DYT1 mutation in Iranian patients affected withprimary dystonia.To determine the frequency of DYT1 gene. DNA extracted from patients’peripheral blood was subjected to PCR-sequencing for exon 5 of the DYT1 gene. The collectionof samples was based on random sampling.In this study, we investigated 60 patients with primary dystoniawho referred to the Tehran Medical Genetics Laboratory (TMGL) to determine thedeletional mutation of 904-906 del GAG in the DYT1 gene (15099 to 15101based on reference sequence: NG_008049.1) was identified in 11 patients (18.33%). Theaverage age of affected patients with this mutation was 13.64 ± 7.4 years.The deletional mutation of 904-906 del GAG in the DYT1 deletional mutation of 904-906 del GAGhas a high frequency in Iranian patients in comparison with other non-Jewish populations.Therefore, this particular mutation may be the main representative of pathogenic DYT1gene for a large proportion of Iranian patients with primary dystonia.It can be concluded that the  DYT1 gene.The DYT1 gene discovered in 1997 is located onchromosome 9 and encodes a protein termed Torsin A. This protein is a member of a superfamilyof ATPases which is a DNA-binding protein withparticular homology to heat shock proteins. It isexpressed in several tissues, in particular in thecentral nervous system (CNS) in the basal ganglia andcerebral cortex. It is also thought to be involvedin cellular trafficking of the dopamine transporterand other membrane-bound proteins (TOR1A) is the only identifiedgene responsible for type 1 dystonia and the 3 bpdeletion (GAG) in exon 5 (15099 to15101 basedon reference sequence: NG_008049.1) is the mostcommon causative mutation. The deletion resultsin the loss of one of a pair of glutamic acid residuesnear the carboxy terminus in a conservedregion of ATP-binding proteins (Torsin A) withunclear function  in exon 5 ofthe DYT1 who referred to the Tehran MedicalGenetics Laboratory (TMGL) were investigatedto determine the deletional mutation of 904-906del GAG in the DYT1 gene. The Ethics Committeeof Pasteur Institute of Iran approved this atudy. Aneurologist diagnosed the type of disorder accordingto the Fahn et al. Dystonia classification criteria suspectedof criteria.DYT1 gene was amplifiedas follows:Utilizing the primers listed in table 1, a 205 bpfragment from exon 5 of the 2, 0.2 mM dNTPs, 10 pM of each of theforward and reverse primers, 0.5 unit Tag DNApolymerase  and 500-1000 ng genomicDNA. Amplification of the 205 bp was monitoredby electrophoresing 10 µl of the PCR producton 1.5% agarose gel, stained with ethidium bromideand visualized by exposing to UV light.94℃ for 5 minutes for initial denaturation followedby 32 cycles at 94℃ for 1 minute, 60℃ for1 minute and 72℃ for 1 minute. Final extensionwas carried out for 5 minutes at 72℃. The PCRfinal volume was 62 µl containing the following:50 mM KCl, 10 mM Tris-HCl at pH=8.3, 50 mMMgClThe sequencing reactions were performed by thechain termination method as described elsewhere, 13 by AQuantitative variables were expressed as means ±SD while qualitative variables were expressed aspercentages.DYT1 gene (205 bp)and DNA sequencing, we analyzed the rate of 3 bpGAG deletional mutation (In this study 60 patients suspected of type 1 dystoniawere selected and after amplifying the specificfragment for exon 5 of the mutation .There were 36 (57%) males and 26 (43%) females.Table 2 summarizes data related to the average ageof the patient population and those with positivefindings for the mutation. Females (10%) had thehighest mutation frequency. The age of the affectedpatients positive for the GAG deletion mutation waslower compared to the total patient population.DYT1 gene in a group of Iranian patientswith PTD was determined by DNA sequencing.This mutation is the most common cause oftype 1 dystonia studied. Its frequency was 18.33%in the studied population of the globus pallidus and pallidotomy. Althoughexperience with this approach is still limited, preliminaryresults in patients with primary generalizeddystonia, especially dystonia , 18. TheDYT1 in Iranian PTD patients compared to other non-Jewish populations is quite outstanding. Therefore,this mutation is responsible for a significant proportionof affected Iranian patients. For genetic diagnosisof the remainder of the patients, more analysesof other genes implicated in primary dystonia, suchas the DYT6 (THAP1) gene, are necessary (The high frequency of the GAG deletional mutationof ecessary , 20.


The efforts to achieve good glycemic control might damage the quality of life (QoL). This issue has not been elaborated in Indonesia because there was no diabetes-specific quesionnaire available in Indonesian language previously. This study would like to find out whether there is a correlation between glycemic control and QoL of adolescents aged 13-18 years with type 1 diabetes mellitus (T1DM).Cross-sectional study was held on patients who came to The Pediatric Endocrinology Clinic of Cipto Mangunkusumo Hospital or who joinned The Endocrinology Working Group Unit of Indonesian Pediatric Society programs during October to December 2010. Glycemic control was measured by hemoglobin A1c (HbA1c) levels. The audit of diabetes dependent quality of life-teen (ADDQoL-Teen) Indonesian questionnaire was used to assess the QoL.A total of 36 adolescents participated in the study. Only seven subjects had a normal HbA1c level. Median HbA1c level was 8.7 %. Eighteen subjects considered their QoL as good or brilliant, while 14 subjects felt that diabetes had a negative impact. The most severe negative impact was felt on the matter of dietary aspect, but could be managed better by subjects with intensive treatment. There was no correlation between HbA1c level on the last examination and average weighted impact (AWI) score  or between mean HbA1c level within 6 months and AWI score .This study did not show any correlation between glycemic control and QoL of adolescents with T1DM.


