Factors affecting embryo production in superovulated Bos taurus cattle.

Despite a long history of bovine superovulation research, significant commercial applications did not start until the early 1970s. For some 20 years thereafter, superovulation represented the primary tool for the production of cattle embryos. In the early 1990s, commercial invitro production (IVP) was initiated in cattle. Although ovum pick-up and IVP are now commercially practiced on a wide scale, superovulation and embryo recovery by flushing remain a widespread and very effective approach to the production of cattle embryos. This review covers both the history and the effects of multiple factors on superovulation in Bos taurus cattle. There are three general protocols for suitable pre-FSH programming of donors so that gonadotrophin-responsive follicles are available. Superovulation protocols vary widely based on the FSH source, the diluent used, the number and timing of FSH injections and the timing and utilisation of various prostaglandins, controlled internal progesterone releasing devices, gonadotrophin-releasing hormone, and other means of controlling follicular development and ovulation. The number of oocytes that can be stimulated to grow and ovulate within any given donor can be estimated by either ultrasound-guided sonography or by measuring concentrations of anti-Müllerian hormone in the blood. Animal-related factors that can influence the efficacy of superovulation include cattle breed, age, parity, genetics, lactational status and reproductive history. In addition, nutrition, stress, season, climate, weather and several semen factors are discussed.

Anti-EDAR Agonist Antibody Therapy Resolves Palate Defects in Pax9-/- Mice.

To date, surgical interventions are the only means by which craniofacial anomalies can be corrected so that function, esthetics, and the sense of well-being are restored in affected individuals. Unfortunately, for patients with cleft palate-one of the most common of congenital birth defects-treatment following surgery is prolonged over a lifetime and often involves multidisciplinary regimens. Hence, there is a need to understand the molecular pathways that control palatogenesis and to translate such information for the development of noninvasive therapies that can either prevent or correct cleft palates in humans. Here, we use the well-characterized model of the Pax9-/- mouse, which displays a consistent phenotype of a secondary cleft palate, to test a novel therapeutic. Specifically, we demonstrate that the controlled intravenous delivery of a novel mouse monoclonal antibody replacement therapy, which acts as an agonist for the ectodysplasin (Eda) pathway, can resolve cleft palate defects in Pax9-/- embryos in utero. Such pharmacological interventions did not reverse the arrest in tooth, thymus, and parathyroid gland development, suggesting that the relationship of Pax9 to the Eda/Edar pathway is both unique and essential for palatogenesis. Expression analyses and unbiased gene expression profiling studies offer a molecular explanation for the resolution of palatal defects, showing that Eda and Edar-related genes are expressed in normal palatal tissues and that the Eda/Edar signaling pathway is downstream of Pax9 in palatogenesis. Taken together, our data uncover a unique relationship between Pax9 and the Eda/Edar signaling pathway that can be further exploited for the development of noninvasive, safe, and effective therapies for the treatment of cleft palate conditions and other single-gene disorders affecting the craniofacial complex.

Quality of life with biweekly docetaxel and capecitabine in advanced gastro-oesophageal cancer.

PURPOSE: This study aimed to evaluate the feasibility and tolerability of biweekly docetaxel with capecitabine as first-line treatment in advanced gastro-oesophageal cancer. METHODS: Fifty-three patients at median age of 61 years with advanced gastric cancer were included in this prospective, non-randomized, multicentre phase II trial to receive intravenous docetaxel 50 mg/m2 on days 1 and 15, and oral capecitabine 1250 mg/m2 every 12 h, on days 1-7 and 15-21 of each 28-day cycle. QOL was assessed using EORTC QLQ-C30, together with the gastric module (QLQ-STO 22). RESULTS: Forty-six patients were evaluable for QOL analyses. No deterioration in global health status was found. Social functioning scores improved, and eating difficulties and pain were alleviated during treatment. The most common grade 3 or 4 toxicity was neutropenia (47%), whereas neutropenic fever was uncommon (6%). The clinical benefit rate was 60%, including complete and partial responses as well as stabilized disease. Median overall survival was 8.8 months (95% CI 5.8-11.9 months), and median time to progression was 6.2 months (95% CI 4.9-7.5 months). CONCLUSIONS: Biweekly docetaxel with capecitabine is a feasible treatment in AGC, delivered on an outpatient basis, with no need for central venous access device. No deterioration of global health status was reported. In addition, pain and eating difficulties were alleviated during study treatment. This trial is registered at ClinicalTrials.gov , number NCT00669370.

