Molecular profiles of mitogen activated protein kinase signaling pathways in orofacial development

Authors

  • Saurabh Singh,

    1. Department of Molecular, Cellular and Craniofacial Biology, University of Louisville Birth Defects Center, ULSD, Louisville, Kentucky 40292
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  • Xiaolong Yin,

    1. Department of Molecular, Cellular and Craniofacial Biology, University of Louisville Birth Defects Center, ULSD, Louisville, Kentucky 40292
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  • M. Michele Pisano,

    1. Department of Molecular, Cellular and Craniofacial Biology, University of Louisville Birth Defects Center, ULSD, Louisville, Kentucky 40292
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  • Robert M. Greene

    Corresponding author
    1. Department of Molecular, Cellular and Craniofacial Biology, University of Louisville Birth Defects Center, ULSD, Louisville, Kentucky 40292
    • Department of Molecular, Cellular and Craniofacial Biology, University of Louisville Birth Defects Center, ULSD, Louisville, Kentucky 40292
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  • This article is a US Government work and, as such, is in the public domain in the United States of America.

Abstract

BACKGROUND:

Formation of the mammalian orofacial region involves multiple signaling pathways regulating sequential expression of and interaction between molecular signals during embryogenesis. The present study examined the expression patterns of members of the MAPK family in developing murine orofacial tissue.

METHODS:

Total RNA was extracted from developing embryonic orofacial tissue during gestational days (GDs) 12–14 and used to prepare biotinylated cDNA probes, which were then denatured and hybridized to murine MAPK signaling pathways gene arrays.

RESULTS:

Expression of a number of genes involved in the (ERK1/2) cascade transiently increased in the embryonic orofacial tissue over the developmental period examined. Numerous members of the SAPK/JNK cascade were constitutively expressed in the tissue. Genes known to play a role in p38 MAPK signaling exhibited constitutive expression during orofacial development. Western blot analysis demonstrated that ERK2/1, p38, and SAPK/JNK kinases are present in embryonic orofacial tissue on each of GD 12, 13, and 14. By using phospho-specific antibodies, active ERK was shown to be temporally regulated during orofacial development. Minimal amounts of active p38 and active SAPK/JNK were detected in orofacial tissue during GDs 12–14.

CONCLUSIONS:

Our study documents specific expression patterns of genes coding for proteins belonging to the ERK1/2, p38, and SAPK/JNK MAPK families in embryonic orofacial tissue. We also demonstrate that active, phosphorylated forms of ERK1/2 only were detected in the embryonic tissue investigated, suggesting a more central role for members of this family in embryonic orofacial development. Birth Defects Research (Part A) © 2006 Wiley-Liss, Inc.

INTRODUCTION

The craniofacial region is one of the most rapidly growing and developing areas in the embryo, and thus it is highly susceptible to malformations. Normal craniofacial development depends on a series of precisely orchestrated morphological and molecular events such that an alteration in any one of these coordinated processes could lead to abnormal development. In mammalian embryos, the orofacial complex that includes palatal processes develops chiefly from the first pharyngeal arch in the form of bilateral extensions of the maxillary processes and contains cranial neural crest–derived mesenchyme surrounded by an epithelial layer (Johnston, 1966; Noden, 1975). The palatal processes, medial projections of the maxillary processes, first grow vertically on either side of the tongue, then undergo a series of morphogenetic movements that result in their reorientation above the tongue thereby bringing this medial edge epithelia (MEE), on the apical surface of each process, into contact. Subsequent fusion of the palatal processes results in formation of the definitive secondary palate (Greene et al., 1995).

Past morphological, biochemical, and molecular analyses of normal orofacial development have provided fascinating insights regarding control of cellular growth and differentiation of orofacial tissue. Normal growth of the entire craniofacial region, via precisely regulated spatio-temporal patterns of proliferation, is known to be critical for proper development in both the human (Greene et al., 1992; Yoneda and Pratt, 1981) and in laboratory animals (Fraser et al., 1956; Shah et al., 1987). In this context, the developing secondary palate has proved to be of inestimable value as a model system for gaining insight into the etiology of palatal clefts and has also provided an excellent paradigm for elucidating the interplay between myriad molecular signals governing cellular proliferation, differentiation, and death during embryogenesis. Exquisite controls are placed upon these processes during morphogenesis. While basic cues and mechanisms are undoubtedly shared by the adult and developing organism, the intricacies involved in regulating these cellular processes in a spatial and temporal continuum, to result in the genesis of new tissues and organs, are likely to include mechanisms unique to the embryo or fetus. Development of the orofacial region involves integration and regulation of the sequential expression and interaction of numerous molecular signals (Greene et al., 1991; Nugent et al., 1995; Potchinsky et al., 1998; Weston et al., 1998). Among the multiple signaling pathways active in the embryogenesis of this region, the mitogen-activated protein kinase (MAPK) pathways are thought to play important roles (Seger and Krebs, 1995).