Eleven characterized allergens of Hevea brasiliensis are available as recombinant proteins for in vitro IgE diagnosis. Hev b 2 and Hev b 13 are only suitable as native proteins. By testing patient sera against this panel of recombinant and native allergens of Hevea brasiliensis individual sensitization profiles and the relevance of single allergens can be assessed.Sera of 30 spina bifida patients with specific IgE to NRL were tested for allergen-specific IgE antibodies (sIgE) to natural rubber latex (NRL) and 13 single Hev b allergens using the ImmunoCAP system. The results of the specific IgE values against single allergens were plotted . Minor and major allergens for spina bifida patients sensitized or allergic against NRL could be identified.Regarding all 30 spina bifida patients with sIgE Hev b 1, 2, 3, 5, 6.01 and 13 were identified as major Hev b allergens. In the patients without latex-related symptoms Hev b 2 and Hev b 6.01 were found only in small percentages of the latex-specific IgE response and low frequencies  whereas in patients with latex-related symptoms these allergens were found in high concentrations and frequencies. Hev b 5 represents the allergen with the highest percentage of the latex specific IgE response in all groups of patients, Hev b 1 is the allergen with the highest frequency of sensitization (about 80%) in all groups.Latex extracts for skin prick testing or in vitro allergosorbents should contain the major allergens Hev b 1, 2, 3, 5, 6.01 and 13. In spina bifida patients Hev b 6.01 and Hev b 2 could be useful to distinguish sensitized from allergic patients.


Tbx20 and Tbx3 cause distinct congenital heart abnormalities in the mouse: Tbx20 mutations result in failure of heart looping, developmental arrest and lack of chamber differentiation, while hearts of Tbx3 mutants progress further, loop normally but show atrioventricular convergence and outflow tract defects. The two genes have overlapping areas of expression in the atrioventricular canal and outflow tract of the heart but their potential genetic interaction has not been previously investigated. In this study we produced compound mutants to investigate potential genetic interactions at the earliest stages of heart development. We find that Tbx20; Tbx3 double heterozygous mice are viable and fertile with no apparent abnormalities, while double homozygous mutants are embryonic lethal by midgestation. Double homozygous mutant embryos display abnormal cardiac morphogenesis, lack of heart looping, expression patterns of cardiac genes and time of death that are indistinguishable from Tbx20 homozygous mutants. Prior to death, the double homozygotes show an overall developmental delay similar to Tbx3 homozygous mutants. Thus the effects of Tbx20 are epistatic to Tbx3 in the heart but Tbx3 is epistatic to Tbx20 with respect to developmental delay.Members of the T-box family of transcription factors are important regulators orchestrating the complex regionalization of the developing mammalian heart. Individual mutations in  Tbx1, Tbx2, Tbx3, Tbx5, Tbx18 and Tbx20 are expressed in the heart and regulate various aspects of embryonic heart development. The importance of unperturbed T-box gene function is highlighted by mutations in mice and human, which are associated with congenital heart disease [TBX20 suffer from diverse cardiac defects, including ventricular septal defects, aberrant valvulogenesis, tetralogy of Fallot and cardiomyopathy [TBX3 result in the ulnar-mammary syndrome in which a subset of patients has ventricular septal defects [The four-chambered mammalian heart develops from a simple linear heart tube by polar elongation, myocardial differentiation and morphogenesis. This complex regionalization of the developing heart is orchestrated by multiple signaling modules and transcriptional circuits. Members of the T-box family of transcription factors are particularly important regulators of myocardial proliferation and patterning. T-box family members myopathy –7. Mutat defects .Tbx20 is expressed throughout the first heart field, in a subset of second heart field progenitors and at later stages in the endocardium and derived mesenchyme of the atrioventricular and outflow tract (OFT) cushions and the atrioventricular septum [Tbx20 expression decreases in the chamber myocardium compared with atrioventricular and cardiac outflow regions [Tbx20 die early during development and display short, severely underdeveloped heart tubes that fail to undergo looping [Tbx20 is compatible with both cell-autonomous and non-cell-autonomous defects of this process. Chamber myocardial genes are not activated in Tbx20 deficient hearts while Tbx2, which is normally restricted to the atrioventricular canal (AVC) and OFT, is ectopically expressed throughout the cardiac crescent and linear heart tube [Tbx20 heterozygous mice survive postnatally but show dilated cardiomyopathy, which phenocopies at least a subset of human TBX20 mutant defects [In the mouse, r septum –13. Foll regions ,12. Seve looping . Failureart tube . Tbx20 hTbx3 expression is first detected in the inflow tract at the onset of heart looping. Its expression delineates the developing cardiac conduction system, endocardial cushions in the AVC, and the mesenchyme of the OFT [Tbx3 enables development of the cardiac