Ectodysplasin A in Biological Fluids and Diagnosis of Ectodermal Dysplasia.

The tumor necrosis factor (TNF) family ligand ectodysplasin A (EDA) is produced as 2 full-length splice variants, EDA1 and EDA2, that bind to EDA receptor (EDAR) and X-linked EDA receptor (XEDAR/EDA2R), respectively. Inactivating mutations in Eda or Edar cause hypohidrotic ectodermal dysplasia (HED), a condition characterized by malformations of the teeth, hair and glands, with milder deficiencies affecting only the teeth. EDA acts early during the development of ectodermal appendages-as early as the embryonic placode stage-and plays a role in adult appendage function. In this study, the authors measured EDA in serum, saliva and dried blood spots. The authors detected 3- to 4-fold higher levels of circulating EDA in cord blood than in adult sera. A receptor binding-competent form of EDA1 was the main form of EDA but a minor fraction of EDA2 was also found in fetal bovine serum. Sera of EDA-deficient patients contained either background EDA levels or low levels of EDA that could not bind to recombinant EDAR. The serum of a patient with a V262F missense mutation in Eda, which caused a milder form of X-linked HED (XLHED), contained low levels of EDA capable of binding to EDAR. In 2 mildly affected carriers, intermediate levels of EDA were detected, whereas a severely affected carrier had no active EDA in the serum. Small amounts of EDA were also detectable in normal adult saliva. Finally, EDA could be measured in spots of wild-type adult or cord blood dried onto filter paper at levels significantly higher than that measured in EDA-deficient blood. Measurement of EDA levels combined with receptor-binding assays might be of relevance to aid in the diagnosis of total or partial EDA deficiencies.

Embryo yield in dairy cattle after superovulation with Folltropin or Pluset.

Two commercial FSH products were compared in a retrospective study on 3990 commercial superovulations and embryo recoveries in dairy heifers and cows. In addition, the 56-day nonreturn rate of 19,400 embryos produced with these two preparations was analyzed. Embryo collections were performed during a 16-year period from donors of Holstein and Ayrshire breeds. Folltropin (Vetoquinol S.A., Lure cedex, France) group (Group F) consisted of 2592 superovulations, of which 80% were performed on heifers and 20% on cows, and Pluset (Laboratorios Calier, S.A., Barcelona, Spain) group (Group P) of 1398 treatments, of which 66% and 34% were on heifers and cows, respectively. Total number of recovered structures, number of transferable embryos, and the proportion of unfertilized ova (UFO) and degenerated embryos were analyzed. Distribution of embryos into quality grades (1-3) and developmental stages (4-9) according to the IETS classification guidelines and means for each collection were evaluated. The proportion of low-responders having fewer than five corpora lutea and yielding fewer than five embryos or ova was investigated for each treatment. Group P yielded 1.1 recovered structures more than Group F (P < 0.001). Consequently, however, the number of transferable embryos did not differ among the groups, being 7.0 and 7.1 in Groups F and P, respectively. Instead, there was an increase in the number of UFO from 2.0 in Group F to 3.0 in Group P (P < 0.001). The quality of embryos and the developmental stages were similar between the groups and there was no difference in the proportion of low-responding donors in Group F and Group P. Also, there was no difference in the nonreturn rate after transfer of embryos originating from donors superovulated with Folltropin or Pluset. It was concluded that equal numbers of transferable embryos and pregnancies can be achieved with Folltropin and Pluset.

Quality and developmental rate of embryos produced with sex-sorted and conventional semen from superovulated dairy cattle.

