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Keywords:

  • Tmem16;
  • mouse;
  • gene;
  • expression;
  • paralog;
  • gene family;
  • phylogeny

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The TMEM16 protein family has recently been identified through several different experimental strategies including bioinformatic and microarray-based approaches. In mice and humans, there exist 10 paralogs with each containing eight putative transmembrane domains and a conserved C-terminal domain of unknown function. Mutation of at least one member of this family is associated with a human disorder, and several members of this gene family are overexpressed in different types of cancer. Despite their apparent relevance to normal development and disease, little is known about the expression of TMEM16 paralogs during embryonic development. Here, we provide a phylogenetic analysis of mouse and human TMEM16 paralogs and report the expression of Tmem16a, Tmem16b, Tmem16c, Tmem16f, Tmem16h, Tmem16j, and Tmem16k during murine embryogenesis with an emphasis on the respiratory, digestive, skeletal, and integumentary systems. These data should encourage investigations into the functions of TMEM16 paralogs in vertebrate development. Developmental Dynamics 237:2566–2574, 2008. © 2008 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The TMEM16 family of genes was first systematically described in vertebrates during in silico characterization of 11q13, a chromosomal region amplified in a variety of tumors (Katoh and Katoh,2003). A novel expressed sequence tag (EST) corresponding to TMEM16A was mapped to human chromosomal region 11q13 and its sequence and predicted topology led to the identification of three paralogous genes (now known as TMEM16B, TMEM16C, and TMEM16D). Subsequently, the same bioinformatics approach led to the identification of other paralogs including TMEM16E, TMEM16F, TP53I5, and TMEM16H (Katoh and Katoh,2004b, c,2005).

Independent studies with diverse objectives have identified the above genes and additional members of the TMEM16 family. For example, in addition to being identified through this bioinformatic approach, TMEM16A was also identified as a transcript expressed at high levels in gastrointestinal stromal tumors and given the name DOG1 (discovered on GIST-1; West et al.,2004). This same gene was given the name TAOS2 (tumor amplified and overexpressed-2), because it was found to be frequently amplified and overexpressed in oral squamous cell carcinomas (Huang et al.,2006). The genes TMEM16E and TMEM16G were also identified by multiple groups and given additional names derived from the contexts in which they were discovered (Bera et al.,2004; Katoh and Katoh,2004a; Kiessling et al.,2005).

We identified murine Tmem16a in a screen for genes expressed in the zone of polarizing activity (ZPA) of the developing limb (Rock et al.,2007). To study the function of Tmem16a in development, we generated a null allele of this gene in mice (J.R.R. and B.D.H., unpublished data). Surprisingly, Tmem16a mutants did not demonstrate a patterning defect in the limbs, but all mutants died within 1 month of parturition demonstrating a requirement for this gene in normal development.

Although high levels of expression of several TMEM16 paralogs are associated with cancer, the mutation of only a single member of this gene family, TMEM16E, has been linked to a human disorder (Tsutsumi et al.,2004; West et al.,2004; Galindo and Vacquier,2005; Huang et al.,2006). Two independent missense mutations in a single cysteine residue of TMEM16E have been linked to the autosomal dominant inherited disease gnathodiaphyseal dysplasia that is characterized by fragility of the long bones and cemento-osseous lesions of the jaw (Tsutsumi et al.,2004; OMIM 166260). Interestingly, this cysteine is conserved throughout TMEM16E orthologs in all species examined and in 7 of the 10 TMEM16 proteins in mice and humans.

In the budding yeast Saccharomyces cerevisiae, a single TMEM16 ortholog has been identified (Entian et al.,1999). Phenotypic data from a high-throughput screen suggested that this protein, Ist2p, might mediate osmotic tolerance. Of the four TMEM16 orthologs identified in Drosophila melanogaster, functional data exist for only Axs (reviewed in Kramer and Hawley,2003). The mutation of this ortholog is associated with a high frequency of X chromosome nondisjunction during female meiosis.

