Case Series of Patients With Hereditary Benign Intraepithelial Dyskeratosis.

PURPOSE: The purpose of this retrospective case series was to compare the outcomes of different treatment options for patients diagnosed with hereditary benign intraepithelial dyskeratosis (HBID). METHODS: The study is designed as a single-institution retrospective chart review of patients who were clinically diagnosed with HBID during their care at the Duke Eye Center. Patient demographics were obtained, and disease course after different therapies was analyzed. RESULTS: Seventeen patients were diagnosed with HBID. 52.9% (9/17) of patients identified with HBID reported Native American ancestry. Medical therapy alone failed to reduce the size or number of corneal lesions in any patient identified in this study. Ten of the 17 patients required surgical intervention. Two eyes received corneal biopsies, 3 eyes received a full conjunctival lesion excision with amniotic membrane grafting, 12 eyes received superficial keratectomy with amniotic membrane grafting, and 1 eye received keratoprosthesis. Lesion recurrence was seen in 9 of the 10 patients treated with surgical excision with an average time to recurrence of 1.5 and 2 months for conjunctival excisions and superficial keratectomy, respectively, when excluding patients who missed scheduled postoperative follow-up appointments. CONCLUSIONS: Hereditary benign intraepithelial dyskeratosis is a rare and poorly understood disorder that predominantly affects people with Native American ancestry. Medical therapy only provides symptomatic relief, and patients who receive surgical excision almost always develop recurrence. As a result, we recommend future investigations focus on identifying the optimal surgical technique and timing to limit the morbidity of HBID and improve outcomes.

Identificação de alterações bioquímicas em células epiteliais bronquiais humanas normais expostas a fumarato / Identification of biochemical changes in normal human bronchial epitelial cells exposed to fumarate

A reprogramação metabólica de células do câncer é apontada como uma característica essencial para o desenvolvimento da doença (cancer hallmark). Estudos mostram que mutações na enzima fumarato hidratase levam ao aumento da concentração intra e extracelular de fumarato, o que ocorre paralelamente à indução da transformação maligna. Neste trabalho, a fim de entender se o excesso de fumarato extracelular pode propiciar a transformação de células normais, foram quantificados alguns endpoints relacionados aos efeitos do fumarato e à transformação maligna, além de alterações metabólicas em células imortalizadas de epitélio brônquico humano normal (BEAS-2B) expostas ao fumarato. Uma vez que fumarato nas concentrações de 0,1 a 10 mM ao longo de 144 h não foi citotóxico, foram selecionadas as concentrações de 1 mM, 5 mM e 10 mM para as incubações. Fumarato induziu a formação de colônias em soft-agar após o período de sete dias (168 h) de exposição, o que indica a indução de transformação celular. Fumarato é um oncometabólito que inibe enzimas que dependem de α-cetoglutarato como co-substrato, dentre as quais as enzimas ten eleven translocation (TET) que catalisam a formação de 5-hidroximetilcitosina (5-hmC) a partir de 5-metilcitosina (5-mC), o primeiro passo da sequência de reações que levam à desmetilação do DNA. Os níveis totais de 5-hmC estavam diminuídos no DNA das células expostas. Colônias retiradas do soft-agar (controle, 1, 5 e 10 mM de fumarato) foram cultivadas e, após 90 dias em cultura, as células foram submetidas ao ensaio de invasão e migração em câmara de Boyden (transwell), tendo sido observada maior capacidade de migração/invasão das células anteriormente expostas ao fumarato. Foi observada indução de estresse redox nas células expostas ao fumarato. A partir da quantificação de metabólitos intracelulares por HPLC-ESI-MS/MS e HPLC-ESI-Q-TOF, verificamos que as células BEAS-2B absorveram o fumarato adicionado ao meio de cultura, o qual foi convertido intracelularmente a malato, aspartato, argininosuccinato, citrato, succinato e glutamato. O oncometabólito 2-L-hidroxiglutarato foi detectado em níveis aumentados nas células expostas a fumarato, assim como adenosina, enquanto que NAD+ e NADP+ apareceram diminuídos. As alterações metabólicas na presença de fumarato contribuíram para a manutenção do balanço energético das células, ou mesmo para um saldo positivo de energia. A exposição das células a [13C4]fumarato permitiu a análise do fluxo inicial do fumarato absorvido pelas células. A partir dessa análise verificamos que o fumarato absorvido entra no ciclo de Krebs, gerando malato, que é em grande parte desviado para reações externas ao ciclo, como a geração de aspartato e argininosuccinato. Citrato proveniente das reações de [13C4]fumarato no ciclo de Krebs foi detectado em níveis inferiores aos endógenos. O uso de [13C4]fumarato permitiu a visualização da geração de [13C4]succinato, que tem como possível fonte a atividade reversa da succinato desidrogenase. Verificamos também a geração de [13C3]glutamato. Supõe-se que as alterações metabólicas induzidas pelo fumarato absorvido pelas células BEAS-2B contribuam para a modulação da expressão de genes e da atividade de proteínas que favorecem o processo tumorigênico

