Cloning and expression of a mouse member of the PLUNC protein family exclusively expressed in tongue epithelium.

Palate, lung, and nasal epithelium clone (Plunc, now renamed Splunc1) is a small secreted protein expressed in the oropharynx and upper airways of humans, mice, rats, and cows. This protein is structurally homologous to known mediators of host defense against gram-negative bacteria. We have characterized the genomic sequence and expression of a novel but closely related gene from rodents, which we call splunc5. Mouse Splunc5 sequence is 60% identical to mouse Splunc1. The genes also share highly conserved genomic elements including intron-exon structure and intronic sequence. Strikingly, splunc5 is expressed exclusively in the interpapillary epithelium of the tongue's dorsal surface. By comparing the expression profiles of splunc5, splunc1, and a third related sequence, lplunc1, in mice, we show that these genes are expressed in unique domains along a continuous corridor of oral, lingual, pharyngeal, and respiratory epithelia. This expression pattern is consistent with the hypothesis that these proteins protect epithelial surfaces colonized by potentially pathogenic microorganisms.

Four reasons to consider a novel class of innate immune molecules in the oral epithelium.

An expanding number of innate immune molecules occupy the "epithelial frontier". This review introduces a recently recognized class of mammalian proteins with similarity to PLUNC (palate, lung and nasal epithelium clone), which is itself related to the host defense protein BPI (bactericidal/permeability-increasing protein). Four emerging lines of evidence unite the PLUNC-like proteins: conserved genetic structure, epithelial expression, three-dimensional protein similarity, and a physiological response to injury or inflammation. By analogy to known proteins of the innate immune system, an emerging hypothesis for this family is that they act as sensors of Gram-negative bacteria in the oral cavity, among other areas.

Four BPI (bactericidal/permeability-increasing protein)-like genes expressed in the mouse nasal, oral, airway and digestive epithelia.

A cluster of related genes whose products show structural identity with bactericidal permeability-increasing protein (BPI) has been identified in the genomes of both mice (on chromosome 2) and humans (on chromosome 20). Genes in the cluster include those encoding parotid secretory protein (PSP), von Ebner minor salivary gland protein (VEMSGP) and sequences in the PLUNC (palate, lung and nasal epithelium clone) family, among others. This mini-review addresses the tissue-specific expression of these genes in the mouse.

Development of an evolutionarily novel structure: fibroblast growth factor expression in the carapacial ridge of turtle embryos.

The turtle shell, an evolutionarily novel structure, contains a bony exoskeleton that includes a dorsal carapace and a ventral plastron. The development of the carapace is dependent on the carapacial ridge (CR), a bulge in the dorsal flank that contains an ectodermal structure analogous to the apical ectodermal ridge (AER) of the developing limb (Burke. 1989a. J Morphol 199:363-378; Burke. 1989b. Fortschr Zool 35:206-209). Although the CR is thought to mediate the initiation and outgrowth of the carapace, the mechanisms of shell development have not been studied on the molecular level. Here, we present data suggesting that carapace formation is initiated by co-opting genes that had other functions in the ancestral embryo, specifically those of limb outgrowth. However, there is divergence in the signaling repertoire from that involved in limb initiation and outgrowth. In situ hybridizations with antisense riboprobes derived from Trionyx spiniferous fibroblast growth factor-10 (tfgf10) and Trachemys scripta (T. scripta) fibroblast-growth factor 8 (tfgf8) cDNAs were performed on sections of early T. scripta embryos (< 30 days). Expression of tfgf10 was localized to the mesenchyme subjacent to the ectoderm of the CR. In the chick limb bud, FGF10 is known to be expressed in the early limb-forming mesenchyme and is capable of inducing FGF8 in the AER to initiate the outgrowth of the limb bud. Although the expression of tfgf8 was found in the AER of the developing turtle limb, it was not seen in the CR. Thus, the initiation of the carapace is in agreement with FGF10 expression in the CR, but FGF8 does not appear to have a role in mediating early carapace outgrowth.

Genomic organization of the mouse plunc gene and expression in the developing airways and thymus.

Few genes have been isolated which display specific expression in the proximal airways. A recently identified mouse cDNA, plunc, appears to be confined to the upper airways and nasopharyngeal epithelium, and may prove a useful marker for these regions. We now report the genomic cloning and characterization of the mouse plunc gene as well as its developmental expression in the nasal and airway epithelium. We also report the novel finding that plunc is also expressed in the medullary compartment of the murine thymus. The mouse gene contains nine exons and the intron-exon boundaries are conserved with those in the human homologue. At e14.5 plunc is expressed in the nasal epithelium and several days later is seen in the thymic lobes, but not in the lining of the tracheobronchial tree. Expression in the trachea and main-stem bronchi first appears at 1--2 days after birth. Tracheobronchial expression persists at high levels throughout adulthood, as do regional areas of nasal and thymic expression. Finally, we show that the human homologue is expressed in bronchial epithelium, suggesting a transcript that is evolutionarily conserved in the mammalian airway.

Expression of angiogenic growth factors in paragangliomas.