This study investigated the effect of sex-sorted semen compared with conventional semen on the outcome of embryo recovery, placing special emphasis on the quality, and developmental stage of embryos. Data were analyzed for 443 embryo collections with sex-sorted semen (SEX group) and 1528 with conventional semen (CONV group) in superovulated dairy heifers and cows. The insemination protocol for conventional semen included two inseminations, comprising a total dose of 30 million sperm passing into the uterine body. For sex-sorted semen, two (30%) to three (70%) deep uterine inseminations were performed, the total dose ranging from eight to 12 million sperm. The data were analyzed separately for heifers and cows. The total number of recovered structures was similar among the groups. The number of viable embryos decreased in the SEX groups compared with the CONV (with 1.4 and 3.2 fewer embryos in heifers and cows, correspondingly, P < 0.001), and correspondingly the proportions of unfertilized ova and degenerated embryos increased in the SEX groups (P < 0.001). The proportion of unsuccessful collections, yielding no transferable embryos, increased in the SEX groups for both heifers (from 7.2% to 11.2%, P = 0.025) and cows (from 9.0% to 20.7%, P < 0.001). Regarding the quality of viable embryos, the quality grades were superior in the CONV group compared with the SEX group for heifers (P < 0.001) and cows (P < 0.001). The proportion of grade 1 embryos decreased by 6.5 percentage points in heifers and 11.9 percentage points in cows when sex-sorted semen was used. Correspondingly, the proportions of grade 2 and 3 embryos increased in heifers and cows when sexed semen was used. The mean developmental stages of embryo collections were numerically slightly lower in the SEX group. In heifers, the delay in developmental stage was statistically significant (P = 0.001), but in cows, there was only a tendency toward that (P = 0.067). In conclusion, sex-sorted sperm decreased the transferable embryo yield and increased the risk of a recovery yielding no transferable embryos. Furthermore, use of sex-sorted semen decreased the proportion of grade 1 embryos. In addition, it also seemed to delay embryonic development, although the delay in embryonic development was minimal and its biological relevance remains undefined. Despite the compromised embryo production, taken into account the optimization of recipient resources, the use of sex-sorted semen is advantageous, especially in superovulated heifers, which are of most importance in the modern breeding strategies using genomic selection.

Transfer of cattle embryos produced with sex-sorted semen results in impaired pregnancy rate and increased male calf mortality.

This study investigated the pregnancy rate and calf mortality after transfer of embryos produced using sex-sorted semen. Data for 12,438 embryo transfers performed on dairy farms were analyzed. Of these, 10,697 embryos were produced using conventional semen (CONV embryos) and 1741 using sex-sorted semen from 97 bulls (SEX embryos), predominantly of Ayrshire and Holstein breeds. Of the CONV embryos, 27.4% were transferred fresh, whereas of the SEX embryos, 55.7% were fresh. Recipient attributes (breed, parity, number of previous breeding attempts, and interval from calving to transfer) were comparable for both embryo types, heifers representing 57.8% of recipients in the CONV group and 54.8% in the SEX group. Recipients that were not artificially inseminated or did not undergo a new embryo transfer after the initial embryo transfer and had registered calving in fewer than 290 days after the transfer were considered pregnant. Pregnancy rate for recipients receiving CONV embryos was 44.1%, and for those receiving SEX embryos, it was 38.8%. The odds ratio for pregnancy in recipients receiving CONV embryos was 1.34 compared with SEX embryos (P < 0.001). The proportion of female calves was 49.6% and 92.3% in CONV and SEX groups, respectively. Overall, calf mortality was comparable in both groups. Mortality was similar in CONV and SEX groups (6.6% and 7.7%, respectively) for female calves. For male calves, mortality was 9.2% in the CONV group but significantly higher, 16.0% (P < 0.05), in the SEX group. This study showed that transfer of embryos produced with sex-sorted semen decreased the pregnancy rate by about 12% compared with embryos produced using conventional semen. Mortality of male calves born from SEX embryos was higher than for those born from CONV embryos.

Age-specific neuromuscular interaction during elderly habitual running.

AIM: It has been reported that advancing age causes tendons to become more compliant and fascicles length shorter. This could then lead to enhancement of movement efficiency provided that the elderly adults can activate their muscles in the same way as the younger adults (YOUNG) during dynamic movements. This study was designed to examine the age-specific behaviour of the medial gastrocnemius (MG) fascicles and tendinous tissues together with lower-leg muscle activities when the well-trained elderly runners ran on the treadmill at preferred speeds. METHODS: The well-trained 11 elderly subjects (ELD) who have running experiences and 11 YOUNG were recruited as subjects. While ELD were running on the treadmill at their preferred speed, the lengths of the MG fascicles and tendinous tissues (Lfa and LTT respectively) were measured by ultrasonography together with kinematics and lower-leg muscle activities. RESULTS: Although the behaviour of the MG muscle-tendon unit did not show any significant differences between both groups during the contact, our results showed significant differences in fascicle-tendinous tissue behaviour as well as muscle activities. The LTT during the entire contact phase was greater in ELD than in YOUNG (P < 0.001). Co-activation of lower-leg muscles from pre-activation to braking phases was higher in ELD than in YOUNG (P < 0.01). The changes of the Lfa during contact were less, and the LTT shortening was greater in ELD than in YOUNG (P < 0.001). CONCLUSION: These results imply that ELD cannot activate their muscles similar to YOUNG during running, and those different activities may modify the Lfa to utilize the tendon elasticity effectively.