conduction system by restricting cell division and repressing the chamber-specific gene expression program. Tbx3 deficient embryos are developmentally retarded and die at midgestation apparently due to yolk sac deficiencies although Tbx3 mutant hearts are also abnormal [In the mouse heart,  the OFT ,17. Tbx3abnormal ,19. Tbx3abnormal .Tbx20 and Tbx3 cause distinct congenital heart abnormalities, but their overlapping expression in the AVC and outflow tract in the developing heart raises the question of whether they act independently or through transcriptional regulation of common target genes. Tbx20 expression appears ectopically in the apex of the ventricular septum of Tbx3 mutant hearts at E12.5 [Tbx3 expression is markedly down regulated at E9.5 in the AVC of embryos with a conditional deletion of Tbx20 [Tbx20; Tbx3 double homozygous mutants to explore possible genetic interaction between these two genes based on their overlapping expression in the AVC and OFT. With respect to heart development, the double homozygous phenotype was indistinguishable from that of the Tbx20 single homozygous mutant in that heart looping and development was arrested early in development. In addition, a general developmental delay characteristic of Tbx3 homozygous mutants was apparent in the double homozygous mutants, apparently independent of the cardiac phenotype.Thus, when individually mutated, at E12.5  and Tbx3of Tbx20 . HoweverTbx20 locus, Tbx20lacZ [Tbx20-, or a null mutation of the Tbx3 locus, Tbx3tm1Pa [Tbx3-, were intercrossed to produce double heterozygotes, which were intercrossed to generate double homozygotes as well as other genetic combinations. Embryos and weanling mice were genotyped for Tbx20 and Tbx3 by PCR from lysates of yolk sac or tail tip, respectively. The mutant allele of Tbx20 was genotyped with primers: Primer 1 (LoxP F2): 5’-GACTGGAGAGGCCATCAAAA-3’ and Primer 2 (LacZR2): 5’-GTTTTCCCAGTCACGACGTT- 3’; and the wild type allele was genotyped with primers: T20 wt sense: 5”-CCCAAGGAGAAGGAGGCAGCAGAGAAC-3’ and T20 wt antisense: 5’- CGCAAGTATAAAATGGGGGTTCCTGACC-3’. PCR conditions were 3 minutes at 94oC, 30 cycles , and 5 minutes at 72oC. The primers and PCR conditions for Tbx3 were as previously described [All experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health under protocols approved by the Columbia University Institutional Animal Care and Use Committee (protocol No. AC-AAAD3302). Mutant alleles were maintained on a mixed genetic background derived from 129, C57BL/6Tac and ICR mice. Mice heterozygous for a null mutation of the x3tm1Pa , hereaftescribed .in situ hybridization [For timed pregnancies, females were placed with males overnight and checked the following morning for the presence of vaginal plugs. Noon on the day of the plug was designated E0.5. Embryos between E8.5 and E9.25 were dissected out of the uteri and their extra-embryonic membranes in phosphate buffered saline (PBS) with 0.2% bovine serum albumin and were fixed in 4% paraformaldehyde in PBS overnight. Standard procedures were used for whole mount dization .http://www.wessa.net/rwasp_notchedbox1.wasp).Statistical analyses were performed using the Chi-square distribution, Mann Whitney U test and notched box plot analysis , Tbx20-/-; Tbx3+/- (n=29), as well as double homozygous mutant embryos (n=14), were morphologically indistinguishable from one another at any given developmental stage: Starting at the heart looping stage, heart tube formation was retarded, hearts were small, and heart looping failed, resulting in two vertically oriented chamber-like swellings with a characteristic hour-glass shape by E9.25  similar to Tbx3-/- embryos (4/4)  from heterozygous matings were scored for heart looping and other morphological and developmental features. Of these, 126 embryos were generated by double heterozygous intercrosses while 84 embryos were generated by crossing Tbx20by E9.25  & 3, as - hearts –15. EmbrTbx3-/- embryos were not observed between E8.5 and E9.25, which is in agreement with previous work [Tbx20+/-; Tbx3-/- embryos at E8.5 and 1 of 6 double homozygous mutant embryos at E9.25   but was upregulated and ectopically expressed throughout the entire Tbx20-/- mutant heart by E9.25. Similar upregulation and ectopic expression of Tbx2 was seen in Tbx20-/-; Tbx3-/- double homozygous mutant hearts, whereas expression in Tbx3-/- hearts was normal .Expression of escribed   of Tbx20-/-, Tbx3-/- or Tbx20-/-; Tbx3-/- embryos compared to stage-matched wild type controls (Tbx5, which is expressed in the atrioventricular and atrial progenitors in an antero-posterior gradient, was slightly reduced in both Tbx20-/- and Tbx20-/-; Tbx3-/- embryos at E8.5 but no difference in expression was evident among the genotypes by E9.25 (We observed no change in expression of Tbx1, Tbx2 and Tbx3 all interact in OFT development with Tbx1 upstream of Tbx2 and Tbx3. Double homozygous mutants for Tbx1 with either Tbx2 or Tbx3 have heart failure more severe than either single mutant alone [Tbx2 and Tbx5 have been shown to repress or activate, respectively the expression of Nppa through competing interactions with the transcriptional co-factor Nkx2-5 [Tbx3 and Tbx20 have also been shown capable of interacting with Nkx2-5 [Tbx20 has been shown to repress Tbx2 expression in the developing heart, as