Dennis R. Warner

Identification of three novel Smad binding proteins involved in cell polarity

A yeast two‐hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue. Three proteins (Erbin, Par‐3, and Dishevelled) were identified that share several similar structural and functional characteristics. Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity. In GST (glutathione S‐transferase) pull‐down assays, Erbin, Par‐3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full‐length Smad 3 protein. Failure of Erbin, Par‐3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain confirms that the binding site resides within the MH2 domain. Erbin, Par‐3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding specificity. Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower affinity. While transforming growth factor β (TGFβ) has been suggested to alter cell polarity through a Smad‐independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFβ could alter cell polarity via a Smad‐dependent signaling mechanism.

Cross‐talk between the TGFβ and Wnt signaling pathways in murine embryonic maxillary mesenchymal cells

The transforming growth factor beta (TGFβ) and Wnt signaling pathways play central roles regulating embryogenesis and maintaining adult tissue homeostasis. TGFβ mediates its cellular effects through types I and II cell surface receptors coupled to the nucleocytoplasmic Smad proteins. Wnt signals via binding to a cell surface receptor, Frizzled, which in turn activates intracellular Dishevelled, ultimately leading to stabilization and nuclear translocation of β‐catenin. Previous studies have demonstrated several points of cross‐talk between the TGFβ and Wnt signaling pathways. In yeast two‐hybrid and GST‐pull down assays, Dishevelled‐1 and Smad 3 have been shown to physically interact through the C‐terminal one‐half of Dishevelled‐1 and the MH2 domain of Smad 3. The current study demonstrates that co‐treatment of murine embryonic maxillary mesenchyme (MEMM) cells with Wnt‐3a and TGFβ leads to enhanced reporter activity from TOPflash, a Wnt‐responsive reporter plasmid. Transcriptional cooperation between TGFβ and Wnt did not require the presence of a Smad binding element, nor did it occur when a TGFβ‐responsive reporter plasmid (p3TP‐lux) was transfected. Overexpression of Smad 3 further enhanced the cooperation between Wnt and TGFβ while overexpression of dominant‐negative Smads 2 and 3 inhibited this effect. Co‐stimulation with TGFβ led to greater nuclear translocation of β‐catenin, providing explanation for the effect of TGFβ on Wnt‐3a reporter activity. Wnt‐3a exerted antiproliferative activity in MEMM cells, similar to that exerted by TGFβ. In addition, Wnt‐3a and TGFβ in combination led to synergistic decreases in MEMM cell proliferation. These data demonstrate a functional interaction between the TGFβ and Wnt signaling pathways and suggest that Wnt activation of the canonical pathway is an important mediator of MEMM cell growth.

Intracellular dynamics of Smad-mediated TGFβ signaling

The transforming growth factor‐β (TGFβ) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGFβ on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFβ action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGFβ. In the current study, elements of the Smad component of the TGFβ intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFβ1, TGFβ2, and TGFβ3 was demonstrated. TGFβ‐initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGFβ‐inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad‐mediated TGFβ signaling system in cells of the developing murine palate. J. Cell. Physiol. 197: 261–271, 2003.

Expression of Wnts in the developing murine secondary palate.

Morphogenesis of the mammalian secondary palate requires coordination of cell migration, proliferation, differentiation, apoptosis and synthesis of extracellular matrix molecules by numerous signal transduction pathways. Recent evidence suggests a role for members of the Wnt family of secreted cytokines in orofacial development. However, no study has systematically or comprehensively examined the expression of Wnts in embryonic orofacial tissue. We thus conducted a survey of the expression of all known Wnt genes in the developing murine secondary palate. Using an RT-PCR strategy to assay gene expression, 12 of the 19 known members of the Wnt family were found to be expressed in embryonic palatal tissue during key phases of its development. The expression of 5 Wnt family members was found to be temporally regulated. Moreover, these Wnts had unique spatio-temporal patterns of expression which suggested possible roles in palatal ontogeny.