Embryo production with sex-sorted semen in superovulated dairy heifers and cows.

The aim of this study was to examine the effect of sex-sorted semen on the number and quality of embryos recovered from superovulated heifers and cows on commercial dairy farm conditions in Finland. The data consist of 1487 commercial embryo collections performed on 633 and 854 animals of Holstein and Finnish Ayrshire breeds, respectively. Superovulation was induced by eight intramuscular injections of follicle-stimulating hormone, at 12-hour intervals over 4 days, involving declining doses beginning on 9 to 12 days after the onset of standing estrus. The donors were inseminated at 9 to 15-hour intervals beginning 12 hours after the onset of estrus with 2 + 2 (+1) doses of sex-sorted frozen-thawed semen (N = 218) into the uterine horns or with 1 + 1 (+1) doses of conventional frozen-thawed semen (N = 1269) into the uterine corpus. Most conventional semen (222 bulls) straws contained 15 million sperm (total number 30-45 million per donor). Sex-sorted semen (61 bulls) straws contained 2 million sperm (total number 8-14 million per donor). Mean number of transferable embryos in recoveries from cows bred with sex-sorted semen was 4.9, which is significantly lower than 9.1 transferable embryos recovered when using conventional semen (P ≤ 0.001). In heifers, no significant difference was detected between mean number of transferable embryos in recoveries using sex-sorted semen and conventional semen (6.1 and 7.2, respectively). The number of unfertilized ova was higher when using sex-sorted semen than when using conventional semen in heifers (P < 0.01) and in cows (P < 0.05), and the number of degenerated embryos in cows (P < 0.01), but not in heifers. It was concluded that the insemination protocol used seemed to be adequate for heifers. In superovulated cows, an optimal protocol for using sex-sorted semen remains to be found.

Effect of superficial collagen patterns and fibrillation of femoral articular cartilage on knee joint mechanics-a 3D finite element analysis.

Collagen fibrils of articular cartilage have specific depth-dependent orientations and the fibrils bend in the cartilage surface to exhibit split-lines. Fibrillation of superficial collagen takes place in osteoarthritis. We aimed to investigate the effect of superficial collagen fibril patterns and collagen fibrillation of cartilage on stresses and strains within a knee joint. A 3D finite element model of a knee joint with cartilage and menisci was constructed based on magnetic resonance imaging. The fibril-reinforced poroviscoelastic material properties with depth-dependent collagen orientations and split-line patterns were included in the model. The effects of joint loading on stresses and strains in cartilage with various split-line patterns and medial collagen fibrillation were simulated under axial impact loading of 1000 N. In the model, the collagen fibrils resisted strains along the split-line directions. This increased also stresses along the split-lines. On the contrary, contact and pore pressures were not affected by split-line patterns. Simulated medial osteoarthritis increased tissue strains in both medial and lateral femoral condyles, and contact and pore pressures in the lateral femoral condyle. This study highlights the importance of the collagen fibril organization, especially that indicated by split-line patterns, for the weight-bearing properties of articular cartilage. Osteoarthritic changes of cartilage in the medial femoral condyle created a possible failure point in the lateral femoral condyle. This study provides further evidence on the importance of the collagen fibril organization for the optimal function of articular cartilage.

Contrast-Enhanced Micro-Computed Tomography in Evaluation of Spontaneous Repair of Equine Cartilage.