in its absence, Tbx2 is upregulated throughout the chamber myocardium of the heart tube [The DNA binding domain of T-box proteins, as well as T-box binding motifs identified in the promoters of downstream target genes, are highly conserved. This leads to the possibility that these transcription factors have targets in common and could compete or cooperate in transcriptional regulation of specific targets in areas of overlapping gene expression. Furthermore, some T-box proteins can bind DNA as dimers, raising the possibility of heterodimerization. In the developing heart at least seven T-box genes are expressed ,23 and ant alone . At the r Nkx2-5 –27. Tbx3h Nkx2-5 ,17. T-boTbx20 and Tbx3 have areas of overlapping expression in the OFT and AVC of the developing heart and thus have the potential to interact either by affecting common downstream target genes or by cross regulation. A potential example of cross regulation was noted in Tbx3 mutant hearts at E13.5-14.5 where Tbx20, which is normally expressed in the base of the developing interventricular septum, appears to be ectopically expressed in the developing conduction system at the apex of the septum where Tbx3 would normally be expressed [Tbx3-expressing tissue in the abnormally developing septum is not clear. On the other hand, Tbx20 regulates Tbx3 in the AVC as deletion of Tbx20 specifically in the AVC leads to a downregulation of Tbx3 expression at E9.5 [xpressed . However at E9.5 .Tbx20 single homozygous mutants: morphologically the double homozygous mutant hearts displayed a vertically oriented hourglass shaped, two-chambered heart with no looping. Molecularly, Tbx2 was upregulated throughout the heart tube and Nppa was severely down regulated in the double homozygous mutants, similar to Tbx20 single mutants, whereas Pitx2 and Tbx5 were unchanged. This phenotype is indistinguishable from the Tbx20-/- phenotype where cardiac development is arrested and chamber differentiation does not occur although the anterior/posterior and left/right axes are established correctly. Because of this early arrest starting at E8.5, cardiac development never reached the stage where phenotypic features characteristic of the Tbx3 mutant heart, such as convergence defects, increased AVC cell proliferation and ventricular septal defects, could be evaluated. Thus, Tbx20 is epistatic to Tbx3 with respect to the early heart phenotype and heart development is arrested at an earlier developmental stage than the Tbx3 phenotype becomes manifest. To further investigate potential genetic interactions in the cardiac conduction system, conditional alleles that allow survival beyond the time of death of the homozygous mutants would be required. For this type of study, the recently developed Tbx3 allelic series, which has been used to demonstrate exquisite dose sensitivity of the cardiac conduction system to levels of Tbx3 in embryos and adults could be combined with a conditional Tbx20 allele to test for genetic interactions throughout development and adult life [Tbx3 homozygous mutant embryos are delayed in their overall development compared with littermates [Tbx20-/-; Tbx3-/- double homozygous embryos that was not seen in Tbx20 single mutants. Thus, developmental delay is associated with the Tbx3 mutant genotype, even though Tbx20 mutants have a more severe and earlier heart abnormality. It was previously postulated that vascular defects and apoptosis in the Tbx3 mutant yolk sac were responsible for this developmental delay [Tbx3 and Tbx20 are expressed in the yolk sac, Tbx20 in the mesoderm layer [Tbx3 in both endoderm and mesoderm [Tbx3 mutants and was not exacerbated in double mutants. Thus the developmental delay associated with the Tbx3 mutant genotype does not appear to be associated with an early yolk sac vasculature phenotype and remains to be elucidated.To investigate the phenotypic consequences of possible interactions between the two genes at the earliest stages of heart development, we produced double mutants using mutant alleles that ablate function and compared the morphological and molecular phenotypes of the single, compound and double mutants. We found no evidence for a genetic interaction in double heterozygotes, which were viable and fertile, or in double homozygotes, which displayed an embryonic heart phenotype indistinguishable from ult life . It has termates . We obseal delay , even thal delay . Both Tbmesoderm , and vasmesoderm . In this


Here, we report that zebrafish embryos having decreased function of the orthologous hoxd4a gene manifest striking perturbations in vasculogenesis, angiogenesis and primitive and definitive hematopoiesis. These defects are preceded by reduced expression of the hemangioblast markers scl1, lmo2 and fli1 within the posterior lateral plate mesoderm (PLM) at 13 hours post fertilization (hpf). Epistasis analysis revealed that hoxd4a acts upstream of meis1.1 but downstream of cdx4 as early as the shield stage in ventral-most mesoderm fated to give rise to hemangioblasts, leading us to propose that loss of hoxd4a function disrupts hemangioblast specification. These findings place hoxd4a high in a genetic hierarchy directing hemangioblast formation downstream of cdx1/cdx4 and upstream of meis1.1. An additional consequence of impaired hoxd4a and meis1.1 expression is the