Nuclear convergence of the TGFβ and cAMP signal transduction pathways in murine embryonic palate mesenchymal cells

Transforming growth factors beta (TGFbeta) and cyclic AMP (cAMP) both participate in growth and differentiation of the developing mammalian secondary palate and elicit similar biological responses. Cross-talk between these two signal transduction pathways in cells derived from the embryonic palate has been demonstrated previously. In the present study, we have examined nuclear convergence of these signalling pathways at the level of transcriptional complex formation. Biotinylated oligonucleotides encoding a consensus Smad binding element (SBE), or a cyclic AMP response element (CRE), were mixed with cell extracts from murine embryonic palate mesenchymal (MEPM) cells that were treated with either TGFbeta or forskolin. Protein-oligonucleotide complexes were precipitated with streptavidin-agarose, and analysed by Western blotting to identify proteins in the complex bound to each consensus oligonucleotide. TGFbeta treatment of MEPM cells increased the levels of phosphorylated Smad2, phosphorylated cAMP response element binding protein (CREB), and the coactivator, CREB binding protein (CBP), that were part of a complex bound to the SBE. Treatment of cells with forskolin, a stimulator of adenylate cyclase, increased the amount of phosphorylated CREB and CBP, but not the amount of phosphorylated Smad2 bound in a complex to the SBE. Additionally, the presence of the co-repressors, c-Ski and SnoN, was demonstrated as part of a complex bound to the SBE (but not the CRE). Amounts of c-Ski and SnoN found in the SBE-containing complex increased in response to either TGFbeta or forskolin. These results demonstrate that phosphorylated CREB forms a complex with the co-activator CBP, phosphorylated Smad2 and the co-repressors c-Ski and SnoN on a consensus SBE. This suggests cooperative regulation of genes with SBE-containing promoters by the cAMP and TGFbeta signalling pathways in the developing palate.

Mesenchymal cell remodeling during mouse secondary palate reorientation

The formation of mammalian secondary palate requires a series of developmental events such as growth, elevation, and fusion. Despite recent advances in the field of palate development, the process of palate elevation remains poorly understood. The current consensus on palate elevation is that the distal end of the vertical palatal shelf corresponds to the medial edge of the elevated horizontal palatal shelf. We provide evidence suggesting that the prospective medial edge of the vertical palate is located toward the interior side (the side adjacent to the tongue), instead of the distal end, of the vertical palatal shelf and that the horizontal palatal axis is generated through palatal outgrowth from the side of the vertical palatal shelf rather than rotating the pre‐existing vertical axis orthogonally. Because palate elevation represents a classic example of embryonic tissue re‐orientation, our findings here may also shed light on the process of tissue re‐orientation in general. Developmental Dynamics 239:2110–2117, 2010.

MicroRNA expression profiling of the developing murine upper lip

Clefts of the lip and palate are thought to be caused by genetic and environmental insults but the role of epigenetic mechanisms underlying this common birth defect are unknown. We analyzed the expression of over 600 microRNAs in the murine medial nasal and maxillary processes isolated on GD10.0–GD11.5 to identify those expressed during development of the upper lip and analyzed spatial expression of a subset. A total of 142 microRNAs were differentially expressed across gestation days 10.0–11.5 in the medial nasal processes, and 66 in the maxillary processes of the first branchial arch with 45 common to both. Of the microRNAs exhibiting the largest percent increase in both facial processes were five members of the Let‐7 family. Among those with the greatest decrease in expression from GD10.0 to GD11.5 were members of the microRNA‐302/367 family that have been implicated in cellular reprogramming. The distribution of expression of microRNA‐199a‐3p and Let‐7i was determined by in situ hybridization and revealed widespread expression in both medial nasal and maxillary facial process, while that for microRNA‐203 was much more limited. MicroRNAs are dynamically expressed in the tissues that form the upper lip and several were identified that target mRNAs known to be important for its development, including those that regulate the two main isoforms of p63 (microRNA‐203 and microRNA‐302/367 family). Integration of these data with corresponding proteomic datasets will lead to a greater appreciation of epigenetic regulation of lip development and provide a better understanding of potential causes of cleft lip.