The metabolic reprogramming of cancer cells is identified as an essential feature for the development of the disease (a cancer hallmark). Studies show that mutations in the enzyme fumarate hydratase lead to increased intra- and extracellular fumarate concentration, which occurs in parallel with the induction of malignant transformation. In this work, in order to understand if excess extracellular fumarate can lead to the transformation of normal cells, some endpoints related to the effects of fumarate and malignant transformation were quantified, as well as metabolic alterations in the immortalized normal human bronchial epithelial cell line BEAS-2B exposed to fumarate. Since fumarate at concentrations from 0.1 to 10 mM over 144 h was not cytotoxic, the concentrations of 1 mM, 5 mM and 10 mM were selected for incubations. Fumarate induced colony formation in soft agar after the seven day (168 h) exposure period, which indicates the induction of cell transformation. Fumarate is an oncometabolite that inhibits α-ketoglutarate-dependent enzymes, among which are ten eleven translocation (TET) enzymes that catalyze the formation of 5-hydroxymethylcytosine (5-hmC) from 5-methylcytosine (5-mC), the first step in the sequence of reactions leading to DNA demethylation. Total 5-hmC levels were decreased in the DNA of exposed cells. Colonies removed from the soft-agar (control, 1, 5 and 10 mM fumarate) were cultured and after 90 days in culture the cells were subjected to the Boyden chamber (transwell) invasion and migration assay, and a greater capacity for migration/invasion of cells previously exposed to fumarate was observed. Redox stress induction was observed in cells exposed to fumarate. From the quantification of intracellular metabolites by HPLC-ESI-MS/MS and HPLC-ESI-Q-TOF, we found that BEAS-2B cells absorbed the fumarate added to the culture medium, which was intracellularly converted to malate, aspartate, argininosuccinate, citrate, succinate and glutamate. The oncometabolite 2-L-hydroxyglutarate was detected at increased levels in cells exposed to fumarate, as well as adenosine, while NAD+ and NADP+ appeared decreased. Metabolic changes in the presence of fumarate contributed to the maintenance of the energy balance of the cells, or even to a positive energy balance. Exposure of cells to [13C4]fumarate allowed the analysis of the initial flow of the fumarate absorbed by the cells. From this analysis we found that the absorbed fumarate entered the Krebs cycle, generating malate, which was largely diverted to reactions outside the cycle, such as the generation of aspartate and argininosuccinate. Citrate from the reactions of [13C4]fumarate in the Krebs cycle was detected at levels lower than endogenous. The use of [13C4]fumarate allowed the detection of [13C4]succinate, which has as its possible source the reverse activity of succinate dehydrogenase. We also observed the generation of [13C3]glutamate. The metabolic changes induced by the absorbed fumarate are supposed to contribute to the modulation of gene expression and protein activity that favor the tumorigenic process

Thymic Epithelium Abnormalities in DiGeorge and Down Syndrome Patients Contribute to Dysregulation in T Cell Development.