The MAPKs comprise a family of highly conserved eukaryotic enzymes that are among the most thoroughly studied of signal transduction systems. At least five distinct MAPK cascades, functioning as separate modules, are known to exist in mammalian cells (Garrington and Johnson, 1999; Widmann et al., 1999). Each cascade can be triggered by a diversity of signaling molecules. The cascades include: (1) the mitogenic extracellular signal regulated kinase (ERK1/2) cascade, which preferentially regulates cell growth and differentiation; (2) the stress-activated protein kinase 1 (SAPK) cascade, comprising the c-jun N-terminal kinase (JNK) family; (3) the SAPK2/3 cascade, comprising the p38 family of proteins; (4) the mitogenic extracellular signal regulated kinase (ERK5/BMK1) cascade; and (5) the ERK3 cascade. Pathways 2 and 3 function primarily in stress response, whereas pathway 4 is preferentially activated in response to growth factors and stress.

Typically, these cascades are organized into a three-kinase pathway consisting of a MAPK, an MAPK activator (MKK or MAP kinase kinase), and an activator of the MAPK activator (MKKK or MAP kinase kinase kinase). MKKs are characteristically dual specificity kinases that phosphorylate MAPKs on both tyrosine and threonine residues, while the MKKs are themselves phosphorylated on serine or threonine residues by the MKKKs (Widmann et al., 1999). Transmission of signals is achieved by a sequential series of phosphorylation reactions wherein each downstream kinase serves as a substrate for the upstream activator. For example, in the ERK1/2 cascade, the two related mammalian MAPKs, ERK1 and ERK2 (p44mapk and p42mapk), are phosphorylated (activated) by MAP kinase/ERK kinase (MEK), which was phosphorylated (activated) primarily by the ser/thr protein kinase Raf-1 after having been recruited to the plasma membrane by Ras. In the ERK5/BMK1 pathway, ERK5/BMK1 is phosphorylated by MEK5, which is activated by MEKK2/MEKK3.

Once activated, MAPKs translocate to the nucleus and effect downstream ser/thr phosphorylation of regulatory molecules such as transcription factors, thereby effecting changes in gene expression. Nuclear translocation is best understood for ERK1 and ERK2 where phosphorylated ERKs dimerize and are actively transported across the nuclear membrane (Brunet et al., 1999; Khokhlatchev et al., 1998).

While MAPK modules appear to signal independently of each other, several of these pathways converge on identical nuclear transcription factors, some of which can be regulated by more than one MAPK cascade (Garrington and Johnson, 1999). Moreover, MAPKs can also integrate signals derived from other signaling pathways such as the STAT and cAMP cascades (Vossler et al., 1997; Xia et al., 1995). Indeed, in embryonic orofacial tissue, MAPK phosphorylation at tyrosine-185 and subsequent activation can be stimulated via multiple pathways, many known to play significant roles in differentiation of orofacial tissue (Potchinsky et al., 1998). Thus, the MAPK pathways can coordinate responses to multiple diverse effectors. This is potentially of critical importance during metazoan development wherein responses to environmental and genetic cues depend on regulation and coordination of multiple signal transduction pathways.

Various growth and differentiation factors control the formation of the orofacial region. Two of the more important of these factors are epidermal growth factor (EGF) and the transforming growth factors beta (TGFβ) (Kaartinen et al., 1995; Potchinsky et al., 1998). The EGF signaling pathway is active during embryogenesis of a number of structures, such as the secondary palate, the development of which is dependent on epithelial-mesenchymal interactions (Doetschman, 1999). Moreover, EGF plays a crucial role in the regulation of cell proliferation and differentiation (Pisano and Greene, 1987). The binding of EGF to its receptor activates receptor tyrosine kinase (RTK) with concomitant tyrosine autophosphorylation (Greene and Pisano, 2000). Although RTK activation leads to a variety of downstream effects, the primary pathway for EGF-induced effects is via ERK1/2 phosphorylation. TGFβ is a member of a large family of multifunctional growth and differentiation factors, many of which are involved in the development of the orofacial region in mammals (Gehris and Greene, 1992). Several lines of evidence support the notion that TGFβ signaling may be mediated in part through the MAPK cascade in the developing embryo (Weston et al., 1998). Indeed, TGFβ-mediated signaling can modulate the effects of EGF on cell growth in cells derived from the embryonic orofacial region (Weston et al., 1998). Hence, a MAPK may lie downstream of TGFβ receptors in much the same way that the RAS/ERK MAPKs lie downstream of tyrosine kinase receptors.

Developmental roles of MAPKs have been demonstrated in vivo (Massague, 1998). Embryos deficient in ERK2 exhibit defects in mesoderm differentiation and placental angiogenesis. Deletion of p38α MAPK results in angiogenic defects in the placenta and peripheral vessels (Wang et al., 2004). ERK5-deficient embryos are embryonic lethal due to defects in angiogenesis and cardiovascular development (Hayashi and Lee, 2004; Kuida and Boucher, 2004; Regan et al., 2002). Because family members mediate the effects of several critical growth factors during embryonic orofacial development, we sought to define the expression profile of MAPK proteins during orofacial ontogeny. We examined the expression of 96 genes associated with MAPK families during the critical period of murine orofacial growth and morphogenesis. Levels of ERK2/1, p38, and SAPK/JNK kinase proteins were also determined by immunoblotting and their active forms analyzed using phospho-specific antibodies.