TMEM16 proteins characteristically comprise eight predicted transmembrane domains with both termini facing the cytoplasm; although, it has been suggested that one isoform of TMEM16G comprises only seven transmembrane domains (Bera et al.,2004; Galindo and Vacquier,2005). In addition, a C-terminal domain of unknown function is found in all TMEM16 proteins (DUF590, InterPro IPR007632). Some members of this family are localized to the plasma membrane (Bera et al.,2004; Juschke et al.,2004; West et al.,2004; Galindo and Vacquier,2005), while others have been described on intracellular membranes (Kramer and Hawley,2003; Mizuta et al.,2007).

Although their precise molecular functions remain to be described, current data suggests that members of the TMEM16 family of proteins are involved in both normal vertebrate development and disease. Unfortunately, only limited expression data for a few members of this protein family has been reported during embryonic development (Hecht et al.,2007; Mizuta et al.,2007; Rock et al.,2007). Here, we report the widespread but tissue-specific expression of members of this family during murine embryogenesis, focusing on several major thoracic and abdominal organ systems.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Phylogeny of the TMEM16 Protein Family

As a first step in our characterization of the TMEM16 protein family, we constructed a neighbor-joining phylogenetic tree (Fig. 1A). The genes and accession numbers of the protein sequences used for this phylogenetic characterization are listed in Supplementary Table 1 (which can be viewed online). In mice and humans, 10 TMEM16 paralogs have been identified; our analysis identified only a single ortholog in Caenorhabditis elegans. The single ortholog from C. elegans was included as an outgroup to root the phylogenetic tree. As expected, this C. elegans ortholog (F56A8.1) was most divergent from the human and mouse TMEM16 proteins (Fig. 1A). For each mouse paralog, its human ortholog was the most similar protein sequence. Within the vertebrate proteins, TMEM16H and TMEM16K comprised one major clade while the other eight human and murine TMEM16 proteins clustered in the second major clade. This suggested that TMEM16H and TMEM16K might constitute a subfamily of TMEM16 proteins with functions distinct from those of the other family members.

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Figure 1. Phylogenetic analysis of TMEM16 proteins. A: A neighbor-joining phylogenetic tree was generated using the available protein sequence data for the 10 identified murine and human TMEM16 paralogs and single identified Caenorhabditis elegans ortholog (see Supplementary Table 1). Bootstrap values (1,000 replicates) are shown on each branch. B: Multiple alignment of murine and human TMEM16 protein sequences in the region of a conserved cysteine residue (asterisk) mutated in TMEM16E in humans with gnathodiaphyseal dysplasia.

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The mutation of a cysteine residue in a predicted extracellular loop of human TMEM16E has been linked to the autosomal dominant disorder gnathodiaphyseal dysplasia (Tsutsumi et al.,2004). It was previously reported that this residue is conserved in TMEM16E from all species examined; our multiple alignment demonstrated that this cysteine is conserved in 7 of the 10 TMEM16 paralogs in both mice and humans (asterisk Fig. 1B). Interestingly, the homologs in the TMEM16H/TMEM16K clade not only lack this conserved cysteine, but lack this entire protein region that might contain a functional domain (Fig. 1B). This data further supports the existence of a functional subfamily including TMEM16H and TMEM16K.

TMEM16A is most similar to TMEM16B with 62% identity at the amino acid level for the murine proteins. Our data suggests that these proteins descended from a common ancestor. Similar evolutionary relationships for TMEM16C/TMEM16D and TMEM16E /TMEM16F can be inferred from our data. As functional data for these proteins is generated, this phylogenetic context might facilitate its interpretation.

Expression of TMEM16 Paralogs in the Developing Respiratory System

The vertebrate respiratory system has evolved to provide the necessary surface area for diffusion of gases required for metazoan terrestrial life. In mice, the respiratory system is first evident as two lung buds emerge from the ventral foregut endoderm (reviewed in Cardoso and Lu,2006). These buds elongate and branch during the pseudoglandular phase of lung development to generate the basic pattern of the respiratory tree. Whole-mount and section RNA in situ hybridizations revealed Tmem16a expression in the epithelium of the trachea and lungs on embryonic day (E) 12.5 and E14.5 (Fig. 2A,B). Notably, Tmem16a expression was not detected in the distal epithelial tips by whole-mount or section RNA in situ hybridization at E12.5 or E14.5 (arrows in Fig. 2A,B). The mesenchyme immediately subjacent to the proximal conducting airway epithelium at E14.5 expressed Tmem16a (arrowheads in Fig. 2B). At E16.5, this mesenchymal expression of Tmem16a was no longer detected, but the epithelial expression of Tmem16a persisted (Fig. 2C). At E18.5, we detected high levels of Tmem16a expression only in epithelial cells of the terminal saccules (Fig. 2D).