deregulation of multiple Hox genes implicated in vasculogenesis and hematopoiesis which may further contribute to the defects described here. Our results add to evidence implicating key roles for Hox genes in their initial phase of expression early in gastrulation.Mice lacking the 4 Starting at 48 hpf, the HSCs then seed what will become the hematopoietic stroma of the caudal hematopoietic tissue (CHT) at 3 dpf, followed by expansion and differentiation The second and definitive wave of hematopoiesis begins in the ventral wall of the DA at 24 hpf clochelycatcdx1 and cdx4cdx4 single mutants and cdx1/cdx4 doubly deficient embryos are accompanied by the down-regulation of the hemangioblast and hematopoietic stem cell (HSC) marker scl1 and a number of Hox genes, and normal hematopoiesis can be rescued by the forced expression of hoxa9aSeveral genes have been implicated in the control of successive steps in the formation of blood and the vasculature. In zebrafish, the earliest acting gene to be identified to date is Hoxb4, leads to defects in blood lineages. Hoxb4 and indeed all remaining Hox genes of paralog group 4  display potent HSC-promoting functions Hox and Meis1 genes in normal hematopoiesis is further reflected in their well-documented contributions to human and murine leukemias Hox genes have also been implicated in hematopoietic and vasculogenic processes in mammals Hox clusters, with zebrafish retaining 7 clusters and 47 genes following evolutionary loss hoxb6b, hoxb7a, and hoxa9a are known to regulate primitive hematopoiesis and are required for hematopoietic stem cell formation pbx and meis function strongly compromise primitive and definitive hematopoiesis as well as the vasculature In teleosts, a genome duplication event generated 8 Hoxd4 function show abnormalities of the anterior-most vertebrae, the atlas and axis, but are viable and fertile hoxd4a loss-of-function morphants. We demonstrate a surprising role for zebrafish hoxd4a in vasculogenesis, angiogenesis and primitive and definitive hematopoiesis. Defects resulting from hoxd4a deficiency can be rescued by capped mRNA for hoxd4a, meis1.1, scl, and fli1 but not by cdx4. Impaired meis1.1 function following hoxd4a knockdown leads to widespread Hox gene deregulation including the reduced expression of Hox genes previously implicated in hematopoiesis, vasculogenesis and angiogenesis. The cdx4, hoxd4a and meis1.1 genes are spatially and temporally co-expressed in shield-stage embryos within the ventral-most presumptive mesoderm, the site from which fate mapping studies have shown the hemangioblast to arise hoxd4a morphants display decreased meis1.1 expression in ventral-most presumptive mesoderm, and this meis1.1 expression is rescued by prior injection of capped hoxd4a mRNA. Thus, our data indicate that hoxd4a functions at the earliest times and near the top of a regulatory hierarchy directing hemangioblast formation, acting downstream or in parallel to cdx genes, but upstream or parallel to meis1.1. Another consequence of impaired hoxd4a and meis1.1 function is the subsequent deregulation of multiple Hox genes previously implicated in vasculogenic and hematopoietic processes. We conclude that hoxd4a has acquired (or retained) a much more important role in hematopoiesis and vasculogenesis than observed for its murine ortholog Hoxd4. Additionally, our results add to a growing body of evidence defining functions for Hox genes during their early phase of expression at gastrulation, well before they are deployed along the antero-posterior axis proper.Mice lacking All animal work conformed to the Institutional Animal Care and Use Committee (IACUC) guidelines at Nanyang Technological University and was reviewed and approved under protocol number ARF SBS/NIE-A 0144 AZ.fli1:EGFP) Wild-type AB and transgenic . Optimal doses for each MO were tested based on phenotypic effects, and each experiment was performed in parallel with a non-specific MO (standard control MO supplied by GeneTools Inc) injected at the same concentration.Antisense morpholino oligonucleotides (MOs) were obtained from Gene Tools Inc and injected into zebrafish embryos at 1–4 cell stage. Splice MOs targeting the 5′ splice site intron-exon junction (splice acceptor) are designated as follows MO1∶5′-GTT CAC TGT GAA GGA CAA AAT CAC A-3′ and exon-intron junction (splice donor), MO2∶5′- GCA AAG AGA GTG GAT CTT ACC CGT A-3′. MOs were diluted in Danieau’s buffer (0.4 mM MgSOhoxd4a was generated by PCR using the primers shown in Table S1in File S1 and cloned into pCR®II-TOPO®. The mMESSAGE mMACHINE Kit (Ambion) was used to synthesize capped mRNA. All mRNAs used for rescue experiments were assessed over a range of concentrations and the following amounts were chosen as the minimum sufficient to induce rescue: scl1 (100 pg), fli1a (30 pg), meis1.1 (100 pg) and hoxd4a (50 pg). All injections were done at the 1-cell stage.Full-length cDNA for File S1.Total RNA was extracted from embryos at 26–28 hpf using the PureLink™ Micro-to-Midi™ Total RNA Purification System (Invitrogen). 