Phenytoin Inhibits Cell Proliferation through microRNA-196a-5p in Mouse Lip Mesenchymal Cells.

Cleft lip (CL) is one of the most common birth defects. It is caused by either genetic mutations or environmental factors. Recent studies suggest that environmental factors influence the expression of noncoding RNAs [e.g., microRNA (miRNA)], which can regulate the expression of genes crucial for cellular functions. In this study, we examined which miRNAs are associated with CL. Among 10 candidate miRNAs (miR-98-3p, miR-101a-3p, miR-101b-3p, miR-141-3p, miR-144-3p, miR-181a-5p, miR-196a-5p, miR-196b-5p, miR-200a-3p, and miR-710) identified through our bioinformatic analysis of CL-associated genes, overexpression of miR-181a-5p, miR-196a-5p, miR-196b-5p, and miR-710 inhibited cell proliferation through suppression of genes associated with CL in cultured mouse embryonic lip mesenchymal cells (MELM cells) and O9-1 cells, a mouse cranial neural crest cell line. In addition, we found that phenytoin, an inducer of CL, decreased cell proliferation through miR-196a-5p induction. Notably, treatment with a specific inhibitor for miR-196a-5p restored cell proliferation through normalization of expression of CL-associated genes in the cells treated with phenytoin. Taken together, our results suggest that phenytoin induces CL through miR-196a-5p induction, which suppresses the expression of CL-associated genes.

Single-cell patterning and axis characterization in the murine and human definitive endoderm.

Defining the precise regionalization of specified definitive endoderm progenitors is critical for understanding the mechanisms underlying the generation and regeneration of respiratory and digestive organs, yet the patterning of endoderm progenitors remains unresolved, particularly in humans. We performed single-cell RNA sequencing on endoderm cells during the early somitogenesis stages in mice and humans. We developed molecular criteria to define four major endoderm regions (foregut, lip of anterior intestinal portal, midgut, and hindgut) and their developmental pathways. We identified the cell subpopulations in each region and their spatial distributions and characterized key molecular features along the body axes. Dorsal and ventral pancreatic progenitors appear to originate from the midgut population and follow distinct pathways to develop into an identical cell type. Finally, we described the generally conserved endoderm patterning in humans and clear differences in dorsal cell distribution between species. Our study comprehensively defines single-cell endoderm patterning and provides novel insights into the spatiotemporal process that drives establishment of early endoderm domains.

Mouth opening is mediated by separation of dorsal and ventral daughter cells of the lip precursor cells in the larvacean, Oikopleura dioica.

Mouth formation involves the processes of mouth opening, formation of the oral cavity, and the development of associated sensory organs. In deuterostomes, the surface ectoderm and the anterior part of the archenteron are reconfigured and reconnected to make a mouth opening. This study of the larval development of the larvacean, Oikopleura dioica, investigates the cellular organization of the oral region, the developmental processes of the mouth, and the formation of associated sensory cells. O. dioica is a simple chordate whose larvae are transparent and have a small number of constituent cells. It completes organ morphogenesis in 7 h, between hatching 3 h after fertilization and the juvenile stage at 10 h, when it attains adult form and starts to feed. It has two types of mechanosensory cell embedded in the oral epithelium, which is a single layer of cells. There are twenty coronal sensory cells in the circumoral nerve ring and two dorsal sensory organ cells. Two bilateral lip precursor cells (LPCs), facing the anterior surface, divide dorsoventrally and make a wedge-shaped cleft between the two daughter cells named the dorsal lip cell (DLC) and the ventral lip cell (VLC). Eventually, the DLC and VLC become detached and separated into dorsal and ventral lips, triggering mouth opening. This is an intriguing example of cell division itself contributing to morphogenesis. The boundary between the ectoderm and endoderm is present between the lip cells and coronal sensory cells. All oral sensory cells, including dorsal sensory organ cells, were of endodermal origin and were not derived from the ectodermal placode. These observations on mouth formation provide a cellular basis for further studies at a molecular level, in this simple chordate.

Morphological investigations on the lips and cheeks of the goat (Capra hircus): A scanning electron and light microscopic study.

