We have identified a novel gene transcript of approximately 1.1 kilobases in length that is expressed in the presumptive nasal epithelium of the mouse embryo. In situ hybridization analysis shows discrete regions of expression associated with the palate, nasal septum, and nasal conchae. This transcript is also expressed strongly in the trachea and bronchi of the adult lung. Screening of a mouse heart cDNA library yielded several overlapping clones to give a continuous sequence of 1113 bases, containing an open reading frame of 278 codons comprising the complete mRNA. No significant homologies with known genes were observed at the nucleotide level; limited amino acid homology with two salivary gland-specific proteins was noted. A search for functionally significant protein motifs revealed consensus sequences for N-glycosylation, protein kinase C and casein kinase phosphorylation, and a leucine zipper. Additionally, we observed a unique amino acid sequence pattern, consisting of the residues Gly-(Leu/Pro/ Gln)-(Pro/Leu)-Leu-Pro-Leu, repeated four times near the amino-terminal portion of the protein with two amino acid residues separating the repeats. Based on these observations, we propose that we have identified a new gene, which we call plunc (for palate, lung, and nasal epithelium clone; GenBank™ accession number U69172).

The transforming growth factor-beta (TGFbeta) 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 TGFbeta on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFbeta 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 TGFbeta. In the current study, elements of the Smad component of the TGFbeta intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFbeta1, TGFbeta2, and TGFbeta3 was demonstrated. TGFbeta-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 TGFbeta-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 TGFbeta signaling system in cells of the developing murine palate.

Evidence is presented for the biochemical diagnosis of the first case of feline mannosidosis. A marked deficiency of acidic alpha-D-mannosidase in the brain, kidney and liver and excessive excretion of mannose-rich oligosaccharides in the urine were found in a kitten suffering from a nervous disorder. Residual acidic alpha-D-mannosidase, ranging from 2 to 5.5% of the normal activity, was observed in the tissues of the affected kitten. It has similar kinetic and physicochemical properties to the normal activity. The amount of mannose in the urine of the affected kitten was 19-fold greater than in a comparable control, and the molar ratio of mannose to N-acetylglucosamine was approx. 6 : 1. High concentrations of neutral oligosaccharides were detected in the urine. The predominant oligosaccharide appeared to be a hexasaccharide. The biochemical features of bovine, feline and human mannosidosis are compared, and it is concluded that feline mannosidosis may be a useful animal model for studying the human disease.

A series of antisense oligonucleotides were developed to inhibit specifically expression of a mutated exogenous gene for collagen without inhibiting expression of an endogenous gene for the same protein. The test system consisted of mouse NIH 3T3 cells that were stably transfected with an internally deleted construct of the human gene for the pro alpha 1(I) chain of type I procollagen [Olsen et al. (1991) J. Biol. Chem. 266, 1117]. The target site was a region at the 3' end of exon 1 and the first few nucleotides of intron 1 of the exogenous human gene that differed in sequence by nine nucleotides from the sequence of the endogenous mouse gene. Expression of the two genes was assayed by Western blot with cross-reacting antibodies and by steady-state levels of mRNAs. None of the oligonucleotides were effective in concentrations up to 25 microM when administered without any carrier. However, when administered with 5 or 10 micrograms/mL lipofectin, one of the oligonucleotides in concentrations of 0.1-0.2 microM inhibited expression of the exogenous gene from 50% to 80% without significant inhibition of expression of the endogenous gene. Also, a missense version of the same oligonucleotide had no significant effect, and the inhibition observed with the most effective oligonucleotide was abolished by a single base change. Time course experiments indicated that, after a 4-h treatment, inhibition appeared at 8 h and persisted for at least 22 h.(ABSTRACT TRUNCATED AT 250 WORDS)