1 µg total RNA was treated with 1 U DNase I (Fermentas) at 37°C for 15 min and used for reverse transcription with SuperScript® III First-Strand Synthesis (Invitrogen). Quantitative reverse transcriptase PCR (qRT-PCR) was performed using SYBR GreenER™ qPCR SuperMix (Invitrogen) on a BioRad iCycler iQ5. The data  were normalized against zebrafish β-actin. The sequences of the oligonucleotides used for qRT-PCR are given in in situ hybridization (WISH) was performed as previously described nkx2.5, a PCR fragment was amplified from 26–28 hpf embryonic cDNA using the primers shown in File S1. The reverse primer incorporated a T7 promoter allowing the PCR product to be used directly for probe production. DIG- labeled antisense RNA probes were transcribed from linearized template using T3, T7 or SP6 RNA polymerase (Roche). Probes for hoxb6b and hoxb7a were derived by RT-PCR-mediated amplification from RNA from 26–28 hpf embryos using primers described in Wan et al Whole mount 2, 100 mM NaCl, 0.1% Tween 20) for three times (each for 15 min) at room temperature. For alkaline phosphatase staining, the embryos were incubated in NBT/BCIP (Roche) solution for 30 min.Embryos at 72 hpf were fixed with 4% paraformaldehyde in PBS  at room temperature for 30 min, followed by treatment with pre-cooled acetone for 30 min at −20°C. After rinsing with PBS twice (5 min each), the embryos were equilibrated with NTMT buffer  embryos (150 embryos each) were dechorionated manually at 48 hpf, rinsed for 15 min in calcium-free Ringer’s solution and passed five times through a 200 µl pipette tip to remove the yolk. The embryos were dissociated in 0.25% trypsin and 1 mM EDTA for 60 minutes at 28.5°C, during which the sample was passed six times through a 200 µl pipette tip every 10 minutes in order to obtain a single cell suspension. The dissociated cell suspension was centrifuged at 1000 g for 9 min at 4°C, the supernatant discarded, and the cells resuspended in ice cold 0.9× PBS plus 5% fetal bovine serum and passed by gravity through a 40 µm nylon mesh filter. Flow cytometry was performed on a BD LSII instrument (BD Biosciences). We used wild type zebrafish embryos as a negative control.hoxd4a during zebrafish development was examined by WISH. Maternal transcripts were seen at the 1 cell stage, and zygotic transcripts were readily detected from 3 hpf to 48 hpf . As observed previously hoxd4a is expressed in the hindbrain with an anterior border at r6/7, in neural crest migrating to the future branchial arches, and in the pectoral fin fields  at the 1–4 cell stage resulted in severely deformed embryos that died by 7 dpf. However, at a reduced dosage of MO (4 ng), embryos exhibited grossly normal morphology up to 30 hpf but with a developmental delay of approximately 9 h. In all experiments reported below, we compared morphants and control embryos at equivalent developmental stages. In embryos of at least 26 hpf, we routinely assessed the length between the eye and the otic vesicle as suggested previously To understand the function of hoxd4a MO, in situ hybridization revealed a marked reduction of hoxd4a transcripts at 26–28 hpf (>26 somites) , nkx2.5  and myod  was unaltered in the morphants, demonstrating that there was no gross defect in the overall patterning of the mesoderm.Following injection of the anti-somites) Fig. 1W.hoxd4a morphants the number of circulating RBCs was considerably reduced (n = 96/100) (S3), but was strongly rescued by co-injection with capped mRNA for hoxd4a (. Reduced RBC production at 48 hph (hoxd4a mRNA (Video S3) suggestive of vascular defects. A lack of RBCs was also apparent by observation of the heart (File S1). Apart from anemia, pericardial edema and areas of edema over the yolk sac were also observed from 36 hpf (File S1). In addition, the heart rate was also mildly but significantly slower in morphants at 26–28 hpf and 48 hpf (File S1), and may have affected definitive hematopoiesis (see Discussion).The onset of blood circulation in zebrafish is at 25 to 26 hpf. In control-injected embryos at 48 hpf, red blood cells (RBCs) streamed normally over the yolk, through the ducts of Cuvier toward the heart, and through the dorsal aorta, posterior cardinal vein and caudal plexus. By contrast, in hoxd4a morphants, we made use of the Tg(gata1:dsRed) transgenic line gata1 promoter drives expression of the Discosoma red fluorescent protein (dsRed) in erythrocytic lineages during primitive and definitive hematopoiesis. While a strong signal was observed in the ICM and PBI of control embryos at 26 hpf, little could be detected in hoxd4a morphants  was analyzed by WISH. Both gata1 and hbbe1 transcripts were diminished in the posterior PLM at 13 hpf (∼ 8 somites) (vs C–D) and the ICM and PBI at 26–28 hpf (File S1), establishing a defect in primitive hematopoiesis. Co-injection of capped hoxd4a mRNA along with the hoxd4a MO significantly rescued the expression of both erythroid markers , specifically implicating hoxd4a in the phenotype.To assess the effect of reduced runx1 and cmyb marking the HSCs runx1 and cmyb in hoxd4a morphants at 26–28 hpf (vs C-D) and 48 hpf (vs G-H) was severely down-regulated. The expression levels of markers of primitive and definitive hematopoiesis were quantitated by qRT-PCR and showed a strong reduction in hoxd4a morphants and significant rescue upon co-injection with capped hoxd4a mRNA .In addition to reduced numbers of blood cells, hoxd4a morphants was investigated through the use of the fli1:EGFP transgenic line which expresses enhanced GFP under the control of the fli1 locus in