The thymus plays a fundamental role in establishing and maintaining central and peripheral tolerance and defects in thymic architecture or AIRE expression result in the development of autoreactive lymphocytes. Patients with partial DiGeorge Syndrome (pDGS) and Down Syndrome (DS) present alterations in size and architecture of the thymus and higher risk to develop autoimmunity. We sought to evaluate thymic architecture and thymocyte development in DGS and DS patients and to determine the extent to which thymic defects result in immune dysregulation and T cell homeostasis perturbation in these patients. Thymi from pediatric patients and age-matched controls were obtained to evaluate cortex and medullary compartments, AIRE expression and thymocyte development. In the same patients we also characterized immunophenotype of peripheral T cells. Phenotypic and functional characterization of thymic and peripheral regulatory T (Treg) cells was finally assessed. Histologic analysis revealed peculiar alterations in thymic medulla size and maturation in DGS and DS patients. Perturbed distribution of thymocytes and altered thymic output was also observed. DGS patients showed lower mature CD4+ and CD8+ T cell frequency, associated with reduced proportion and function of Tregs both in thymus and peripheral blood. DS patients showed increased frequency of single positive (SP) thymocytes and thymic Treg cells. However, Tregs isolated both from thymus and peripheral blood of DS patients showed reduced suppressive ability. Our results provide novel insights on thymic defects associated with DGS and DS and their impact on peripheral immune dysregulation. Indeed, thymic abnormalities and defect in thymocyte development, in particular in Treg cell number and function could contribute in the pathogenesis of the immunodysregulation present in pDGS and in DS patients.

Non-muscle myosin II deletion in the developing kidney causes ureter-bladder misconnection and apical extrusion of the nephric duct lineage epithelia.

In kidney development, connection of the nephric duct (ND) to the cloaca and subsequent sprouting of the ureteric bud (UB) from the ND are important for urinary exit tract formation. Although the roles of Ret signaling are well established, it remains unclear how intracellular cytoskeletal proteins regulate these morphogenetic processes. Myh9 and Myh10 encode two different non-muscle myosin II heavy chains, and Myh9 mutations in humans are implicated in congenital kidney diseases. Here we report that ND/UB lineage-specific deletion of Myh9/Myh10 in mice caused severe hydroureter/hydronephrosis at birth. At mid-gestation, the mutant ND/UB epithelia exhibited aberrant basal protrusion and ectopic UB formation, which likely led to misconnection of the ureter to the bladder. In addition, the mutant epithelia exhibited apical extrusion followed by massive apoptosis in the lumen, which could be explained by reduced apical constriction and intercellular adhesion mediated by E-cadherin. These phenotypes were not ameliorated by genetic reduction of the tyrosine kinase receptor Ret. In contrast, ERK was activated in the mutant cells and its chemical inhibition partially ameliorated the phenotypes. Thus, myosin II is essential for maintaining the apicobasal integrity of the developing kidney epithelia independently of Ret signaling.

Effect of folic acid in a modified experimental model of anorectal malformations adriamycin-induced in rats.

PURPOSE: To determine the effect of a single dose of adriamycin (ADR) to induce anorectal malformations (ARMs) and determine the effect of folic acid (FA) in this model. METHODS: Ten female Wistar rats were divided randomly in two groups. Group A - ADR; Group B - FA+ADR. Dams from group B received daily, since two weeks before the pregnancy to the end of pregnancy, FA (50mg/kg) by gavage. Dams from both groups received ADR (6mk/kg) by intraperitoneal injection on gestational day (GD) 8. Their fetuses were harvested by cesarean section on GD21 and were examined looking for ARMs. The thickness of anal stratified squamous epithelium (ASSE) and intestinal epithelium (IE) were analyzed. p≤0.05*. RESULTS: 81 fetuses were harvested. The number of fetuses; number of ARMs; mean (∆%) (± SD) were determined to be, respectively: ADR - 41[29;65%(±37%)] versus FA+ADR - 40[04;16%(±36%)] (p=0.05). AMRs were significantly lower in FA+ADR group than in ADR group (p=0.05). The thickness (µm) of ASSE (± SD) and IE (± SD) were measured, respectively: ADR - [25.98(±0.74) and 19.48(±1.68)] versus FA+ADR - [24.74(±0.91) and 24.80(±0.81)] (p<0.005). The thickness of IE was significantly enlarged when FA was given (p<0.005). CONCLUSIONS: Single dose of adriamycin on D8 was able to induce anorectal malformations. Folic acid reduces the number and enlarged the IE of ARMs ADR-induced.

