshold (Livak and Schmittgen, 2001). Expression levels with two-fold or greater changes in expression level between the 2 developmental stages were subjected to paired Student’s t test; p values less than 0.01 were considered statistically significant.
In situ Hybridization of RNA
Embryos (10
days old) were harvested from C57BL/6 timed-pregnant females mated and subjected to in situ hybridization as described by Gong and Guo (2003). All hybridizations were performed at 60°C with the use of gene-specific riboprobes derived from cDNA in plasmid vectors. Fgf8 cDNA was a gift from Gail Martin (UCSF), and the Dkk1 cDNA was a gift from C. Niehrs (Germany). The K18 and Tbx14/15 riboprobes were generated by PCR amplification with gene-specific primers (sequences in Appendix Table IIIb).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Up-regulation of Tbx15 during Midfacial Development
Our initial screening by Clontech Atlas Array analysis indicated that Tbx14/15 was expressed at a higher level at TS12 compared with that at TS 6-8. We verified differential expression of Tbx15 by RT-qPCR using gene-specific primers. Among 3 independent sample pairs of TS 6-8 and TS 12-14, there was a two- to 4.3-fold change in Tbx15 mRNA. The magnitude of change detected by RT-qPCR was comparable with the 3.1-fold change determined by microarray analysis. In situ hybridization clearly demonstrated differential expression of Tbx15 between these 2 stages of midfacial development. In 10-day-old embryos with a tail somite (TS) count of 8 or fewer, the Tbx14/15 gene was not expressed at a visible level in the 3 facial processes (Figs. 1A
, 1B). By TS 14, Tbx14/15 transcripts were visible in the mesenchymal tissues behind the LNP (white arrowhead in Fig. 1D, black arrows in Fig. 1E). Expression in the maxillary primordium was initiated at this stage, being restricted to the mesenchymal tissues at its caudal half, and increasing with growth (TS 14 - Fig. 1C; TS > 22 - Fig. 1F).
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, 2B). By TS14, expression of the gene had decreased and was more restrictive, being absent from most epithelium, except that lining the stomadeum (arrowheads in Fig. 2C), nasal pit, and the eye primordium (Fig. 2D). By 11.5 dpc, K18 was expressed in much of the ectodermal layer covering the presumptive olfactory cells in the nasal pit (Fig. 2E) and the epithelial layer of the tongue and oral cavity (Fig. 2F).
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), appearing over a broad domain further anteriorly around the putative site of fusion in the LNP and MNP (arrow, Fig. 3A). The expression level was lower in tissues further from the site of fusion, and higher in the MNP compared with the LNP (TS6, arrow in Fig. 3B). A similar pattern, but higher level, of expression in the site of fusion was observed in the TS11 embryo, where fusion of the midfacial processed had already occurred, and the epithelial seam (bilayer epithelium) formation was evident (es in Figs. 3C, 3D). By TS16, expression of the gene was strong in the medial surface of the MxP (Fig. 3E) and along the site of fusion (fusion line denoted by smaller black arrowheads in F). By this stage, Dkk1 transcripts were also found immediately underlying the ectodermal layer of the MNP and LNP (Fig. 3F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Of the 4 genes that we analyzed, the expression pattern of Fgf8 in the midfacial region has been the most well-documented (Bachler and Neubuser, 2001; Firnberg and Neubuser, 2002). The absence of change in expression levels at the 2 stages was not surprising, in view of the fact that the rapid growth of the midfacial processes that occurs from TS6 to 12 would require that the expression levels of a molecule involved in directing outgrowth, such as Fgf8, be maintained during the period. Unlike Fgf8, the expression of Dkk1 in the midface has not been described previously. The Dkk1 gene is a family member of secreted proteins and is a potent inhibitor of wingless (Wnt)/beta-catenin signaling (Glinka et al., 1998), and appears to play a key role in embryonic patterning and specification of anterior structures (Glinka et al., 1998; Mukhopadhyay et al., 2001). Dkk1 is also believed to mediate epithelial-mesenchymal interactions and Bmp4-induced apoptosis during limb development (Mukhopadhyay et al., 2001; Grotewold and Ruther, 2002a, b). Bmp4 appeared to be expressed in the ectoderm overlying mesenchymal expression of Dkk1 in the fusion site (Gong and Guo, 2003). The increase in Dkk1 expression in the midface from TS6 to TS12, a period where extensive changes in both the growth and fusion of the midfacial processes occur, suggests a role for this gene in midfacial development. Functional studies must be performed to determine whether Dkk1 plays a role in mediating apoptosis during midfacial development.