The current study was done to provide comprehensive information on the anatomical features of the lips and cheeks of the goat by gross examination and morphometric analysis in addition to scanning electron microscope (SEM). Samples from 12 normal healthy adult goat's heads of both sexes were collected directly after slaughtering. The lips and cheeks were dissected, and specimens were collected for both light and SEM. The lips of goat were soft and mobile. The free border of both lips was characterized rostrally by the presence of labial projections. The number, size, and arrangement of labial projections differed in the upper and lower lips. On the other hand, the buccal papillae were arranged into 6-8 longitudinal rows parallel to the cheek teeth. The length of these papillae decreased caudally while they were absent on the most caudal part of the cheek. Presence of several types and shapes of labial projections and papillae, and buccal papillae suggest a high degree of mechanical adaptation of the lips and cheeks of the goat. This study provides baseline data for clinical studies. This study is the first report to shed light on the morphology of the lips and cheeks of the goat by gross and scanning electron microscopy.

TGFß1-Smad canonical and -Erk noncanonical pathways participate in interleukin-17-induced epithelial-mesenchymal transition in Sjögren's syndrome.

Interleukin-17 (IL-17) is a pleiotropic cytokine that plays a primary role in triggering epithelial-mesenchymal transition (EMT) in many chronic inflammatory diseases. EMT plays a critical role in the progression of salivary gland (SG) fibrosis in primary Sjögren's syndrome (pSS). This study focused on the activation of the canonical TGF-ß1/Smad2/3 and noncanonical TGF-ß1/Erk1/2 pathways in IL-17-dependent TGFß1-induced EMT in human SG epithelial cells (SGEC) derived from healthy subjects. The expression of phosphorylated Smad2/3 and Erk1/2 during IL-17 treatment-stimulated EMT was evaluated in healthy SGEC. Cotreatment with IL-17 and specific TGFß receptor type I kinase inhibitor SB431542, or Erk 1/2 inhibitor U0126, abrogates the corresponding morphological changes and EMT phenotypic markers expression in healthy SGEC. Interestingly, inhibition of canonical TGFß1/Smad2/3 signal transduction had no effect on activation of the noncanonical TGFß1/Erk1/2/EMT pathway, suggesting that the two pathways act independently in activating IL-17-dependent EMT in SGEC.

MicroRNA-124-3p suppresses mouse lip mesenchymal cell proliferation through the regulation of genes associated with cleft lip in the mouse.

BACKGROUND: Cleft lip (CL), one of the most common congenital birth defects, shows considerable geographic and ethnic variation, with contribution of both genetic and environmental factors. Mouse genetic studies have identified several CL-associated genes. However, it remains elusive how these CL-associated genes are regulated and involved in CL. Environmental factors may regulate these genes at the post-transcriptional level through the regulation of non-coding microRNAs (miRNAs). In this study, we sought to identify miRNAs associated with CL in mice. RESULTS: Through a systematic literature review and a Mouse Genome Informatics (MGI) database search, we identified 55 genes that were associated with CL in mice. Subsequent bioinformatic analysis of these genes predicted that a total of 33 miRNAs target multiple CL-associated genes, with 20 CL-associated genes being potentially regulated by multiple miRNAs. To experimentally validate miRNA function in cell proliferation, we conducted cell proliferation/viability assays for the selected five candidate miRNAs (miR-124-3p, let-7a-5p, let-7b-5p, let-7c-5p, and let-7d-5p). Overexpression of miR-124-3p, but not of the others, inhibited cell proliferation through suppression of CL-associated genes in cultured mouse embryonic lip mesenchymal cells (MELM cells) isolated from the developing mouse lip region. By contrast, miR-124-3p knockdown had no effect on MELM cell proliferation. This miRNA-gene regulatory mechanism was mostly conserved in O9-1 cells, an established cranial neural crest cell line. Expression of miR-124-3p was low in the maxillary processes at E10.5, when lip mesenchymal cells proliferate, whereas it was greatly increased at later developmental stages, suggesting that miR-124-3p expression is suppressed during the proliferation phase in normal palate development. CONCLUSIONS: Our findings indicate that upregulated miR-124-3p inhibits cell proliferation in cultured lip cells through suppression of CL-associated genes. These results will have a significant impact, not only on our knowledge about lip morphogenesis, but also on the development of clinical approaches for the diagnosis and prevention of CL.

Detection of cellular material in lip-prints.