Twist as a new prognostic marker in hematological malignancies

Twist proteins are members of basic helix-loop-helix family and are major regulators of embryogenesis. In adult humans, Twist proteins are mainly expressed in precursor cells, including myogenic, osteoblastic, chondroblastic and myelomonocytic lineages, maintaining their undifferentiated state. In addition, they play important roles in lymphocyte function and maturation. Recently, several studies have reported regulatory roles for Twist in the function and development of hematopoietic cells as well as in survival and development of numerous hematological malignancies. It is activated by numerous signal transduction pathways, including Akt, nuclear factor κB, Wnt, signal transducer and activator of transcription 3, mitogen-activated protein kinase and Ras signaling. Activated Twist has an anti-apoptotic role and protects cancer cells from apoptotic cell death. In addition, overexpression of Twist promotes the process of epithelial-mesenchymal transition, which has an essential role in cancer metastasis. Hereby, we review the aberrant expression of Twist in hematopoietic malignancies such as leukemias, lymphomas and myelodysplastic syndrome, which is related with poor prognosis and drug resistance in these disorders. Inactivation of Twist by small RNAs technology or chemotherapeutic inhibitors targeting Twist and upstream or downstream molecules of Twist signaling pathways may be helpful in management of disease to improve treatment strategies in malignancies (AU)

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Effect of folic acid in a modified experimental model of anorectal malformations adriamycin-induced in rats

PURPOSE: To determine the effect of a single dose of adriamycin (ADR) to induce anorectal malformations (ARMs) and determine the effect of folic acid (FA) in this model. METHODS: Ten female Wistar rats were divided randomly in two groups. Group A - ADR; Group B - FA+ADR. Dams from group B received daily, since two weeks before the pregnancy to the end of pregnancy, FA (50mg/kg) by gavage. Dams from both groups received ADR (6mk/kg) by intraperitoneal injection on gestational day (GD) 8. Their fetuses were harvested by cesarean section on GD21 and were examined looking for ARMs. The thickness of anal stratified squamous epithelium (ASSE) and intestinal epithelium (IE) were analyzed. p≤0.05*. RESULTS: 81 fetuses were harvested. The number of fetuses; number of ARMs; mean (∆%) (± SD) were determined to be, respectively: ADR - 41[29;65%(±37%)] versus FA+ADR - 40[04;16%(±36%)] (p=0.05). AMRs were significantly lower in FA+ADR group than in ADR group (p=0.05). The thickness (µm) of ASSE (± SD) and IE (± SD) were measured, respectively: ADR - [25.98(±0.74) and 19.48(±1.68)] versus FA+ADR - [24.74(±0.91) and 24.80(±0.81)] (p<0.005). The thickness of IE was significantly enlarged when FA was given (p<0.005). CONCLUSIONS: Single dose of adriamycin on D8 was able to induce anorectal malformations. Folic acid reduces the number and enlarged the IE of ARMs ADR-induced.

Hereditary Benign Intraepithelial Dyskeratosis: Report of a Case and Re-examination of the Evidence for Locus Heterogeneity.

BACKGROUND: Hereditary benign intraepithelial dyskeratosis (HBID) is a rare autosomal-dominant disorder of the conjunctiva and oral mucosa first described in and predominantly affecting descendents of Haliwa-Saponi Native Americans. We report a spontaneous case of histopathologically-confirmed HBID affecting an individual not of Native American ancestry. MATERIALS AND METHODS: Report of a case with histopathologic examination of an excised conjunctival specimen as well as molecular and cytogenetic analysis. RESULTS: A Caucasian boy with a history of oral lesions and conjunctival injection from birth developed bilateral corneal opacities at age 5 and underwent penetrating keratoplasty, with recurrence of the corneal opacification shortly after surgery. Examination of a conjunctival biopsy specimen revealed features consistent with HBID. Copy number variant (CNV) analysis revealed a de novo 4q35 duplication that overlapped the duplication previously associated with HBID, although no genes were identified in the common interval. NLRP1 gene sequencing failed to reveal a presumed pathogenic variant. CONCLUSIONS: HBID may develop de novo in individuals who are not of Native American ancestry. The absence of coding regions in a duplicated region of 4q35 common to both the individual that we report and previously associated with HBID raises questions regarding the significance of this CNV in the pathogenesis of HBID.