endothelial cells The state of the vasculature in hoxd4a knockdown on vasculogenesis and angiogenesis were also assessed by exploiting the high endogenous levels of alkaline phosphatase characteristic of endothelial cells. In control embryos, intense signals revealed well-formed ISV, subintestinal vessels (SIVs) and vertebral artery (VTA), whereas such vessels were indiscernible or highly reduced in hoxd4a morphants . Similar hemorrhaging has been observed in fli1 morphants hoxd4a morphants.The effects of  and VTA Fig. 4I.hoxd4a knockdown on genetic programs directing vasculogenesis and angiogenesis, we performed in situ hybridization for the endothelial markers fli1 and flk1. Both genes were significantly reduced in the LPM of hoxd4a morphants at 13 hpf (vs C–D), and in the major trunk vessels and ISVs at 26 hpf (File S1), implying that hoxd4a morphants are defective in axial vasculature. Co-injection of capped mRNA for hoxd4a, rescued both fli1 and flk1 expression, establishing a specific role for hoxd4a in the altered gene expression pattern .To evaluate the effect of efnb2a and ephb4ahoxd4a expression . Likewise, the expression of the vein marker ephb4a was reduced at 13 hpf . Results for fli1, flk1, vegf and efnb2a were quantified by qRT-PCR on RNA extracted from 26–28 hpf embryos. The results confirm the decreased expression of these genes in hoxd4a morphants, and the ability of co-injected mRNA for hoxd4a to rescue their expression . The impaired expression of scl and lmo2 persisted at 26 hpf (File S1), suggesting that their later independent functions in definitive hematopoiesis were also compromised. Co-injection of capped hoxd4a mRNA rescued scl and lmo2 expression at 13 hpf (File S1).The hemangioblast is the common precursor to the blood and endothelial lineages and is defined by early expression of such genes as hoxd4a MO was co-injected with capped mRNA for either scl or fli1. The expression of scl and lmo2 was rescued in both cases , placing the action of hoxd4a above these early acting effectors of hemangioblast specification To establish the hierarchy between these key regulators, the gata1 and hbbe1 at 13 and 26 hpf was rescued by co-injection of scl and fli1 mRNA , though rescue by fli1 at 13 hpf was incomplete. Expression of the vasculogenic markers fli1 and flk1 was likewise rescued by scl and fli1 mRNA at 13 hpf (File S1). Together, these results reveal that hoxd4a occupies a very high position in the transcriptional hierarchy governing hemangioblast formation or function.Consistent with the above findings, expression of Meis homologs have been implicated in the earliest stages of hematopoiesis and vasculogenesis in mice and zebrafish meis1.1 in hoxd4a morphants.A number of studies have demonstrated major regulatory roles for Hox transcription factors in hematopoietic cell fate decisions meis1.1 expression was significantly reduced in the PLM of hoxd4a morphants (File S1), while at 26–28 hpf meis1.1 expression was reduced throughout the embryo  in hoxd4a morphants by co-injection with mRNA for meis1.1. All three markers were strongly rescued at 13 hpf .At 13 hpf, nscripts Fig. 8C.clochecdx1 and cdx4 genes are among the earliest effectors of the hemangioblast lineage cdx1/cdx4 embryos lack midline vasculature and show a complete failure of hematopoiesis hoxd4a morphants, cdx4 expression is unaltered at 13 hpf  and 26 hpf . We conclude that hoxd4a acts downstream or parallel to cdx1/cdx4 but upstream or parallel to meis1.1.Along with cdx4, hoxd4a and meis1.1 are ever temporally and spatially co-expressed so as to directly implement this hierarchy in a cell-autonomous fashion. Prior fate-mapping studies have established that precursors to the PLM hemangioblasts arise from the ventral-most presumptive mesoderm of shield-stage embryos hoxd4a is expressed . These results validate the suggestion that the early loss of meis1.1 function leads to the deregulation of several, though not all, Hox genes, thereby providing a fuller explanation for how the initial impairment of a single Hox gene, hoxd4a, can lead to massive defects in hematopoiesis, vasculogenesis, and angiogenesis.Members of the MEIS family regulate Hox gene expression in both invertebrates and vertebrates e RT-PCR Fig. 10Ahoxd4a near the top of a regulatory cascade directing hematopoiesis, vasculogenesis and angiogenesis in zebrafish embryos  and vasculogenic defects, and impaired expression of multiple Hox genes Hox gene expression is not affected in meis1.1 or meis3 morphants cdx4 expression in hoxd4a morphants, and the hoxd4a morphant phenotype cannot be rescued by the injection of capped mRNA for cdx4. It may be that hoxd4a is an immediate downstream effector of cdx1/4 function. Alternatively, hoxd4a may act in parallel with cdx1 and cdx4 such that their function, but not expression, is compromised. Such a mechanism is implied by recent studies showing that products of murine CDX1 and HOXD4 form functional heterodimers cdx1 and cdx4 in definitive hematopoiesis could not be established with certainty due to major effects in the development of the dorsal aorta in doubly deficient embryos hoxd4a morphants do form distinctly separate DA and PCV, and these vessels are open to circulation. Nonetheless, we did observe impaired expression of the arterial marker efnb2a, suggesting