We report on the visualization of cellular material within lip-prints using Diamond™ dye (DD). The transfer of cellular material via the lips can occur in cases of contact with food or drinking items as well as cases of alleged sexual assault involving oral contact. DD can effectively detect cellular material transferred by touch. Here we investigate if lip-prints can be detected and whether there is consistency within, or variability between, a person's propensity to shed cells within lip-prints. Ten volunteers were asked to press their lips against a glass slide with medium pressure for 15 s after not eating or drinking for at least 30 min. Both upper and lower lips were observed, and all tests were performed in five replicates, giving in total 900 observed areas. Consistency in the amount of cellular material deposited by lip-prints for each of the 10 individuals was observed, with each individual being associated with a 'lip shedder' status between the extremes of heavy and light. The majority of females shed more cells than the majority of males. No correlation was observed between the lip-prints shedder-status compared to deposition of cellular material from a thumb. Further, no correlation was observed between lip morphology and the 'lip shedder' status. Visualization of cellular material was not affected by lip-balm but was adversely affected by cosmetics such as lipstick. This technique demonstrates the visualization of deposited cells from parts of the body other than fingers and how cellular material can be visualized allowing targeted collection of DNA.

Oral exfoliative cytology and corrosion of metal piercings. Tissue implications.

OBJECTIVES: A group of adolescents with oral piercings was studied to determine the presence of metallic particles in cells exfoliated from the mucosa surrounding their metal oral piercings and the association between such particles and the metal jewelry, and to evaluate subsequent tissue implications. MATERIALS AND METHODS: Sixteen teenage patients who had tongue and/or lip piercings were included. The clinical features of the oral mucosa and lip skin were evaluated. Exfoliative cytology was performed in the area surrounding the piercing. The surface of used and unused jewelry was studied by scanning electron microscopy and energy dispersive X-ray analysis. RESULTS: Hyperplastic, leukoedematous, and lichenoid lesions were observed in the mucosa, as well as lesions associated with metallosis of the lip skin. Cytological smears showed the presence of particles inside the epithelial cells; the particles were found to contain aluminum, tungsten, and molybdenum. In one case requiring surgical removal of the piercing, histological examination of the tissue associated with the piece of jewelry showed the presence particles containing aluminum, iron, and tin inside multinucleated giant cells. Although surface finish defects were observed on both unused and used piercing jewelry, they were more evident on the used pieces. CONCLUSIONS: Ion particles are released from the metal piercings and could have been adjuvant factors in the development of the observed lesions. Cells exfoliated from the oral mucosa surrounding metal piercings may serve as bioindicators of corrosion processes. CLINICAL RELEVANCE: We propose the use of exfoliative cytology to monitor corrosion processes and for routine clinical follow up.

Filling Procedures for Lip and Perioral Rejuvenation: A Systematic Review.

The ideal perioral and lip rejuvenation technique provides the longest period of efficacy, lowest complication rate, and best esthetic results. Genetics, intrinsic aging, sun exposure, and repetitive muscle twitching of the orbicularis oris produce angular, radial, and vertical lines of the perioral lines and, for this reason, the needs of patients in the treatment of this anatomical area can range from simple lip enhancement to a broader and more comprehensive treatment with simultaneous correction of perioral wrinkles. A myriad of materials have been described for rejuvenation of this area. At present, the most popular and commonly used lip enhancers are dermal fillers, but there is still no agreement on what the best material for filling soft tissue of the face and in particular of the perioral region is. This systematic review will focus on the various dermal fillers, of different materials approved by the US Food and Drug Administration (FDA) namely poly-L-lactic acid, calcium hydroxylapatite, and hyaluronic acid and also different grafts, for perioral rejuvenation, with the goal of determining the optimal approach. A systematic search for English studies involving perioral rejuvenation was performed using these databases: PubMed, Google Scholar, and Ovid, using a combined keyword search or medical subject headings. At the end of our study selection process, 17 relevant publications were included. For each study, year of publication, type of material used for filling, number of patients, subject of study assessment, and efficacy of the filler procedure for lip rejuvenation were analyzed.

Analysis of the cell populations composing the mononuclear cell infiltrates in the labial minor salivary glands from patients with rheumatoid arthritis and sicca syndrome.