Increased nuclear ß-catenin expression in oral potentially malignant lesions: A marker of epithelial dysplasia.

BACKGROUND: Deregulation of ß-catenin is associated with malignant transformation; however, its relationship with potentially malignant and malignant oral processes is not fully understood. The aim of this study was to determine and compare the nuclear ß-catenin expression in oral dysplasia and oral squamous cell carcinoma (OSCC). MATERIAL AND METHODS: Cross sectional study. Immunodetection of ß-catenin was performed on 72 samples, with the following distribution: 21 mild dysplasia, 12 moderate dysplasia, severe dysplasia 3, 36 OSCC including 19 well differentiated, 15 moderately differentiated and 2 poorly differentiated. Through microscopic observation the number of positive cells per 1000 epithelial cells was counted. For the statistical analysis, the Kruskal Wallis test was used. RESULTS: Nuclear expression of ß-catenin was observed in all samples with severe and moderate dysplasia, with a median of 267.5, in comparison to mild dysplasia whose median was 103.75. Only 10 samples (27.7%) with OSCC showed nuclear expression, with statistically significant differences between groups (p < 0.05). CONCLUSIONS: Our results are consistent with most of the reports which show increased presence of ß-catenin in severe and moderate dysplasia compared to mild dysplasia; however the expression of nuclear ß-catenin decreased after starting the invasive neoplastic process. This suggests a role for this protein in the progression of dysplasia and early malignant transformation to OSCC. Immunodetection of ß-catenin could be a possible immune marker in the detection of oral dysplasia.

Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis.

Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1(+/-) embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure.

Congenital lesions of epithelial origin.

Defects of embryologic development give rise to a variety of congenital lesions arising from the epithelium and are among the most common congenital lesions of the head and neck in the pediatric population. This article presents several congenital lesions of epithelial origin, including congenital midline cervical cleft, pilomatrixoma, dermoid, foregut duplication cysts, and preauricular sinuses and pits. In addition, the management of these lesions is reviewed.

Abnormal WT1 expression in human fetuses with bilateral renal agenesis and cardiac malformations.

BACKGROUND: Bilateral renal agenesis has multiple etiologies. Animal models have provided useful information on possible causes of this condition, but its etiology in humans is less clear. We recently described autopsy findings of two human fetuses with bilateral renal agenesis and abnormal expression of WT1 (Wilms tumor 1) in liver mesothelium. METHODS: We have identified 14 additional fetuses with bilateral renal agenesis from autopsies performed in our institution over the past 10 years and subjected archival liver biopsy specimens from these cases to immunohistochemistry for WT1, as well as α-smooth muscle actin (α-SMA) and desmin to assess liver mesenchymal abnormalities. RESULTS: Six of seven fetuses with combined bilateral renal agenesis and cardiac anomalies showed abnormalities of WT1 expression in liver mesothelial cells, which was not seen in other fetuses with bilateral renal agenesis. Except in one case, the fetuses with renal agenesis and cardiac defects also showed liver mesenchymal anomalies (assessed by increased α-SMA expression), which was not present in other renal agenesis fetuses. CONCLUSIONS: WT1 is widely expressed in mesothelial cells during development, and we hypothesized that some of the defects are caused by abnormal function of mesenchyme derived from mesothelial cells, similar to the mesothelium-derived defects proposed in animal models. The methods we used are available to many laboratories and can be applied to archival paraffin tissue blocks. We suggest that future similar studies could help to expand the understanding of renal agenesis in humans and could help to subclassify this condition. This would be useful in patient management and counseling.