OBJECTIVE/HYPOTHESIS: To determine if angiogenic growth factors including vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) are expressed in human paragangliomas. STUDY DESIGN: A histopathologic and molecular examination of paraganglioma specimens obtained from surgical cases or retrieved from the Pathology Department of the Massachusetts Eye and Ear Infirmary. METHODS: Fresh tumor or archival, paraffin-embedded paraganglioma specimens were analyzed by immunohistochemistry, Western blotting, and ELISA. RESULTS: Positive immunohistochemical staining for VEGF was observed in five of nine surgical specimens and in six of eight archival specimens (11/17, or 65%). PD-ECGF immunoreactivity was detected in four of five surgical specimens and six of eight archival specimens (10/13, or 77%). The presence of PD-ECGF was confirmed by Western blot assay and ELISA confirmed the presence of VEGF in tumor extract. CONCLUSIONS: Both VEGF and PD-ECGF are expressed in paragangliomas and may contribute to the extreme vascularity of these tumors. Key Words. Vascular endothelial growth factor, platelet-derived, endothelial cell growth factor, hypoxia, tumor vasculature.

Differential display identification of plunc, a novel gene expressed in embryonic palate, nasal epithelium, and adult lung.

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; GenBankTM accession number U69172).

Expression of the paired-box genes Pax-1 and Pax-9 in limb skeleton development.

Vertebrate Pax genes encode a family of transcription factors that play important roles in embryonic patterning and morphogenesis. Two closely related Pax genes, Pax-1 and Pax-9, are associated with early axial and limb skeleton development. To investigate the role of these genes in cartilage formation we have examined the expression profiles of Pax-1 and Pax-9 in developing chick limb mesenchyme in vivo and in vitro. Both transcripts are detected by reverse transcription polymerase chain reaction and Northern blotting throughout chick limb development, from the early bud stages (Hamburger-Hamilton 20-23) to fully patterned appendages (stage 30). Whole-mount in situ hybridization reveals complex, nonoverlapping expression domains of these two genes. Pax-1 transcripts first appear at the anterior proximal margin of the limb buds, while Pax-9 is expressed more distally at what will be the junction of the autopod and the zeugopod. In situ hybridization to serial sections of the girdles reveals that in the pectoral region Pax-1 is expressed proximally in condensed mesenchyme surrounding the junction of the developing scapula, humerus, and coracoid. In the pelvis, Pax-1 is expressed between the femur and the developing acetabulum and along the ventral edge of the ischium; this transcript was also found in the distal hindlimb along the posterior edge of the fibula. Pax-9 transcripts were not detected in the pectoral girdle at any stage, and only weakly in the pelvis along the ventral ischial margin. In the distal parts of both wings and legs, however, Pax-9 is strongly expressed between the anterior embryonic cartilages (e.g., distal radius or tibia) and the anterior ectodermal ridge. The expression of both genes was strongest in undifferentiated cells of precartilage condensations or at the margins of differentiated cartilages, and was absent from cartilage itself. In micromass cultures of chondrifying limb bud mesenchyme expression of Pax-1 and Pax-9 is maintained for up to 3 days in vitro, most strongly at the end of the culture period during chondrogenic differentiation. As seen in vivo, transcripts are found in loose mesenchyme cells at the outer margins of developing cartilage nodules, and are absent from differentiated chondrocytes at the nodule center. Taken together, these investigations extend previous studies of Pax-1 and Pax-9 expression in embryonic limb development while validating limb bud mesenchyme culture as an accessible experimental system for the study of Pax gene function and regulation. Our in vivo and in vitro observations are discussed with reference to 1) the relationship between somitic and limb expression of these two Pax genes, 2) what regulates this expression in different regions of the embryo, and 3) the putative cellular functions of Pax-1 and Pax-9 in embryonic skeletogenesis.

An in vivo Comparative Study of Intersegmental Flexibility in the Ophiuroid Arm.

We present the first in vivo measurements of intersegmental rotation in the ophiuroid arm, comparing lateral bending performance in seven epifaunal species from Discovery Bay, Jamaica. The species studied include suspension-feeders, deposit-feeders, and scavengers, and also represent two major types of vertebral ossicle morphology. Animals were photographed with strobe illumination, and the angular deflections between arm segments were recorded. Despite considerable variation in vertebral morphology, ecology, and behavior, Discovery Bay ophiuroids show similar, overlapping distributions of maximal intersegmental rotations. Although interspecific differences in mean lateral flexibility can be statistically significant, absolute differences among species are small and of unknown functional significance. These quantitative data challenge long-standing assumptions about how the ophiuroid vertebral skeleton affects intersegmental flexibility, and how intersegmental flexibility per se affects an ophiuroid's ecological style or success.

Microstructural roughness of skeletal calcite in ophiuroid vertebral ossicles: evidence of wear?

Local areas of roughened skeletal calcite are reported from the otherwise smooth, imperforate skeletal articulations of the ophiuroid vertebral ossicle (Echinodermata: Ophiuroidea). Complementary patterns of roughness on both proximal and distal articulating surfaces suggest local points of wear between adjacent ossicles, presumably caused by repeated rotation of the intervertebral joint. This surface feature is discussed with respect to its possible origin (mechanical action, resorption of skeletal material, experimental artifact) and the functional morphology of the ophiuroid arm skeleton.