OBJECTIVE: Contrast-enhanced computed tomography (CECT) has been introduced for the evaluation of cartilage integrity. Furthermore, CECT enables imaging of the structure and density of subchondral bone. In this laboratory study, we investigate the potential of microCECT to simultaneously image cartilage and subchondral bone for the evaluation of tissue healing. DESIGN: Osteochondral lesions (Ø = 6 mm) were surgically created in equine intercarpal joints (n = 7). After spontaneous healing for 12 months, the horses were sacrificed and osteochondral plugs (Ø = 14 mm), including the repair cartilage and adjacent intact tissue, were harvested. The nonfibrillar and fibrillar moduli and the permeability of cartilage were determined using indentation testing. Contrast agent diffusion into the samples was imaged for 36 hours using high-resolution CT. Results from CECT, mechanical testing, and microscopic analyses were compared and correlated. RESULTS: The contrast agent diffusion coefficient showed a significant (P < 0.05) difference between the repair and adjacent intact tissue. MicroCECT revealed altered (P < 0.05) bone volume fraction, mineral density, and microstructure of subchondral bone at the repair site. The contrast agent diffusion coefficient correlated with the moduli of the nonfibrillar matrix (R = -0.662, P = 0.010), collagen fibril parallelism index (R = -0.588, P = 0.035), and glycosaminoglycan content (R = -0.503, P = 0.067). The repair cartilage was mechanically and structurally different from adjacent intact tissue (P < 0.05). CONCLUSIONS: MicroCECT enabled simultaneous quantitative evaluation of subchondral bone and monitoring of cartilage repair, distinguishing quantitatively the repair site from the adjacent intact tissue. As the only technique able to simultaneously image cartilage and determine subchondral bone mineral density and microstructure, CECT has potential clinical value.

Keratinocyte-specific ablation of the NF-κB regulatory protein A20 (TNFAIP3) reveals a role in the control of epidermal homeostasis.

The ubiquitin-editing enzyme A20 (tumor necrosis factor-α-induced protein 3) serves as a critical brake on nuclear factor κB (NF-κB) signaling. In humans, polymorphisms in or near the A20 gene are associated with several inflammatory disorders, including psoriasis. We show here that epidermis-specific A20-knockout mice (A20(EKO)) develop keratinocyte hyperproliferation, but no signs of skin inflammation, such as immune cell infiltration. However, A20(EKO) mice clearly developed ectodermal organ abnormalities, including disheveled hair, longer nails and sebocyte hyperplasia. This phenotype resembles that of mice overexpressing ectodysplasin-A1 (EDA-A1) or the ectodysplasin receptor (EDAR), suggesting that A20 negatively controls EDAR signaling. We found that A20 inhibited EDAR-induced NF-κB signaling independent from its de-ubiquitinating activity. In addition, A20 expression was induced by EDA-A1 in embryonic skin explants, in which its expression was confined to the hair placodes, known to be the site of EDAR expression. In summary, our data indicate that EDAR-induced NF-κB levels are controlled by A20, which functions as a negative feedback regulator, to assure proper skin homeostasis and epidermal appendage development.

Transplantation of normal boar testicular cells resulted in complete focal spermatogenesis in a boar affected by the immotile short-tail sperm defect.

Transplantation of testicular cells, also known as spermatogonial stem cell transplantation, is a relatively new approach in the field of male infertility. We used this technique to determine whether donor-derived sperm production in unrelated porcine recipients is possible following ultrasound-guided transfer of testicular cells. This study was undertaken because we had a strain of Finnish Yorkshire boars with a hereditary recessive gene defect rendering all spermatozoa immotile and anatomically abnormal in homozygous boars. Thus, monitoring of the focal success of colonization of donor spermatogonia with subsequent production of progressively motile spermatozoa was extremely sensitive. Testicular cells from young normal crossbred boars were transplanted into the testes of two boars affected with the immotile short-tail sperm (ISTS) defect. Prior to the transplantations, busulfan was used to suppress recipients' endogenous spermatogenesis. The ejaculates were collected and analysed for the presence of motile spermatozoa. In one of the two recipient boars transplanted with testicular cells from normal donors, motile spermatozoa appeared in the ejaculates 12 weeks after the transplantation. Spermatozoa manually selected under a microscope from a frozen aliquot of ejaculate collected 27 weeks after transplantation were genotyped. In two of the 20 vials the donor-derived genotype was visible. The genotyping results substantiated the success - as indicated by the appearance of motile spermatozoa after the spermatogonial transfer. Thus, donor-derived sperm production in unrelated recipients is possible. In addition, the production after transplantation of progressively motile spermatozoa with normal tail lengths shows that the ISTS defect in Finnish Yorkshire boars apparently results from defective transcription of an essential gene for sperm motility in germline cells. To conclude, the transplantation of donor testicular cells can, at least in boars with the ISTS defect, result in complete focal spermatogenesis.