The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled.

Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)-family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of null alleles. We generated null alleles of the single Drosophila ZO protein Polychaetoid (Pyd). Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic null mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

On the role of Eph signalling in thymus histogenesis; EphB2/B3 and the organizing of the thymic epithelial network.

In the current study, we extend our own previous results on the thymocyte phenotype of EphB2 and/or EphB3 deficient mice by analyzing the phenotype and the histological organization of their thymic epithelial stroma. All studied adult EphB-deficient thymi showed profound alterations with respect to the wild-type (WT) ones. Each mutant exhibited a specific phenotype, but also showed common features including occurrence of K5+K8+MTS10+ immature medullary epithelial cells, numerous K5-K8-MTS20+ cells and K5+K8+ cells in the thymic cortex and cortical and medullary K5-K8- areas devoid of epithelial cell markers. In addition, comparative analysis of WT and EphB-deficient embryonic and newborn thymi demonstrated that the observed adult phenotype was a consequence of the gradual accumulation of early phenotypic and morphological defects, becoming more severe at the end of embryonic life and in newborn animals. Together, these results confirm a role for EphB2 and EphB3 in thymus morphogenesis. The obtained data are discussed from the point of view of the recognized role played by these two Ephs in the homeostasis of other epithelia and their possible relationships with molecules known to be involved in thymic epithelial cell development.

Cytogenic abnormalities in buccal cells during spinal deformities in children.

Evaluation of the incidence of nucleus abnormalities in buccal epithelium allows detecting the presence and intensity of the effect of various ecological conditions and pathologies of the musculoskeletal system. Two coefficients were used: mean number of NA per cell and ratio of cells with karyolysis to the total number of cells with NA. Coefficient of karyolysis decreases with increasing anthropogenic load In pupils of a special school in Moscow these coefficients were similar. Analysis of coefficients showed that karyolysis coefficient was reduced in mothers of children with spinal deformities.

A dominant negative allele of the Drosophila leucine zipper protein Bunched blocks bunched function during tissue patterning.

The bunched (bun) gene encodes the Drosophila member of the TSC-22/GILZ family of leucine zipper transcriptional regulators. The bun locus encodes multiple BUN protein isoforms and has diverse roles during patterning of the eye, wing margin, dorsal notum and eggshell. Here we report the construction and activity of a dominant negative allele (BunDN) of the BUN-B isoform. In the ovary, BunDN expression in the follicle cells (FC) resulted in epithelial defects including aberrant accumulation of DE-cadherin and failure to rearrange into columnar FC cell shapes. BunDN expression in the posterior FC led to loss of epithelial integrity associated with extensive apoptosis. BunDN FC phenotypes collectively resemble loss-of-function bun mutant phenotypes. BunDN expression using tissue-specific imaginal disk drivers resulted in characteristic cuticular patterning defects that were enhanced by bun mutations and suppressed by co-expression of the BUN-B protein isoform. These data indicate that BunDN has dominant negative activity useful to identify bun functions and genetic interactions that occur during tissue patterning.

Combined loss of Hey1 and HeyL causes congenital heart defects because of impaired epithelial to mesenchymal transition.

Congenital heart defects affect almost 1% of human newborns. Recently, mutations in Notch ligands and receptors have been found to cause a variety of heart defects in rodents and humans. However, the molecular effects downstream of Notch are still poorly understood. Here we report that combined inactivation of Hey1 and HeyL, two primary target genes of Notch, causes severe heart malformations, including membranous ventricular septal defects and dysplastic atrioventricular and pulmonary valves. These defects lead to congestive cardiac failure with high lethality. We found both genes to be coexpressed with Notch1, Notch2 and the Notch ligand Jagged1 in the endocardium of the atrioventricular canal, representing the primary source of mesenchymal cells forming membraneous septum and valves. Atrioventricular explants from Hey1/HeyL deficient mice exhibited impaired epithelial to mesenchymal transition. Although epithelial to mesenchymal transition was initiated regularly, full transformation into mesenchymal cells failed. This was accompanied by reduced levels of matrix metalloproteinase-2 expression and reduced cell density in endocardial cushions in vivo. We further show that loss of Hey2 leads to very similar deficiencies, whereas a Notch1 null mutation completely abolishes epithelial to mesenchymal transition. Thus, the Hey gene family shows overlap in controlling Notch induced endocardial epithelial to mesenchymal transition, a process critical for valve and septum formation.