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Figure 2. Tmem16a, Tmem16f, and Tmem16j expression in the developing respiratory system. A: Whole lung RNA in situ hybridization showing Tmem16a expression in the epithelium of the trachea and lung at embryonic day (E) 12.5. Arrow indicates epithelium of a distal tip devoid of Tmem16a transcripts. B–D: Section RNA in situ hybridizations showing Tmem16a expression in the epithelium of the lung at (B) E14.5, (C) E16.5, and (D) E18.5. Arrow in B indicates distal epithelium not expressing Tmem16a. Arrowheads in B indicate Tmem16a expression in the mesenchyme surrounding bronchiolar epithelium. D: At E18.5, expression of Tmem16a is restricted to cells in the distal epithelium of the lung. Inset shows higher magnification of Tmem16a expression in E18.5 lung. E:Tmem16f is expressed in the epithelium of the lung at E14.5 and at lower levels in the surrounding mesenchyme. F: Expression of Tmem16j is restricted to the epithelium of the lung at E14.5. ao, aorta; br, bronchiole; bv, blood vessel; epi, epithelium; es, esophagus; ll, left lung; mes, mesenchyme; r, rib; rl, right lung; tr, trachea. Scale bars = 100 μm in B–D, 50 μm in inset in D,E,F.

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We detected the expression of two paralogs, Tmem16f and Tmem16j, by RNA in situ hybridization during lung development (Fig. 2E,F). At E14.5, Tmem16f expression was evident in the epithelium of the lung and diffuse staining was also observed in the lung mesenchyme (Fig. 2E). Because this pattern was not observed using a sense RNA control probe (data not shown), we believe that this staining is not an artifact of the RNA in situ procedure. The expression of the paralog Tmem16j was restricted to the epithelium of the lung at E14.5 (Fig. 2F). In particular, the mesenchymal domains expressing Tmem16a and Tmem16f at this stage were devoid of Tmem16j transcripts. The paralog Tmem16c was not detected in the lung at E14.5 or E16.5 (data not shown).

Expression of TMEM16 Paralogs in the Developing Gastrointestinal Tract

The vertebrate gastrointestinal tract is differentiated along its anteroposterior axis to perform particular functions; however, its radial pattern is generally conserved from the esophagus to the colon (Sukegawa et al.,2000). The innermost epithelium of the gut is surrounded by concentric rings of specialized mesenchyme. RNA in situ hybridization on transverse sections showed that Tmem16a is expressed in the esophageal mesenchyme in a domain that is restricted to the submucosa (Fig. 3A). RNA in situ hybridization on transverse sections through the caudal portion of the gastrointestinal tract revealed a similar distribution of Tmem16a expression at E14.5 (Fig. 3B,C). Unlike the esophageal epithelium, where Tmem16a expression was not detected by RNA in situ hybridization, we detected Tmem16a expression in the epithelium of the posterior stomach (arrowhead in Fig. 3B) and lower levels of expression in the intestinal epithelium (Fig. 3B,C). In addition to expression in the gastrointestinal tract, we noted Tmem16a expression in the epithelium of the trachea, the mesenchyme immediately dorsal to the trachea, the thymus and the aorta at E16.5 (Fig. 3A) as well as the kidneys, pancreas, and ureters at E14.5 (Fig. 3B).