MATERIALS AND METHODS

Animals

Mature male and female ICR mice (Harlan, Indianapolis, IN) were housed in a climate-controlled room with a 12 h alternating dark-light cycle and were mated overnight. The presence of a vaginal plug the following morning (gestational day [GD 0]) was considered as evidence of mating. On GDs 12, 13, and 14, female mice were euthanized by asphyxiation, and embryos were dissected from uteri in sterile Ca++- and Mg++-free PBS. Extraembryonic membranes were removed, and maxillary tissue, including primary palatal tissue and secondary palatal processes, was excised, minced, and stored at −20°C in RNALater solution (Qiagen, Chatsworth, CA) for subsequent extraction of total RNA. Tissue from five to eight embryos (on each of the 3 days of gestation) was pooled into single samples for RNA extraction. Moreover, three distinct RNA samples from fetal orofacial tissue were processed for each day of gestation in order to prepare triplicate sets of target RNAs for hybridization to SuperArray nylon membranes (SuperArray Inc., Bethesda, MD) (nine samples and nine chips total).

RNA Extraction

Total RNA from excised tissue samples was isolated using the RNeasy Mini Kit (Qiagen) following the manufacturer's recommendations. The quality and quantity of the extracted total RNA were assessed by spectrophotometric UV absorbance ratio at 260/280 nm and absorbance at 260 nm, respectively. Absorbance ratios measured for total RNA samples were from 1.98 to 2.10 in Tris-EDTA buffer solution (pH 7.5), indicating the quality of the samples.

cDNA Expression Array Analysis

Nonradioactive Mouse MAP Kinase Signaling Pathways Gene Array (GEArray Q Series MM-017; SuperArray, Inc.) was used to analyze the gene expression profile of members of the MAPK signaling families during orofacial development. Procedures were carried out according to the manufacturer's protocol. Briefly, 3 μg of total RNA was used as template for biotinylated cDNA probe synthesis. RNA was reverse-transcribed by gene-specific primers (supplied with the SuperArray kit) with biotin-16-dUTP. Biotinylated cDNA probes were denatured and hybridized to MAPK signaling pathway gene-specific cDNA fragments spotted on the membranes. The GEArray membranes were then washed and blocked with GE-blocking solution, and incubated with alkaline phosphatase–conjugated streptavidin. The hybridized biotinylated probes were detected by chemiluminescence using the alkaline phosphatase substrate, CDP-Star.

Images of membranes were acquired and quantitated using the Kodak 1D image analysis software on a Kodak Imaging Station, model 440 CF. Raw image data were transferred to the online GEArray Expression Analysis Suite (www.SuperArray.com). All signal intensities were corrected for background by subtracting the minimum value, defined as the numerical value of spots with least intensity. Relative expression levels of different genes were estimated by comparing their signal intensity with that of the internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The average of three data sets for each of the 3 days of gestation was used for results analysis. Genes were considered as not being expressed if their expression level was <20% of the control gene. Genes were considered to be constitutively expressed if the change in their relative expression levels between 2 gestational days was <1.5 fold. If the fold change was >1.5 or <0.67, then the genes were considered as having increased or decreased levels of expression, respectively.

Confirmation of Gene Expression Data

Expression data were confirmed using the real time PCR-based pathway focused gene profiling “RT2 profiler system” from SuperArray, Inc. (MAP Kinase Signaling Pathway PCR Array, APM-061, corresponds to the GEArray Q Series MM-017 used for gene expression profiling above). Procedures were carried out according to the manufacturer's protocol. Briefly, 1 μg of total RNA was used as template for reverse-transcription of cDNA. This was then diluted and added to a master mix containing the fluorescent SYBR Green dye. Aliquots from this mix were added to a 96-well plate, where each well contained predispensed gene-specific primer sets. The plates (one for each day of gestation) were then placed in a TaqMan ABI Prism 7000 Sequence Detector System (Applied Biosystems, Foster City, CA) and real-time PCR analysis was performed.

Cycling parameters were as follows: 95°C for 10 min for activation of HotStart DNA polymerase, followed by 40 cycles of denaturation at 95°C for 15 s each, and finally, primer extension at 60°C for 1 min. Each plate contained a panel of housekeeping gene primers for normalization of the PCR array data, as well as estimation of the linear dynamic range of the assay. Further, for each reaction, both “no reverse transcription control” and “no template” samples were included as negative controls. Raw data were acquired and processed with ABI Sequence Detector System software version 1.0 (Applied Biosystems, UK) to calculate the threshold cycle (Ct) value and relative gene expression values subsequently determined according to the standard ΔΔCt method (Singh et al., 2005).