Epithelial and ectomesenchymal role of the type I TGF-beta receptor ALK5 during facial morphogenesis and palatal fusion.

Transforming growth factor beta (TGF-beta) proteins play important roles in morphogenesis of many craniofacial tissues; however, detailed biological mechanisms of TGF-beta action, particularly in vivo, are still poorly understood. Here, we deleted the TGF-beta type I receptor gene Alk5 specifically in the embryonic ectodermal and neural crest cell lineages. Failure in signaling via this receptor, either in the epithelium or in the mesenchyme, caused severe craniofacial defects including cleft palate. Moreover, the facial phenotypes of neural crest-specific Alk5 mutants included devastating facial cleft and appeared significantly more severe than the defects seen in corresponding mutants lacking the TGF-beta type II receptor (TGFbetaRII), a prototypical binding partner of ALK5. Our data indicate that ALK5 plays unique, non-redundant cell-autonomous roles during facial development. Remarkable divergence between Tgfbr2 and Alk5 phenotypes, together with our biochemical in vitro data, imply that (1) ALK5 mediates signaling of a diverse set of ligands not limited to the three isoforms of TGF-beta, and (2) ALK5 acts also in conjunction with type II receptors other than TGFbetaRII.

Dicer function is essential for lung epithelium morphogenesis.

DICER is a key enzyme that processes microRNA and small interfering RNA precursors into their short mature forms, enabling them to regulate gene expression. Only a single Dicer gene exists in the mouse genome, and it is broadly expressed in developing tissues. Dicer-null mutants die before gastrulation. Therefore, to study Dicer function in the later event of lung formation, we inactivated it in the mouse lung epithelium using a Dicer conditional allele and the Sonic Hedgehogcre (Shhcre) allele. Branching arrests in these mutant lungs, although epithelial growth continues in distal domains that are expanded compared with normal samples. These defects result in a few large epithelial pouches in the mutant lung instead of numerous fine branches present in a normal lung. Significantly, the initial phenotypes are apparent before an increase in epithelial cell death is observed, leading us to propose that Dicer plays a specific role in regulating lung epithelial morphogenesis independent of its requirement in cell survival. In addition, we found that the expression of Fgf10, a key gene involved in lung development, is up-regulated and expanded in the mesenchyme of Dicer mutant lungs. Previous studies support the hypothesis that precise localization of FGF10 in discrete sites of the lung mesenchyme serves as a chemoattractant for the outgrowth of epithelial branches. The aberrant Fgf10 expression may contribute to the Dicer morphological defects. However, the mechanism by which DICER functions in the epithelium to influence Fgf10 expression in the mesenchyme remains unknown.

Requirement for chitin biosynthesis in epithelial tube morphogenesis.

Many organs are composed of branched networks of epithelial tubes that transport vital fluids or gases. The proper size and shape of tubes are crucial for their transport function, but the molecular processes that govern tube size and shape are not well understood. Here we show that three genes required for tracheal tube morphogenesis in Drosophila melanogaster encode proteins involved in the synthesis and accumulation of chitin, a polymer of N-acetyl-beta-D-glucosamine that serves as a scaffold in the rigid extracellular matrix of insect cuticle. In all three mutants, developing tracheal tubes bud and extend normally, but the epithelial walls of the tubes do not expand uniformly, and the resultant tubes are grossly misshapen, with constricted and distended regions all along their lengths. The genes are expressed in tracheal cells during the expansion process, and chitin accumulates in the lumen of tubes, forming an expanding cylinder that we propose coordinates the behavior of the surrounding tracheal cells and stabilizes the expanding epithelium. These findings show that chitin regulates epithelial tube morphogenesis, in addition to its classical role protecting mature epithelia.