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Figure 3. Tmem16a, Tmem16c, Tmem16f, and Tmem16j expression in the embryonic digestive system. A: RNA in situ hybridization showing expression of Tmem16a in the esophageal mesenchyme at embryonic day (E) 16.5. B: Transcription of Tmem16a is observed in the mesenchyme and epithelium of the stomach and small intestine at E14.5. Arrowhead indicates stomach epithelium. C: Higher magnification of boxed region in B showing Tmem16a expression in the small intestine. D: RNA in situ hybridization demonstrating expression of Tmem16c in a discontinuous ring of cells in the esophageal mesenchyme at E16.5. E: Expression of Tmem16c in the Organ of Zuckerkandl and in the mesenchyme of the small intestine, stomach, and intestinal epithelium at E14.5. F: Higher magnification of boxed region in E showing Tmem16c expression in the mesenchyme of the small intestine. G:Tmem16f expression in the esophageal epithelium and mesenchyme at E16.5. H: RNA in situ hybridization showing robust expression of Tmem16f in the intestinal epithelium and lower levels of expression in the intestinal mesenchyme, stomach, kidneys, and ovary at E14.5. I,J: Epithelial expression of Tmem16j in the bronchi, esophagus, pancreas, stomach, and intestine at E16.5 (I) and E14.5 (J). ao, aorta; br, bronchus; es, esophagus; k, kidney; me, muscularis externa; ov, ovary; OZ, Organ of Zuckerkandl; pa, pancreas; si, small intestine; st, stomach; th, thymus; tr, trachea; ur, ureter. Scale bars = 100 μm in A,B,D,E,G–J; 50 μm in C,F.

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In the esophagus at E16.5, RNA in situ hybridization revealed expression of Tmem16c in an incomplete ring of cells in the mesenchyme (Fig. 3D). Transcription appeared limited to a subset of cells within the myenteric plexus of the muscularis externa of the esophagus. This pattern of expression was also observed in the caudal portion of the gastrointestinal tract where mesenchymal expression of Tmem16c was detected in the stomach and small intestine (Fig. 3E,F). In addition to the mesenchymal expression in the small intestine, we also detected expression of Tmem16c in the intestinal epithelium at E14.5 (Fig. 3E,F). The only other site of Tmem16c expression observed on these sections was located ventrally to the dorsal aorta. We believe that this site of expression overlaps with the developing Organ of Zuckerkandl (Fig. 3E), a cluster of neuroendocrine cells implicated in the synthesis of catecholamines (Subramanian and Maker,2006).

Tmem16f transcripts were detected in both the epithelium and mesenchyme of the esophagus at E16.5 (Fig. 3G). Particularly robust expression of Tmem16f was detected at this stage in the muscularis externa of the esophageal mesenchyme (Fig. 3G). In the caudal digestive tract at E14.5, Tmem16f transcripts were detected in the epithelium of the small intestine (Fig. 3H). Lower levels of Tmem16f expression were detected in the mesenchyme of the small intestine and in both the epithelial and mesenchymal components of the trachea (Fig. 3G), ovary, kidney, and stomach (Fig. 3H).

Tmem16j expression in the esophagus, small intestine, stomach, and pancreas was confined to the epithelium at both E14.5 and E16.5 (Fig. 3I,J). Consistent with the restriction of Tmem16j expression to the epithelium of the embryonic gastrointestinal tract and lungs (Figs. 2F, 3J), expression of Tmem16j was restricted to the epithelia of the bronchi at E16.5 (Fig. 3I).

Expression of TMEM16 Paralogs in the Axial Skeleton and Associated Structures

Previously, we reported the expression of Tmem16a in the periostea and articular cartilages of the long bones of the limbs at E15.5 (Rock et al.,2007). The expression of Tmem16e was also previously reported during murine embryogenesis (Tsutsumi et al.,2005; Mizuta et al.,2007). In those studies, Tmem16e expression was detected in the somites and later in differentiating musculoskeletal tissues. For these reasons, we examined the expression patterns of TMEM16 paralogs during the development of the axial skeleton.

RNA in situ hybridization on a transverse section through the spinal column demonstrated weak expression of Tmem16a in the dorsal neural tube at E14.5 (Fig. 4A). At this stage, Tmem16a expression was also detected in the perichondria of the neural arch of the developing vertebrae (arrowhead in Fig. 4A). RNA in situ hybridization on a sagittal section demonstrated expression of Tmem16a in several tissues at E14.5, including the thymus, the wall of the aorta, and the mesenchyme dorsal to the trachea that will form the trachealis muscle (Fig. 4B). At E16.5, expression of Tmem16a persisted in the perichondria of the developing vertebrae and ribs as well as the thymus, trachea, and esophagus (Figs. 2C, 4C).