OBJECTIVES: Sicca symptoms occur in around 30% of rheumatoid arthritis (RA) patients. Herein, we examined the characteristics of RA patients bearing sicca symptomatology (RA-sicca) with a special focus on the immunohistopathological features of their labial minor salivary gland (LMSG) biopsies. METHODS: Our cohort included 100 consecutive RA patients which were interrogated using a sicca symptoms questionnaire. Positive responders were evaluated for ocular and oral dryness and underwent an LMSG biopsy. All samples were immunohistochemically evaluated for the presence and distribution of specific leukocyte subsets using appropriate markers and for the expression of certain immunoregulatory molecules by salivary gland epithelial cells. Positively stained and total mononuclear cells (MNC) were counted in the entire section. Counts were expressed as cell frequency (percentage of cell type number/total infiltrating MNC number). RESULTS: In the majority (86.1%) of the 44 RA-sicca cases, periductal infiltrates were observed in LMSG biopsies. The frequencies of infiltrating cell subtypes and their correlation with lesion severity were different from that previously described in primary Sjögren's syndrome (pSS). Moreover, DCs and ΜΦs frequencies were increased in RA-sicca patients who had a biopsy focus score <1 and absence of anti-Ro/anti-La autoantibodies, in contrast to what was observed for B cells. In about half of the biopsies, salivary gland epithelial cells expressed CD80/B7.1 molecules, most commonly in patients with a positive biopsy or anti-Ro/anti-La autoantibodies. CONCLUSION: LMSG infiltrates composition in RA-sicca patients is distinct from that described in pSS. These differences, further attest to diverse pathophysiologic processes operating in these two entities.

Ultrastructural characterization of normal Merkel cells in the dog.

BACKGROUND: Involvement of Merkel cells (MKs) in different cutaneous diseases as well as in the growth, differentiation and homeostasis of the skin has been previously documented. HYPOTHESIS/OBJECTIVES: The aim was to assess the ultrastructural features of MKs in canine skin, including morphometrics, highlighting their similarities with and differences from those described for other mammals. ANIMALS: Hard palate, nasal planum, lower lip and whisker pad samples were taken from two healthy young dogs destined for academic purposes. METHODS: Ultrathin sections of samples fixed in osmium tetroxide and embedded in Epon 812 resin were stained with uranyl acetate and lead citrate and examined using a JEOL JEM 2010 transmission electron microscope. RESULTS: Ultrastructural characteristics included the following: (i) arrangement in clusters in the basal layer of the epidermis, oral mucosa and external follicular root sheath; (ii) inconstant link with nerve terminal; (iii) oval (10.27 ± 1.64 µm major axis) cell shape with large lobulated nuclei (5.98 ± 1.16 µm major axis); (iv) spine-like and thick cytoplasmic processes interdigitating with surrounding keratinocytes; (v) presence of desmosomes in the cell body or at the base of spine-like processes attaching to neighbouring keratinocytes; and (vi) cytoplasm containing loosely arranged intermediate filaments (10.04 ± 1.17 nm) and numerous dense-core granules (100.1 ± 17.12 nm) arranged in the basal portion of the cytoplasm. CONCLUSIONS AND CLINICAL IMPORTANCE: This study provides the first complete description of the ultrastructural characteristics of MKs in the dog, enhancing our knowledge of the skin structure in this species and providing a basis for future physiological and pathological studies of the role of these cells in normal and damaged canine tissues.

[Comparative ultrasonographic and histomorphologic examination of the lips]. / Vergleichende sonographische und histomorphologische Untersuchungen der Lippen.

BACKGROUND: High-resolution ultrasonography of the lips offers the opportunity to investigate the orbicularis oris muscle (OOM) and evaluate its morphology and function. OBJECTIVES: The goals of this paper are verification of the lip structures visible on ultrasound images by using histological section preparations and recommendation of uniform standards for sonographic examinations of the lips. MATERIALS AND METHODS: The lips of 78 healthy volunteers (age 4-77 years) where scanned with a Hitachi Hi Vision Avius ultrasound device equipped with a linear transducer (L75, variable frequency range 5.0-18.0 MHz). Systematic B-mode examination was performed at five defined points, and the lips where also scanned dynamically in multiple directions. The ultrasonography findings were verified by using histologic samples from five male body donors (age 72-83 years). RESULTS: All parts of the OOM could be well distinguished from one another both histologically and ultrasonographically. Sonographically visible lip structures could be verified histologically. Labial glands and blood vessels of the mucosa could be identified with both methods. CONCLUSION: Ultrasonography allows identification of lip structures and all parts of the OOM. Scars, injuries and atrophy of the lip musculature are well detectable. Functional examinations can visualize muscular dysfunctions and may support the diagnosis of dystonic or hypotonic functional deficits. The following parameters are mandatory for a standardized examination of the lips: sagittal and transverse images of upper and lower lips; use of anatomical "landmarks"; functional diagnostics in tensed and relaxed conditions.