Mutational analysis of the human SLC26A8 gene: exclusion as a candidate for male infertility due to primary spermatogenic failure.

SLC26A8 is an anion transporter that is solely expressed in the testes. It interacts with MgcRacGAP that shows strong structural similarity with the Drosophila protein RotundRacGAP, which is established to have an essential role for male fertility in the fruit fly. To explore whether the SLC26A8 gene has a role in human male infertility, we performed mutational analysis in the coding region of the SLC26A8 gene in 83 male infertility patients and two groups of controls using single-strand conformational polymorphism and direct sequencing methods. We found six novel coding sequence variations, of which five lead to amino acid substitutions. All variants were found with similar frequencies in both patients and controls, thus suggesting that none of them may be causally associated with infertility. We conclude that the SLC26A8 mutations are not a common cause of male infertility.

Oral treatment of acute myeloid leukaemia with etoposide, thioguanine, and idarubicin (ETI) in elderly patients: a prospective randomised comparison with intravenous cytarabine, idarubicin, and thioguanine in the second and third treatment cycle.

A randomised multicentre study was conducted among patients over 65 yr of age with newly diagnosed acute myeloid leukaemia (AML) to compare oral treatment with etoposide 80 mg/m(2) and thioguanine 100 mg/m(2) twice daily on 5 d and idarubicin 15 mg/m(2) on 3 d (ETI) to a mainly i.v. combination of cytarabine 100 mg/m(2) twice daily on 5 d, idarubicin 12 mg/m(2) x 1, and thioguanine (TAI). Ninety-two patients were enrolled. Their median age was 72 yr, range 65-84 yr. Sixty-five patients had de novo AML, 21 AML subsequent to myelodysplastic syndrome, and six treatment-related AML. They received at first a 6-d i.v. treatment with cytarabine and idarubicin. After the first treatment, 68 patients were randomised to receive two cycles of ETI (n = 36) or TAI (n = 32) and thereafter maintenance with mercaptopurine and methotrexate. Of the 92 patients, 52 (57%) achieved remission at some stage. The median survival was 10 months. There were no significant differences between the patients randomised to ETI or TAI in the remission rate (67% vs. 72%), survival (12 months from randomisation in both arms), event-free survival or relapse rate. The patients randomised to receive ETI spent significantly fewer days at hospital during the two randomised cycles (20 vs. 41 d, P = 0.010), and they had fewer days with infusions, shorter neutropenias and thrombocytopenias and fewer and less severe infections. In conclusion, treatment with oral ETI resulted in a similar antileukaemic effect as obtained with mainly i.v. TAI, with less toxicity and reduced need for hospitalisation.

Cochlear distribution of Na,K-ATPase and corticosteroid receptors in two mouse strains with congenital hearing disorders.

As corticosteroid hormones, via their receptors, and Na,K-ATPase are thought to be involved in the regulation of endolymph production, two mouse models were used to investigate whether degeneration of the stria vascularis (SV) and disturbed endolymph composition are correlated with changes in the amounts and distribution of corticosteroid receptors and Na,K-ATPase in the cochlea. Both the shaker-2 mouse and the newly discovered mix mouse are deaf at birth and show vestibular dysfunction. In both mouse strains, the SV is degenerated and endolymph production is severely disturbed. In the shaker-2 mouse, using the C57Bl mouse as a normal control, immunohistochemical staining of mineralo- and glucocorticoid receptors (MR and GR) and the Na,K-ATPase subunits alpha1, alpha3 and beta1 showed a weaker reaction in all structures of the cochlea. The inner ear morphology of the mix mouse is described and compared to that of asymptomatic littermates. Immunostaining of MR, GR and the different Na,K-ATPase subunits in this mouse was considerably weaker in the SV, while staining intensities were normal in the remaining cochlea. The reduced corticosteroid receptor levels may lead to a reduction in Na,K-ATPase expression in the same tissues, although this conclusion should be treated with caution. The conclusion that reduced Na,K-ATPase levels in both mouse strains may be an important mechanism of the disturbed endolymph production is less controversial.

Signaling and subcellular localization of the TNF receptor Edar.