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Figure 4. Expression of Tmem16a, Tmem16b, Tmem16c, Tmem16f, Tmem16h, Tmem16j, and Tmem16k in and around the axial skeleton. A: RNA in situ hybridization showing Tmem16a expression in the neural tube and perichondrium of the neural arch of a vertebra (arrowhead) at embryonic day (E) 14.5. B: Sagittal section showing Tmem16a expression in several tissues at E14.5, including the thymus, aorta, and mesenchyme dorsal to the trachea. C:Tmem16a expression in the perichondria of veterbrae, esophagus, thymus, tracheal epithelium, and the mesenchyme dorsal to the trachea at E16.5. D: RNA in situ hybridization showing Tmem16c expression in the perichondria of the neural arch of a vertebra, a rib, and the vertebral body at E14.5. E: Robust expression of Tmem16c was detected in the perichondria of the ribs on a sagittal section at E14.5. F:Tmem16c expression continued in the vertebral perichondrium as well as the clavicles (arrowheads) and dorsal root ganglia at E16.5. G: RNA in situ hybridization showing Tmem16f expression in the neural tube and dorsal root ganglia at E14.5. H: Sagittal section showing Tmem16f expression in the perichondria of ribs at E14.5. I:Tmem16j is also expressed in the perichondria of ribs at E14.5. (Expression of Tmem16f and Tmem16j in the lung as described in Fig. 2 is also obvious in Fig. 4H and I.) J–L:Tmem16b (J), Tmem16h (K), and Tmem16k (L) were all detected in the neural tube and dorsal root ganglia at E14.5. ao, aorta; drg, dorsal root ganglion; es, esophagus; na, neural arch; r, rib; th, thymus; tr, trachea; ve, vertebra. Scale bars = 100 μm in A,B,D,E,G–L; 200 μm in C,F.

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Like Tmem16a, Tmem16c was expressed in the perichondria of the neural arch and vertebral body at E14.5 and E16.5 (Fig. 4D,F). Also at E14.5, we detected robust expression of Tmem16c in the perichondria of the developing ribs (Fig. 4D,E) and in the clavicles at E16.5 (arrowheads in Fig. 4F). At E14.5 and E16.5, we detected expression of Tmem16c in the dorsal root ganglia and at low levels in the neural tube (Fig. 4D,F).

We detected a low level of transcription of Tmem16f by RNA in situ hybridization in the neural tube as well as the dorsal root ganglia at E14.5 (Fig. 4G). Similarly to Tmem16a and Tmem16c, RNA in situ hybridization on sagittal sections showed strong expression of Tmem16f and Tmem16j in the perichondria of the developing ribs at E14.5 (Figs. 2E,F, 4H,I). Three paralogs, Tmem16b, Tmem16h, and Tmem16k, were detected with similar distributions in the mantle layer of the neural tube and in the dorsal root ganglia at E14.5 (Fig. 4J–L).

Ectodermal Expression of TMEM16 Paralogs

The development of the skin involves stratification of the epidermis (reviewed in Fuchs and Raghavan,2002), and the pathways underlying this process are an active area of research (Lechler and Fuchs,2005; Yi et al.,2008). RNA in situ hybridization revealed that the expression of Tmem16a, Tmem16b, and Tmem16f was restricted to more basal layers of the epidermis at E16.5 (Fig. 5A,B,D), while the paralogs Tmem16c and Tmem16j were detected in the most suprabasal layers of the skin at this stage of development (Fig. 5C,E).

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Figure 5. Tmem16a, Tmem16b, Tmem16c, Tmem16f, and Tmem16j expression in the skin and apical ectodermal ridge (AER), A–E: RNA in situ hybridizations showing Tmem16a (A), Tmem16b (B), Tmem16c (C), Tmem16f (D), and Tmem16j (E) expression in the epidermis at embryonic day (E) 16.5. Tmem16a, Tmem16b, and Tmem16f were present in more basal layers of the epidermis while Tmem16c and Tmem16j were detected in the most suprabasal layers of the skin at this stage of development. F: Whole-mount RNA in situ hybridization showing Tmem16c expression in the posterior AER at E10.5. Scale bars = 50 μm in A–E.