Tissue Preparation for Protein Collection

Pregnant female mice were euthanized by carbon dioxide asphyxiation, and gravid uteri were removed and placed in PBS on ice. Embryonic orofacial tissues from the midfacial region (maxillary tissue, including primary palatal tissue and secondary palatal processes) were dissected, minced, and washed twice with Ca++- and Mg++-free PBS. Embryonic tissues were homogenized in ground glass combination conical/cylindrical tissue grinders at 4°C in an appropriate volume of lysis buffer (250 mM NaCl, 50 mM Tris [pH 7.4], 5 mM EDTA, 0.1% [v/v] Triton X-100, 1 mM phenylmethylsulfonyl fluoride [PMSF], 50 mM NaF, 1 mM sodium orthovanadate, 10 μg/mL leupeptin, and 50 μg/mL aprotinin). Adult tissues were homogenized in the above buffer using a Tekmar Tissumizer at half maximal speed for 30 to 45 s at 4°C. All homogenates were sonicated for 30 s at 4°C and allowed to sit on ice for 30 min with vigorous vortexing every 5 min, then cleared by centrifugation for 10 min at 13,000×g at 4°C. Total protein in the extracts was determined according to the method of Bradford [1976) using bovine serum albumin as a standard.

Immunoblotting

Primary antibodies against phospho and total ERK1/2 and β-actin were obtained from Cell Signaling (Beverly, MA) while those for phospho and total SAPK/JNK and p38 were obtained from New England Biolabs (Beverly, MA). Appropriate amounts of lysate were denatured by boiling in 2× Laemmli sample buffer (Laemmli, 1970), and the proteins were separated by SDS-PAGE electrophoresis at 125 V using 8–16% polyacrylamide Tris-Glycine gels (Novex, San Diego, CA) followed by electrophoretic transfer (30 V for 2 h) of the proteins to PVDF membranes. Gels were subsequently stained with Coomassie blue dye and membranes with 0.1% fast green to visualize proteins and ensure the efficiency of protein transfer. Blots were blocked by incubation in 5% nonfat dry milk in TBST buffer (50 mM Tris, pH 7.6; 150 mM NaCl; 0.1% Tween 20) for 1 h at room temperature. Antibodies were diluted in blocking solution and blots incubated with primary antibody (1:1,000) for 1–1.5 h at room temperature and then washed extensively. An HRP-conjugated goat antirabbit IgG1 (Zymed Labs, Carlsbad, CA) was used as the secondary antibody at 1:10,000 dilution. Immune complexes were detected using the ECL-Plus chemiluminescent detection system (Amersham Pharmacia Biotech, Arlington, IL) according to the manufacturer's instructions. “No primary antibody” control immunodetection was performed on each blot prior to blotting with the primary antibody in order to distinguish between specific immunoreactive bands and nonspecific protein bands due to interaction of the secondary antibody with endogenous proteins. Immunoblots were replicated with similar results on a minimum of three complete sets of tissue samples.

Densitometric Analysis

Densitometric analyses of total and phospho p42/44 bands were performed with NIH Image (v 1.62) software.

RESULTS

MAPK Pathway Gene Expression Profile

Murine orofacial tissue was excised on each of GD12–14, the critical period of orofacial growth and morphogenesis, and total RNA was prepared from the tissues. Three distinct RNA samples for each GD were prepared and subsequently hybridized to SuperArray cDNA membranes. The nonradioactive Mouse MAP Kinase Signaling Pathways Gene Array was used to probe for expression profiles of genes known to be members of MAPK signaling family.

Among the 96 genes tested on the SuperArray cDNA membrane, 39 genes exhibited constitutive expression on each of GDs 12, 13, and 14 (Table 1). The relative expression level of each of these genes did not change >1.5-fold from GD 12 to 13, and from GD 13 to 14. Ten genes that were not expressed (their expression levels were <20% of the positive control genes) are listed in Table 2. Of the 47 genes that exhibited differential expression during orofacial development (Table 3), 46 genes showed at least a 1.5-fold increase from GD 12 to GD 13. However, half of these genes exhibited a diminished level of expression from GD 13 to GD 14. Moreover, no genes exhibited increased expression levels between GD 13 and GD 14. Gene expression data were confirmed by the RT-PCR-based gene profiling “RT2 profiler system” (SuperArray). Each array is a 96-well plate containing SYBR Green-optimized primer sets corresponding to a total of 96 genes, including five housekeeping genes and two negative controls. The particular array used for confirmation of gene expression data contained primer sets that paralleled those imprinted on the nylon membrane used for gene expression profiling above. Table 4 contains representative genes from the profiler array used for this study. As can be seen, there is an overwhelming concordance with the data presented in Tables 1 (constitutively expressed genes) and 3 (differentially expressed genes).