We also saw a four-fold decrease in K18 gene expression. K18 is also a substrate for caspase digestion during the course of epithelial cell apoptosis (Oshima, 2002), a cellular event that is believed to occur when the midfacial processes fuse (Gong and Guo, 2003). Analysis of our Tbx14/15 data suggests that it likely plays a role in the patterning process of the maxillary primordium and is probably not involved in mediating the fusion event of the midface. In this regard, it is interesting to note that several members of the T-box gene family, of which Tbx14/15 is a member, are involved in craniofacial development. Several human disorders have been linked to mutations in T-box genes, e.g., cleft palate with ankyloglossia/TBX22 (Packham and Brook 2003). Also, the mutation in the Dancer spontaneous mouse mutant, in which homozygotes present with clefts of the lip and palate, has been mapped to the Tbx10 gene (Bush et al., 2004).
The 4 genes can be used as markers for further studies of midfacial growth and development. It is highly likely that these genes can act in concert to modulate the growth and fusion of the midfacial region. Analyses of the possible functions of these and other genes will lead to a better understanding of and appreciation for the biological events and molecular players and pathways involved in normal and abnormal midfacial morphogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received February 5, 2004; Last revision September 30, 2004; Accepted October 4, 2004
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Bush JO, Lan Y, Jiang R (2004). The cleft lip and palate defects in Dancer mutant mice result from gain of function of the Tbx10 gene. Proc Natl Acad Sci USA 101:7022–7027.
Firnberg N, Neubuser A (2002). FGF signaling regulates expression of Tbx2, Erm, Pea3, and Pax3 in the early nasal region. Dev Biol 247:237–250.[ISI][Medline]
Francis-West P, Ladher R, Barlow A, Graveson A (1998). Signalling interactions during facial development. Mech Dev 75:3–28.[ISI][Medline]
Glinka A, Wu W, Delius H, Monaghan AP, Blumenstock C, Niehrs C (1998). Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391:357–362.[Medline]
Gong SG, Guo C (2003). Bmp4 gene is expressed at the putative site of fusion in the midfacial region. Differentiation 71:228–236.[ISI][Medline]
Grotewold L, Ruther U (2002a). Bmp, Fgf and Wnt signalling in programmed cell death and chondrogenesis during vertebrate limb development: the role of Dickkopf-1. Int J Dev Biol 46:943–947.[ISI][Medline]
Grotewold L, Ruther U (2002b). The Wnt antagonist Dickkopf-1 is regulated by Bmp signaling and c-Jun and modulates programmed cell death. EMBO J 21:966–975.[ISI][Medline]
Livak KJ, Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408.[ISI][Medline]
Lomax MI, Huang L, Cho Y, Gong TL, Altschuler RA (2000). Differential display and gene arrays to examine auditory plasticity. Hear Res 147:293–302.[ISI][Medline]
Mukhopadhyay M, Shtrom S, Rodriguez-Esteban C, Chen L, Tsukui T, Gomer L, et al. (2001). Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse. Dev Cell 1:423–434.[ISI][Medline]
Oshima RG (2002). Apoptosis and keratin intermediate filaments. Cell Death Differ 9:486–492.[ISI][Medline]
Packham EA, Brook JD (2003). T-box genes in human disorders. Hum Mol Genet 12:R37–R44.
Young DL, Schneider RA, Hu D, Helms JA (2000). Genetic and teratogenic approaches to craniofacial development. Crit Rev Oral Biol Med 11:304–317.[Abstract]
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| Journal of Dental Research ® | Critical Reviews (1990-2004) |