The ins and outs of breathing.

Distinct populations of neurons within the brainstem are responsible for generating and coordinating the rhythmic patterns of neural activity that underlie breathing.

Atoh1-dependent rhombic lip neurons are required for temporal delay between independent respiratory oscillators in embryonic mice.

All motor behaviors require precise temporal coordination of different muscle groups. Breathing, for example, involves the sequential activation of numerous muscles hypothesized to be driven by a primary respiratory oscillator, the preBötzinger Complex, and at least one other as-yet unidentified rhythmogenic population. We tested the roles of Atoh1-, Phox2b-, and Dbx1-derived neurons (three groups that have known roles in respiration) in the generation and coordination of respiratory output. We found that Dbx1-derived neurons are necessary for all respiratory behaviors, whereas independent but coupled respiratory rhythms persist from at least three different motor pools after eliminating or silencing Phox2b- or Atoh1-expressing hindbrain neurons. Without Atoh1 neurons, however, the motor pools become temporally disorganized and coupling between independent respiratory oscillators decreases. We propose Atoh1 neurons tune the sequential activation of independent oscillators essential for the fine control of different muscles during breathing.DOI: http://dx.doi.org/10.7554/eLife.02265.001.

Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.

The differing adipocyte morphologies of deep versus superficial midfacial fat compartments: a cadaveric study.

BACKGROUND: Anatomical studies show that facial fat is partitioned into distinct compartments, with the nasolabial fat pad in a superficial compartment and the deep medial cheek fat in a deep compartment. Gross morphologic differences may exist between these fat depots, but this has never been established at the cellular level. METHODS: Adipose tissue specimens from nasolabial fat and deep medial cheek fat pads were obtained from 63 cadaveric specimens (38 female and 25 male cadavers) aged 47 to 101 years (mean, 71 years). Thirty-seven cadavers had a normal body mass index (≤25 kg/m) and 26 cadavers had a high body mass index (>25 kg/m). Cross-sectional areas of individual adipocytes were calculated digitally and averaged from histologic sections of the adipose tissue samples. RESULTS: The average adipocyte size of nasolabial fat is significantly (p < 0.0001) larger than that of deep medial cheek fat. The average adipocyte size in both nasolabial and deep medial cheek fat is significantly (p < 0.0001) larger in subjects with high compared with low body mass index. Although the overall average adipocyte size is significantly (p < 0.0001) larger in female than in male subjects, this sexual dimorphism is lost in the nasolabial fat depots of overweight subjects and in the deep medial cheek depots of normal-weight subjects. CONCLUSIONS: The significantly smaller adipocyte size in deep medial cheek fat relative to nasolabial fat in elderly subjects supports the theory that deep and superficial facial fat pads are morphologically different. Future investigation of the metabolic and structural properties of these fat compartments will help us understand the different patterns of volumetric facial aging.

Minor salivary glands of the lips: a novel, easily accessible source of potential stem/progenitor cells.