Table 1. Genes Constitutively Expressed during GDs 12–14 of Orofacial Development
Gene symbolUnigeneGenBank no.Fold change in expression
d13_vs_d12d14_vs_d13
  1. Relative expression levels of different genes were estimated by comparing their signal intensity with that of the internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Genes were considered to be constitutively expressed if the change in their relative expression levels between two gestational days was less than 1.5-fold.

Atf2Mm.209903NM_0097151.4760.849
Ccna2Mm.4189NM_0098281.1250.828
Ccnb1Mm.260114NM_1723011.0031.010
Ccnd1Mm.273049NM_0076311.2110.889
Ccnd2Mm.333406NM_0098291.3690.743
Ccnd3Mm.246520NM_0076321.2010.944
Ccne1Mm.16110NM_0076330.9791.015
Cdc42Mm.1022NM_0098611.2240.712
Cdk4Mm.6839NM_0098701.0241.002
Cdk6Mm.278464NM_0098731.2850.914
Cdkn1aMm.195663NM_0076691.1570.922
Cdkn1cMm.168789NM_0098760.9641.020
Cdkn2aMm.4733NM_0098771.0720.912
Cdkn2bMm.269426NM_0076700.9901.017
Cdkn2cMm.1912NM_0076711.1740.898
CrebbpMm.132238XM_1486991.2750.867
Dlk1Mm.157069NM_0100520.9871.034
E2f1Mm.18036NM_0078911.0990.870
Elk4Mm.254233NM_0079231.1690.967
Enpp2Mm.250256NM_0157440.9391.108
Kcnn1Mm.32074NM_0323971.1820.944
Map2k3Mm.18494NM_0089281.0081.018
Map3k1Mm.15918NM_0119451.3800.933
Map3k14Mm.158981NM_0168961.2980.793
Map3k3Mm.27041NM_0119471.0800.904
Map3k7Mm.258589NM_1726881.4550.887
Mapk13Mm.27970NM_0119501.2640.909
Mapk8Mm.21495NM_0167001.1470.811
Mapk8ipMm.2720NM_0111621.2750.786
Mapk8ip3Mm.43081NM_0139311.0340.845
Mapkapk2Mm.221235NM_0085511.0510.884
Mknk1Mm.209327NM_0214611.0061.006
SfnMm.44482NM_0187541.3980.737
MosMm.317339NM_0200211.3560.780
Nfatc4Mm.27908NM_0236991.3571.076
NrasMm.256975NM_0109371.0080.986
Rac1Mm.292510XM_3575221.0230.938
Rac2Mm.1972NM_0090081.3630.849
Stk4Mm.234472NM_0214200.9950.908
Control genes
GapdMm.333399NM_0080840.9940.999
GapdMm.333399NM_0080841.0061.001
PpiaMm.5246NM_0089071.1050.993
PpiaMm.5246NM_0089071.0381.003
PpiaMm.5246NM_0089071.0250.976
PpiaMm.5246NM_0089071.0060.990
Rpl13aMm.180458NM_0094380.9960.972
Rpl13aMm.180458NM_0094381.0130.958
ActbMm.297NM_0073931.0041.003
ActbMm.297NM_0073930.9891.015
Table 2. Genes “Not Expressed” during GDs 12–14 of Orofacial Development*
Gene symbolUnigeneGenbank no.
  • *

    Genes were considered to be “not expressed” if their expression level during the developmental time period was <20% of that of the control (GAPDH) gene.

Ccna1Mm.4815NM_007628
Cdk2Mm.111326NM_016756
Cdkn2dMm.29020NM_009878
JunMm.275071NM_010591
Map3k11Mm.185026NM_022012
Map4k2Mm.25860NM_009006
Mapk10Mm.39253NM_009158
Al874665Mm.222612NM_178907
MycMm.2444NM_010849
Trp53Mm.222NM_011640
Table 3. Genes “Not Expressed” during GD's 12–14 of Orofacial Development*
Gene symbolUnigeneGenBank no.Fold change in expression
d13 vs d12d14 vs d13
  • *

    Genes were considered to be “not expressed” if their expression level during the developmental time period was <20% of that of the control (GAPDH) gene.