BMP signaling dynamics in embryonic orofacial tissue

The bone morphogenetic protein (BMP) family represents a class of signaling molecules, that plays key roles in morphogenesis, cell proliferation, survival and differentiation during normal development. Members of this family are essential for the development of the mammalian orofacial region where they regulate cell proliferation, extracellular matrix synthesis, and cellular differentiation. Perturbation of any of these processes results in orofacial clefting. Embryonic orofacial tissue expresses BMP mRNAs, their cognate proteins, and BMP‐specific receptors in unique temporo‐spatial patterns, suggesting functional roles in orofacial development. However, specific genes that function as downstream mediators of BMP action during orofacial ontogenesis have not been well defined. In the current study, elements of the Smad component of the BMP intracellular signaling system were identified and characterized in embryonic orofacial tissue and functional activation of the Smad pathway by BMP2 and BMP4 was demonstrated. BMP2 and BMP4‐initiated Smad signaling in cells derived from embryonic orofacial tissue was found to result in: (1) phosphorylation of Smads 1 and 5; (2) nuclear translocation of Smads 1, 4, and 5; (3) binding of Smads 1, 4, and 5 to a consensus Smad binding element (SBE)‐containing oligonucleotide; (4) transactivation of transfected reporter constructs, containing BMP‐inducible Smad response elements; and (5) increased expression at transcriptional as well as translational levels of Id3 (endogenous gene containing BMP receptor‐specific Smad response elements). Collectively, these data document the existence of a functional Smad‐mediated BMP signaling system in cells of the developing murine orofacial region. J. Cell. Physiol. 216: 771–779, 2008,

EXPRESSION OF THE NUCLEAR COACTIVATORS CBP AND p300 IN DEVELOPING CRANIOFACIAL TISSUE

SummarycAMP regulatory element-binding protein (CREB)-binding protein (CBP) and its functional homolog, the adenovirus E1A-associated 300-kDa protein (p300) are nuclear coactivators and histone acetyltransferases that integrate signals from disparate pathways by bridging specific transcription factors to the basal transcription apparatus. Their role in patterning and development was suggested by studies in mice in which CBP and p300 expression was disrupted and by the human Rubinstein-Taybi syndrome, which is associated with mutations of CBP. The cAMP signal transduction pathway plays a critical role during development of the palate. The linkage between cAMP and expression of specific genes is mediated via activation of trans-acting deoxyribonucleic acid-binding proteins such as the nuclear CREB. For genes regulated by CBP- or p300-containing transcriptional complexes, rates of transcription will depend in part on cellular levels and distribution of CBP/p300. We have thus determined the temporal and spatial expression of CBP and p300 in murine embryonic palatal tissue. Both CBP and p300 proteins and messenger ribonucleic acids are expressed in palatal tissue on each d of palate development (days 12–14 of gestation), as measured by Western blotting and reverse-transcription polymerase chain reaction. Expression of both CBP and p300 was greatest on day 12 of gestation, suggesting that these transcriptional coactivators are developmentally regulated. Immunohistochemical analysis of CBP and p300 expression in the murine embryonic craniofacial region revealed a ubiquitous distribution for both proteins. These studies lay the groundwork for further investigations into the role of CBP and p300 in cellular signaling during craniofacial development.

PRDM16 expression in the developing mouse embryo.

PRDM16 is a member of the PR domain-containing protein family and is associated with various disease states including myelodysplastic syndrome and adult T-cell leukemia, as well as developmental abnormalities such as cleft palate. It is also known to act as a regulator of cell differentiation. Expression analysis of PRDM16 is limited, especially within the developing embryo. The current study evaluated the temporal and spatial localization of PRDM16 during early mouse development (embryonic days 8.5-14.5). PRDM16 was first detected on E9.5 in a limited number of tissues and by E14.5, was expressed in a broad range of developing tissues including those of the brain, lung, kidney, and gastrointestinal tract. The expression pattern is consistent with a role for PRDM16 in the development of multiple tissues. Collectively, these studies are the first to characterize the expression of the PRDM16 gene during early murine development.