The TGF beta family of genes has been shown to play an important role in regulating various aspects of development, although the mechanisms by which TGF beta exerts its effects have not yet been clarified. Growth and differentiation of both murine embryonic palate mesenchymal (MEPM) cells and palatal epithelium can be regulated by the TGF beta s. We therefore examined the expression of mRNAs encoding TGF beta 1, TGF beta 2, and TGF beta 3 in developing embryonic palatal tissue as well as factors that modulate their levels of expression. Northern blot analysis of RNA isolated from murine embryonic palatal tissue on gestational days (GD) 12, 13, and 14 demonstrated the presence of one mRNA transcript for TGF beta 1 (2.5 kb), two transcripts for TGF beta 2 (4.4 kb, 6.0 kb), and one transcript for TGF beta 3 (3.5 kb). Although steady-state levels of TGF beta 1 mRNA showed no changes during development of the palate, TGF beta 2 mRNA levels were maximal on both GD13 and GD14 and TGF beta 3 mRNA levels transiently increased on GD 13. In addition, levels of TGF beta 3 mRNA seemed much higher than either TGF beta 1 or TGF beta 2. both TGF beta 1 and TGF beta 2 were able to increase, in a dose-related manner, the expression of TGF beta 3 mRNA in murine embryonic palate mesenchymal cells in vitro. In contrast, epidermal growth factor (EGF) down-regulated the expression of TGF beta 3 mRNA even in the presence of TGF beta 1 or TGF beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of retinoic acid (RA) on TGF-beta mRNA expression and protein production in murine embryonic palate mesenchymal (MEPM) cells was examined by Northern blotting and TGF-beta bioassay in association with TGF-beta isoform-specific neutralizing antibodies. Heat or acid activation was used to distinguish between latent and active TGF-beta protein released into the culture medium. RA had little or no effect on TGF-beta1 mRNA expression and protein production. In contrast, RA increased TGF-beta2 and beta3 protein released into the culture medium, the protein being mostly in an inactive or latent form. The amount of active TGF-beta released was increased relative to the total increase in TGF-beta released, suggesting that RA treatment stimulated activation of latent TGF-beta. RA also increased TGF-beta2 mRNA expression; we have previously shown that RA upregulates TGF-beta3 mRNA in these cells. RA and TGF-beta individually inhibited 3H-thymidine incorporation into MEPM cell DNA, while, when administered simultaneously, they inhibited proliferative activity to a greater extent. Heat- or acid-activated conditioned medium (CM) from MEPM cells treated with RA was able to inhibit 3H-thymidine incorporation into MEPM cell DNA to an extent greater than seen with RA treatment alone. Coincubation of heat-activated CM from RA-treated MEPM cells with pan-specific or TGF-beta2 or beta3-specific neutralizing antibodies partially relieved the inhibitory effect on 3H-thymidine incorporation, suggesting that this proliferative response was due to RA-induced TGF-beta. Simultaneous treatment with RA and TGF-beta also stimulated gycosaminoglycan (GAG) synthesis to an extent greater than that seen with TGF-beta treatment alone, this despite the ability of RA to inhibit GAG synthesis. These data demonstrate a role for RA and RA-induced TGF-beta in the regulation of palate cell proliferation and GAG synthesis and suggest a role for TGF-beta in retinoid-induced cleft palate.

The palatal cleft seen in Msx-1 knock-out mice suggests a role for this gene in normal palate development. The cleft is presumed secondary to tooth and jaw malformations, since in situ hybridization suggests that Msx-1 mRNA is not highly expressed in developing palatal tissue. In this study we demonstrate, by Northern blot analysis, the expression of Msx-1, but not Msx-2, in the developing palate and in primary cultures of murine embryonic palate mesenchymal cells. Furthermore, we propose a role for Msx-1 in retinoic acid-induced cleft palate, since retinoic acid inhibits Msx-1 mRNA expression in palate mesenchymal cells. We also demonstrate that transforming growth factor beta inhibits Msx-1 mRNA expression in palate mesenchymal cells, with retinoic acid and transforming growth factor beta acting synergistically when added simultaneously to these cells. These data suggest a mechanistic interaction between retinoic acid, transforming growth factor beta, and Msx-1 in the etiology of retinoic acid-induced cleft palate.

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