ArafMm.220946NM_0097031.7520.713
Ccnb2Mm.22592NM_0076301.5260.639
Ccne2Mm.35867NM_0098301.6070.688
Cdkn1bMm.2958NM_0098751.6650.690
ChukMm.3996NM_0077001.7690.629
Col1a1Mm.277735NM_0077422.3400.809
Creb1Mm.1376NM_1338282.3890.324
EgfrMm.8534NM_0079122.3600.408
Egr1Mm.181959NM_0079131.7570.836
Elk1Mm.347420NM_0079221.9030.771
Ets1Mm.292415NM_0118081.8380.370
Ets2Mm.290207NM_0118092.7160.251
FosMm.246513NM_0102341.8330.667
Grb2Mm.6900NM_0081631.6190.728
Hras1Mm.334313NM_0082841.8160.858
Hspb1Mm.13849NM_0135602.0080.737
Hspa5Mm.330160NM_0223102.7850.670
Kras2Mm.207540NM_0212841.8080.412
KsrMm.4745NM_0135711.7040.595
Smad4Mm.100399NM_0085402.1300.474
Map2k1Mm.248907NM_0089271.6850.822
Map2k1ip1Mm.331392NM_0199201.9340.754
Map2k2Mm.275436NM_0231382.0870.777
Map2k4Mm.27491NM_0091571.7440.715
Map2k5Mm.192651NM_0118401.8910.522
Map2k6Mm.14487NM_0119431.5960.645
Map2k7Mm.3906NM_0119442.9540.392
Map3k2Mm.211762NM_0119462.1460.599
Map3k4Mm.28587NM_0119482.0660.498
Map3k5Mm.6595NM_0085802.3990.386
Map4k1Mm.148278NM_0082791.5590.879
Mapk1Mm.196581NM_0119492.2100.625
Mapk11Mm.91969NM_0111611.6270.807
Mapk12Mm.38343NM_0138711.6660.791
Mapk14Mm.246203NM_0119511.6190.675
Mapk3Mm.8385NM_0119521.7540.587
Mapk6Mm.18856NM_0158061.4770.653
Mapk7Mm.38172NM_0118412.0350.682
Mapk8ip2Mm.173337NM_0219211.5330.707
Mapk9Mm.68933NM_0169611.5530.663
Mapkapk5Mm.272206NM_0107652.2710.740
MaxMm.268548NM_0085582.7160.363
Mef2cMm.24001NM_0252822.2230.712
Pak1Mm.260227NM_0110351.5370.841
Pla2g4aMm.4186NM_0088691.9701.160
Raf1Mm.184163NM_0297801.5690.747
Rb1Mm.273862NM_0090291.9950.557
Table 4. Real-Time PCR Verification of Genes Expressed Constitutively (A) and Differentially (B) during GDs 12–14of Orofacial Development Using the RT2 Profiler System
Gene symbolUnigeneGenBank no.Concordance in gene expression
GD 13_vs_GD12GD 14_vs_GD12
A:
Atf2Mm.209903NM_009715++
Ccnd2Mm.333406NM_009829++
Cdc42Mm.1022NM_009861++
Cdnk2cMm.1912NM_007671++
CrebbpMm.132238XM_148699++
E2f1Mm.18036NM_007891++
Map3k1Mm.15918NM_011945++
Mapk13Mm.27970NM_011950++
SfnMm.44482NM_018754
Nfatc4Mm.27908NM_023699++
Rac2Mm.1972NM_009008++
B:
ArafMm.220946NM_009703++
Col1a1Mm.277735NM_007742++
Hspb1Mm.13849NM_013560++
Kras2Mm.207540NM_021284++
Smad4Mm.100399NM_008540++
Map2k2Mm.275436NM_023138++
Map3k5Mm.6595NM_008580++
MaxMm.268548NM_008558++
Pak1Mm.260227NM_011035++
Rb1Mm.273862NM_009029++

The ERK pathway is active in embryonic orofacial tissue during its growth and differentiation. Mapk1 (ERK2), Mapk3 (ERK1), MEK1, MEK2, Araf, and Raf1 as well as the Raf regulating proteins, Hras1, Kras2, and Ksr, displayed a transient increase in expression during GDs 12–14 (Table 3). The activated transcription factors Creb1, Egfr, Elk1, Ets1, Ets2, Madh4, Mapkapk5, Max, and Mef2c also displayed a transient increase in expression during GDs 12–14 (Table 3), as did several genes known to be induced by MAPK pathways: Col1α1, Egr1, and Hspa5 (Grp78). Expression of several genes encoding cell cycle proteins regulated by the Erk1/2 pathway showed constitutive levels of expression (Table 1). Cyclin B2, Cyclin E2, and p27kip1, however, displayed increasing levels of expression during GDs 12–14 of orofacial development.

MAPKs Mapk8 (JNK1) and Mapk9 (JNK2), involved in the SAPK/JNK cascade, were constitutively expressed (Table 1), while expression of MKK Map2k4 (MKK4, JNKK1) and Map2k7 (JNKK2) increased transiently between GD 12 and 14 (Table 3). Expression of Map3k1 (MEKK1) and MAPK8 interacting proteins Mapk8ip (JIP-2), Mapk8ip2 (JIP-1), and Mapk8ip3 (JIP-3) was constitutive (Table 1), while Map3k11 (MLK3) was not expressed.

MAPKs p38, p38β, and p38γ showed modestly increased expression from GD 12 to GD 13, and expression remained elevated until GD 14. Genes known to play a role in p38 MAPK pathways, such as Rac, Cdc42, Mapkap-2, and Mnk-1, exhibited constitutive expression during orofacial development (Table 1).