Tabby and downless mutant mice have identical phenotypes characterized by deficient development of several ectodermally derived organs such as teeth, hair, and sweat glands. Edar, encoded by the mouse downless gene and defective in human dominant and recessive forms of autosomal hypohidrotic ectodermal dysplasia (EDA) syndrome, is a new member of the tumor necrosis factor (TNF) receptor superfamily. The ligand of Edar is ectodysplasin, a TNF-like molecule mutated in the X-linked form of EDA and in the spontaneous mouse mutant Tabby. We have analyzed the response of Edar signaling in transfected cells and show that it activates nuclear factor-kappaB (NF-kappaB) in a dose-dependent manner. When Edar was expressed at low levels, the NF-kappaB response was enhanced by coexpression of ectodysplasin. The activation of NF-kappaB was greatly reduced in cells expressing mutant forms of Edar associated with the downless phenotype. Overexpression of Edar did not activate SAPK/JNK nor p38 kinase. Even though Edar harbors a death domain its overexpression did not induce apoptosis in any of the four cell lines analyzed, nor was there any difference in apoptosis in developing teeth of wild-type and Tabby mice. Additionally, we show that the subcellular localization of dominant negative alleles of downless is dramatically different from that of recessive or wild-type alleles. This together with differences in NF-kappaB responses suggests an explanation for the different mode of inheritance of the different downless alleles.

Ectodysplasin is released by proteolytic shedding and binds to the EDAR protein.

Anhidrotic ectodermal dysplasia (EDA) is an X-linked disorder characterized by abnormal development of ectoderm and its appendices. The EDA gene encodes different isoforms of ectodysplasin, a transmembrane protein. The two longest isoforms, ectodysplasin-A1 and -A2, which differ by an insertion of two amino acids, are trimeric type II membrane proteins with an extracellular portion containing a short collagenous domain and a TNF ligand motif in the C-terminal region. We show that ectodysplasin is released from cells to the culture medium. Deletion constructs were used to localize the cleavage site and show that the putative recognition sequence of a furin-like enzyme is needed for the cleavage. Some EDA patients have missense mutations affecting this recognition sequence, suggesting that cleavage has biological significance in vivo. EDAR, a recently cloned member of the TNFR family and the product of the downless gene, is able to co-precipitate ectodysplasin, confirming that they form a ligand-receptor pair. In situ hybridization and immunostaining studies show that ectodysplasin and EDAR are expressed in adjacent or partially overlapping layers in the developing human skin. We conclude that as a soluble ligand, ectodysplasin is able to interact with EDAR and mediate signals needed for the development of ectodermal appendages.

TNF signaling via the ligand-receptor pair ectodysplasin and edar controls the function of epithelial signaling centers and is regulated by Wnt and activin during tooth organogenesis.

Ectodermal dysplasia syndromes affect the development of several organs, including hair, teeth, and glands. The recent cloning of two genes responsible for these syndromes has led to the identification of a novel TNF family ligand, ectodysplasin, and TNF receptor, edar. This has indicated a developmental regulatory role for TNFs for the first time. Our in situ hybridization analysis of the expression of ectodysplasin (encoded by the Tabby gene) and edar (encoded by the downless gene) during mouse tooth morphogenesis showed that they are expressed in complementary patterns exclusively in ectodermal tissue layer. Edar was expressed reiteratively in signaling centers regulating key steps in morphogenesis. The analysis of the effects of eight signaling molecules in the TGFbeta, FGF, Hh, Wnt, and EGF families in tooth explant cultures revealed that the expression of edar was induced by activinbetaA, whereas Wnt6 induced ectodysplasin expression. Moreover, ectodysplasin expression was downregulated in branchial arch epithelium and in tooth germs of Lef1 mutant mice, suggesting that signaling by ectodysplasin is regulated by LEF-1-mediated Wnt signals. The analysis of the signaling centers in tooth germs of Tabby mice (ectodysplasin null mutants) indicated that in the absence of ectodysplasin the signaling centers were small. However, no downstream targets of ectodysplasin signaling were identified among several genes expressed in the signaling centers. We conclude that ectodysplasin functions as a planar signal between ectodermal compartments and regulates the function, but not the induction, of epithelial signaling centers. This TNF signaling is tightly associated with epithelial-mesenchymal interactions and with other signaling pathways regulating organogenesis. We suggest that activin signaling from mesenchyme induces the expression of the TNF receptor edar in the epithelial signaling centers, thus making them responsive to Wnt-induced ectodysplasin from the nearby ectoderm. This is the first demonstration of integration of the Wnt, activin, and TNF signaling pathways.