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We previously reported expression of Tmem16a in a domain that overlaps the ZPA in the murine limb bud (Rock et al.,2007). The mesenchymal cells of the ZPA produce SHH which polarizes the limb along its anteroposterior axis (Riddle et al.,1993). A second signaling center, the apical ectodermal ridge (AER), is required for the outgrowth of the limb bud (Saunders,1998). The functions of the ectodermal cells of the AER are at least partly mediated by the secretion of members of the fibroblast growth factor (FGF) family of proteins (Sun et al.,2002). Of interest, we detected expression of Tmem16c in the posterior AER at E10.5 by whole-mount RNA in situ hybridization (Fig. 5F).

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

In mice and humans, at least 10 TMEM16 paralogs have evolved. It has been suggested that the existence of multiple TMEM16 paralogs in mice and humans might have evolved to allow tissue-specific expression of proteins with similar functions (Galindo and Vacquier,2005). Alternatively, the multiple paralogs might have divergent functions as a result of their duplication and subsequent evolution. Our phylogenetic analysis of the TMEM16 family suggests an ancient divergence of a TMEM16H/TMEM16K common ancestor from the other TMEM16 proteins and these proteins might represent a functional subfamily of TMEM16 proteins.

It has previously been reported that Tmem16g is expressed only in the prostate and the embryonic expression pattern of Tmem16e has been reported as well (Bera et al.,2004; Mizuta et al.,2007). We therefore focused our efforts on describing the embryonic expression of the remaining family members. Future characterizations of this family should include Tmem16d for which we have not yet been able to generate in situ data. We chose to limit our descriptions of the other seven family members from E10.5 to E16.5, but have detected expression of Tmem16a and other paralogs before and after these stages.

We show that expression domains of TMEM16 paralogs overlap in several tissues during murine development. For example, in the epithelium of the lung, transcription of Tmem16a, Tmem16f, and Tmem16j was detected. In addition, we also found that the developing skeleton expressed Tmem16a, Tmem16c, Tmem16f, and Tmem16j in overlapping domains. In the developing nervous system, Tmem16b, Tmem16c, Tmem16f, Tmem16h, and Tmem16k were detected in the neural tube and in the dorsal root ganglia. The functions of the TMEM16 proteins in these tissues might be redundant or unique.

We currently do not know if TMEM16 proteins are involved in signal transduction, cell fate specification, cell cycle regulation, or some other developmental mechanism(s). However, this study demonstrates that the TMEM16 paralogs are expressed in several sites of active signaling and morphogenetic events and, therefore, might directly influence these processes. It is also possible that members of this protein family have a more general influence on cell biology. One intriguing hypothesis is that the TMEM16 family mediates aspects of cell adhesion. In support of this idea, the human ortholog TMEM16G has been shown to promote adhesion between prostate cancer cells in vitro (Das et al.,2007). Furthermore, preliminary data from our laboratory suggests that the morphology of several cell types is abnormal in Tmem16a mutant mice.

The expression data presented here suggest that the TMEM16 proteins might have roles in multiple developmental paradigms. In combination with their potential clinical relevance, we hope that this report will inspire further investigation into their functions in normal development as well as disease.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

For phylogenetic tree construction, the murine and human protein sequences in Supplementary Table 1 were aligned in ClustalX (v. 2.0.5) using default parameters. A neighbor-joining tree was generated from this alignment and viewed in NJPlot (v. 2.2). 1,000 bootstrap replicates were performed and values are shown on branches.

For RNA in situ hybridization, embryos were harvested from timed pregnant females and fixed in 4% paraformaldehyde in DEPC-treated PBS at 4°C overnight. Embryos for cryosectioning were washed in PBS and washed in 30% sucrose in PBS overnight at 4°C before embedding in OCT (Sakura, Torrance, CA).

Whole embryos, dissected lungs, or 14-μm sections were processed according to a standard RNA in situ hybridization protocol (Nieto et al.,1996). Plasmids, restriction enzymes, and RNA polymerases used to generate digoxigenin-labeled antisense riboprobes are listed in Supplementary Table 1. Images were acquired using a Lecia DFC300 FX camera (Leica Microsystems, Bannockburn, IL).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The authors thank Dr. Martin Cohn for assistance with phylogenetic analysis of the TMEM16 proteins and Dr. Amel Gritli-Linde for critical reading of the manuscript. J.R.R. was supported by a University of Florida Alumni Graduate Fellowship and B.D.H. was supported by startup funds from the University of Florida College of Medicine.

REFERENCES

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  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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DVDY21676SuppTableS1.xls16KTable S1

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