Activated ERK, SAPK/JNK, and p38 MAPK Protein Analysis

Embryonic orofacial tissue was dissected on GDs 12, 13, and 14, and protein (total cell lysates) was extracted and analyzed for the presence of MAPK proteins by Western blotting. Figures 1 and 2 demonstrate Western blot analysis of active and total ERK, p38, and SAPK/JNK in embryonic orofacial tissue on GDs 12, 13, and 14. Western blot images were quantified by densitometric scanning using the NIH Image (version 1.62) software. MAPKs p42/44 (ERK2/1), p38, and SAPK/JNK were expressed in embryonic orofacial tissue on each of GDs 12, 13, and 14 (Figs. 1 and 2).

Figure 1.

Immunoblot analysis of p42/44 (ERK1/2) in embryonic orofacial tissue on GDs 12, 13, and 14. A total of 60 μg of total protein lysate per lane was separated by SDS-PAGE electrophoresis on 8–16% polyacrylamide Tris-Glycine gels. Western blot analysis was carried out using antibodies specific for phospho-p42/44 (upper panel) and total p42/44 (middle panel). Note that ERKs are temporally regulated with higher activity on GD 13 and 14 as compared to GD 12 (upper panel). Lower panel shows the Western blot of the loading control β-actin.

Figure 2.

Immunoblot analysis of total and active p38 MAPK and SAPK/JNK in embryonic orofacial tissue on GDs 12, 13, and 14. A total of 60 μg of total protein lysate was separated by SDS-PAGE electrophoresis on 8–16% Tris-Glycine gels. (A) Active, phosphorylated (upper panel) and total (lower panel) SAPK/JNK were analyzed by immunoblotting with specific antibodies. (B) Active, phosphorylated (upper panel) and total (lower panel) p38 MAPK were analyzed by immunoblotting with specific antibodies. Minimal amounts of active, phosphorylated p38 were detected in embryonic orofacial tissue during GDs 12–14.

More revealing was determination of the active MAPK modules during development of embryonic orofacial tissue. Phospho-specific p44/p42 MAPK rabbit polyclonal antibody cross-reacts with murine ERKs 1 and 2 phosphorylated at tyrosine 202 and 204, and does not cross-react with either tyr-phosphorylated SAPK/JNK or p38. Thus, this antibody allows determination of the presence of active ERK. To confirm equal loading of proteins, the blots were stripped and reprobed for β-actin, a housekeeping protein. As shown in Figure 1 (lower panel), the abundance of β-actin in the three lanes of the blot was similar. Total and phospho p42/44 bands obtained using the Western blot method were also densitometrically analyzed. It was observed that phospho p42 expression increased by ∼2.5-fold on both GDs 13 and 14, compared to GD 12. In the case of phospho-p44, the expression increased by ∼6.5-fold on GDs 13 and 14, compared to GD 12. Further, the expression of total p42 increased ∼1.4- and 2.3-fold on GDs 13 and 14, respectively. The increase in the case of total p44 was 1.1- and 2.0-fold on GDs 13 and 14, respectively. ERK activity thus appears to be temporally regulated with greater ERK activity on GDs 13 and 14 as compared to GD 12 (Fig. 1). It can thus be concluded that expressions of total and phospho p42/44 increase in the secondary palate of the developing embryo between GDs 12 and 14. Phospho-specific p38 MAPK antibody detects p38 MAPK only when activated by phosphorylation. Phospho-specific SAPK/JNK antibody detects only active (phosphorylated) SAPK/JNK. Minimal amounts of active p38 and active SAPK/JNK were detected in embryonic orofacial tissue during GDs 12–14 (Fig. 2).

DISCUSSION

Development of the embryonic orofacial region involves multiple signaling pathways regulating the sequential expression and interaction of numerous molecular effectors during embryogenesis. The present study analyzed the expression of members of the MAPK family of enzymes in developing embryonic orofacial tissue during GDs 12–14. The MAPKs are represented as five distinctive MAPK cascades: ERK1/2, SAPK/JNK, p38/SAPK2/3, BMK1/ERK5, and ERK3. All are expressed in developing embryonic orofacial tissue during GDs 12–14 and several are differentially expressed. Expression of genes involved in the ERK1/2 cascade, such as Mapk1 (ERK2), Mapk3 (ERK1), Map2k1 (MEK1), Map2k2 (MEK2), Araf, Raf1, and Raf regulating proteins (Hras1, Kras2, and Ksr) increased from GD 12 to GD 13, and then decreased during later stages of orofacial development, that is, from GD 13 to GD 14. This change corresponds to the rapid growth of the secondary palate during this critical period of orofacial development. The epidermal growth factor receptor (EGFR) gene exhibited a significant increase in expression from GD 12 to 13, suggesting enhanced EGFP-mediated signaling during this period. The EGFR is known to be involved in embryonic orofacial development (Kronmiller et al., 1991). During secondary palate development in the hamster, a correlation between MAPK activity and cell proliferation was observed, suggesting involvement of MAPK in regulation of embryonic palatal cell proliferation (Young et al., 1997). EGF signaling is known to be mediated via the ERK1/2 pathway. Our results demonstrating increased expression of a series of transcriptional activators known to promote growth and cell proliferation (creb1, elk1, Ets1/2, madh4, max, and mef2c) are also consistent with enhanced proliferation during GDs 12–14.