OBJECTIVES: Cells with stem/progenitor properties have been detected in major salivary glands, but no data are available on their presence within minor salivary glands (MSGs). This study aimed to isolate and characterize potential stem/progenitor cells from human MSGs. MATERIALS AND METHODS: MSGs of the lower lip were surgically obtained during biopsy for Sjogren's syndrome investigation that finally proved to be histologically normal. The established MSG cultures were assessed for morphology, proliferation, colony-forming-unit efficiency, multipotentiality, and immunophenotypic characteristics. RESULTS: A mixed population of fibroblast-like and a few flat-shaped epithelial-like cells was obtained. These cells were capable for osteogenic, adipogenic, and neurogenic differentiation. Evidence for strong stem cell potency was observed by the detection of early stem cell markers, like Nanog, Oct-3/4, and SSEA-3. These cells also expressed characteristic mesenchymal stem cell markers, including CD90-Thy1, CD105, CD49f, CD81, nestin, CD146, and Stro-1, but were negative for CD117/C-KIT, CD45, and CD271/NFG. In addition, positivity for keratins 7/8 in part of the population was indicative of an epithelial phenotype, whereas these cells were negative for aquaporin-1 expressed in acinar/myoepithelial cells during development. CONCLUSIONS: Based on these data, a cell population with stem/progenitor characteristics was primarily isolated from labial MSGs. The morphologic and immunophenotypic features indicated that this population is mixed with mesenchymal (mainly) and epithelial characteristics. CLINICAL RELEVANCE: Due to their large number and superficial distribution in labial mucosa, MSGs may be proposed as a potential easily accessible source of adult stem/progenitor cells for regenerative therapies of glandular organs with parenchymal pathology.

The aging perioral region -- Do we really know what is happening?

OBJECTIVES: The perioral region is subject to a myriad of different treatments for rejuvenation, many of which are applied without a clear understanding of the underlying physiological processes of perioral aging. The results of these procedures are therefore sometimes not optimal and do not achieve a natural youthful appearance. The aim of this study was to put the results of three investigations into the perioral aging process into relation to clinical application in aesthetic medicine. DESIGN: Three different investigations were performed to evaluate the complex 3-dimensional changes during the perioral aging process. Perioral proportions of 182 standardized subject photographs were measured in a photomorphometric study and correlated to age. In cranial MRI scans of 30 women aged 20-35 and 30 women aged 65-80 relevant anatomical dimensions were measured. Histological cross cuts of the upper lip complex of 20 individuals in two age groups, young (< 40 years, n = 10) and old (> 80 years, n = 10), were analysed. The results were then set into relation to today's lip rejuvenation procedures. RESULTS: All studies showed a statistically significant lengthening of the aging upper lip. The photomorphometric study further showed an increase of prolabium skin at the cost of a decreasing visible upper lip vermilion. The MRI scans showed a decrease in thickness and redistribution towards a length increase but no total volume loss. Histomorphometric analysis revealed statistically significant thinning of the cutis, thickening of the subcutis and a degeneration of elastic and collagen fibers. The orbicularis oris muscle becomes thinner and shows a decrease of the forward curve defining the vermilion border. The results show that the main processes of lip aging are redistribution from thickness to length without total volume loss and a decrease of structural components of the lip, which leads to the decrease of pouting, an inversion of the vermilion and a ptosis of the lip. CONCLUSION: A new and better understanding of the underlying physiological changes of perioral aging is essential and will lead to a better and more specific implementation of perioral rejuvenation procedures which will lead to more natural results.

Isolation of epithelial cells in the developing primary lip and palate.

Failure of the primary lip and palate to fuse leads to clefts of the lip, a birth defect with an incidence of 1 for every 500 in some races. Epithelial cells lining the facial processes of the primary lip and palate, the lateral and medial nasal processes (LNP and MNP), must first make contact to go through a series of highly regulated and coordinated sequence of events to form the normal midface. As yet, many of the basic mechanisms underlying the fusion events of the epithelial-lined surfaces are not known. This is due in part to the difficulty associated with the isolation of the epithelial cells for further study and analysis. The objective of this study was to test the use of laser capture microdissection to collect clean populations of primary lip and palate epithelial cells destined to fuse. Fusing and nonfusing epithelial cell populations of the MNP and LNP were isolated by laser capture microdissection and assayed for gene expression of Bmp-4, Tgfß-2, and their type 1 receptors, Alk-3 and Alk-5, respectively. Transcripts of Bmp-4/Alk-3 and Tgfß-2/Alk-5 were restricted to the epithelial seam of the fusion site, and the epithelium of the prefusion site, in patterns previously reported. Data indicated our ability to isolate clean populations of epithelial and mesenchymal cells around the primary palate fusion site, allowing precise analysis of tissue and site-specific gene expression at high resolution. This study provides the basis of further analysis of the potential molecular players of MNP and LNP fusion and nonfusion of epithelial cells.