Fusion of the MEE cells of the developing secondary palate is preceded by a cessation of MEE DNA synthesis prior to the initial contact of opposing palatal shelves (Ferguson, 1988; Greene, 1989). Exogenous EGF has been shown to inhibit the cessation of MEE DNA synthesis and induce cleft palate (Abbott et al., 1998; Hassell, 1975; Tyler and Pratt, 1980). Moreover, it has been shown that EGF-induced inhibition of palatal MEE fusion is dependent on nuclear ERK1/2 activation and that this mechanism must be tightly regulated during normal palatal fusion (Tyler and Pratt, 1980). Differential expansion of ERK1/2 kinases in developing orofacial tissue is consistent with this notion. ERK1/2 can be activated by various means, all of which result in the activation of various transcriptional factors and ser/thr kinases that contribute to cellular proliferation, differentiation, cell cycle regulation, and cell survival (Chen et al., 1992; Gonzalez et al., 1993; Lenormand et al., 1993). It is evident that MAPK cascades are involved in growth factor–regulated cell proliferation during morphogenesis of quail secondary palate, where EGF-stimulated proliferation of primary cell cultures was accompanied by proliferation and activation of 42-kDa MAPK (Hehn et al., 1998; Yamamoto et al., 2003).

Murine embryonic maxillary mesenchymal (MEMM) cells are known to be responsive to activation of the cyclic AMP (cAMP) and the TGFβ signal transduction pathways (Gehris et al., 1994). These may modulate the proliferative response of MEMM cells to EGF (Weston et al., 1998), which stimulates MAPK phosphorylation and activity in these cells (Bhattacherjee et al., 2004). Thus, it is reasonable to surmise that a convergence between the TGFβ, cAMP, and/or EGF signaling pathways exists in embryonic orofacial tissue. Moreover, modulation of cellular signaling brought about by such cross-talk is likely to be mediated by changing patterns of expression or activation of ERK1/2 family members.

In contrast to the ERK1/2 cascade, genes involved in the SAPK/JNK cascade, such as MAPKs Mapk8 (JNK1), Mapk9 (JNK2), and Mapk8 interacting proteins Mapk8ip (JIP-2), Mapk8ip2 (JIP-1), Mapk8ip3 (JIP-3) were constitutively expressed during GDs 12–14. Western blot analysis demonstrated that while SAPK/JNK is present in embryonic orofacial tissue on each of GDs 12, 13, and 14, it is not phosphorylated, suggesting that SAPK/JNK is not active in orofacial tissues during this developmental period.

MAPKs p38, p38β, and p38γ also exhibited differential expression from GD 12 to GD 13. Genes known to play a role in p38 MAPK pathways such as Rac, Cdc42, Mapkap-2, and Mnk1 also were constitutively expressed during orofacial development. Western blot analyses demonstrated that p38 kinases are present in embryonic orofacial tissue on each of GDs 12, 13, and 14. Minimal amounts of active p38 were detected, however, suggesting that, like SAPK/JNK, p38 is also not active in the tissue during this developmental period.

The p38 pathway controls and enhances the activity of many transcription factors, including ATF1/2, CREB, ELK-1, Ets-1, MEF2A, MEF2C, and SAP-1 (Delghandi et al., 2005; Goedert et al., 1997; Tanaka et al., 1998; Yang et al., 1999). ATF-2 phosphorylation, for example, is necessary for dimerization with Jun (phosphorylated by JNK) to form activating protein-1 (AP-1), which regulates the expression of many genes (Jochum et al., 2001). Phosphorylation of MEF2C leads to increased c-jun transcription, providing additional linkage with p38 and JNK pathways.

Although much less is known about the ERK3 and ERK5 signaling pathways, they also may be involved in orofacial development. ERK3, constitutively expressed in orofacial tissue during GDs 12–14 (Turgeon et al., 2000), has been linked to MAPK-activated protein kinase-5 (MK5), identifying MK5 as a downstream target of ERK3 (Schumacher et al., 2004). Expression of ERK5 and MEK5, the upstream kinase of ERK5, was transiently increased from GD 12 to GD 14, suggesting their involvement in orofacial growth and/or differentiation. ERK5 is known to be essential for early embryonic development and is required for normal development of the vascular system and cell survival (Liu et al., 2003). ERK5 knockout mice die at ∼GD 10.5 (Yan et al., 2003). In situ hybridization of ERK5 revealed strong expression in the head and trunk of the embryo during this stage of development.

In summary, our results show that constituents of five MAPK pathways are expressed in developing orofacial tissue. Further, active (phosphorylated) forms of ERK1/2 were also detected in this embryonic tissue during the critical stages of its growth and morphogenesis, suggesting an important role for members of this MAPK family during orofacial development. The exact relationship between ERK1/2 and development of the embryonic orofacial region requires further investigation.

Acknowledgements

This work was supported in part by NIH from the COBRE Program of the National Center for Research Resources.