that the DA may not be appropriately specified in hoxd4a morphants, an event that could lead to defective HSC formation. The mildly reduced heart rate of morphants may contribute to decreased efnb2a expression and impaired specification of HSCs due to sub-optimal levels of nitrous oxide (NO) signaling hoxd4a and meis1.1 in this process. Nonetheless, depressed NO signaling cannot explain all of the defects noted here, since the reduced flk1 expression that we observe at 13 and 26 hpf is not an expected consequence of decreased heart rate hoxd4a morphants show reduced efnb2a expression in tissues that should not be affected by NO levels consistent with a direct role for hoxd4a or meis1.1 and similar observations in meis1.1 morphants The phenotype we report here is in some ways closer to that reported for scl, lmo2 and fli1, and the ability of the capped mRNAs for these genes to rescue development. However, other aspects of the phenotype may represent later and considerably more downstream functions. For example, decreased expression of efnb2a points to a defect in arterial specification downstream of hemangioblast formation and function, as has been noted in meis1.1 morphants fli1, vegf and flk1 in these processes. The vegf gene is also known to play a role in the initiation of hematopoiesis Some of the phenotypic defects observed here can be explained by effects on relatively early embryonic events, such as impaired formation and function of hemangioblast precursors, as indicated by the loss of hemangioblast markers gata1 commits hematopoietic precursors to erythropoiesis during primitive hematopoiesis in the ICM. Later in development, runx1 and cmyb are required for myeloid development during definitive hematopoiesis in the equivalent of the AGM hoxd4a knockdown, decreased expression of scl and lmo2 is likely to contribute to a failure in the induction of gata1 and defective primitive hematopoiesis as evidenced by a loss of expression of hbbe1; but loss of scl and lmo2 function would also account for the observed defect in runx1 and cmyb expression and the subsequent failure of definitive hematopoiesis. Interestingly, we observe the expression of hoxd4a at 26–28 hpf in the PBI and at 48 hpf in the AGM and caudal vein plexus. The caudal vein plexus is the site of the future CHT hoxd4a might play a direct role in primitive and definitive hematopoiesis apart from its indirect role through meis1.1 earlier in development. Both early and later functions of meis1.1 in hematopoiesis and vasculogenesis are also likely, given very early effects on the expression of hemangioblast markers and later expression of meis1.1 in the ICM as described by others meis1.1 downstream of scl for the induction of primitive hematopoiesis meis1.1 upstream of scl at an earlier time point during hemangioblast specification.Expression of Xenopus, timed interactions between the organizer and non-organizer mesoderm induce Hox expression and pattern the AP axis hoxd4a knockdown could be attributed to defects in gastrulation, two observations argue against this. First, not all the Hox genes we tested were affected, an observation inconsistent with a global gastrulation defect. Second, our demonstration that the expression of markers of paraxial mesoderm (myod), cardiac mesoderm (nkx2.5) and intermediate mesoderm (pax2.1) was unaffected does not support a major impairment of gastrulation movements. Nonetheless, our findings emphasize the importance of Hox gene function in the early peri-gastrulation phase Hox gene expression in vertebrates is implemented in two phases, an early phase in presumptive mesoderm around gastrulation and a late phase in the body proper Hoxd4 function are viable and fertile, and display defects of the anterior vertebral skeleton Hoxd4 null mice, their viability suggests that there is no severe impairment of these processes. By contrast, mutation of the paralogous Hoxb4 gene does lead to hematopoietic defects and some loss of viability, though these mice are still able to complete embryogenesis and many survive to adulthood hoxd4a function has severely deleterious consequences for hematopoiesis, vasculogenesis and angiogenesis. We suggest that this may be due to a dependency of meis1.1 expression on hoxd4a function that was either acquired by teleosts (or simply acquired by zebrafish) or lost in mammals. It may also be that while Hoxb4 has the predominant role in these processes in mammals, evolution has selected for hoxd4a to take on these functions in teleosts. This situation may be partly analogous to that of the midkine orthologs in mice and zebrafish; the two teleost midkine genes are strongly expressed in the adult brain, whereas the mouse ortholog is not Mice lacking File S1Supporting information. Contains supplementary (PDF)Click here for additional data file.Figure S1Patterning and morphology in hoxd4a morphants. (A–H) Knockdown of hoxd4a does not perturb overall patterning of the mesoderm. (A–D) Expression of pax2.1 shows that intermediate mesoderm forms and matures normally in hoxd4a morphants at 13 hpf (B) and 26–28 hpf (D) vs controls .  nkx2.5 expression in the precardiac lateral plate mesoderm at 13 hpf is normal in control (E) and morphant embryos (F).  myod expression in paraxial mesoderm is normal in control (G) and morphant embryos (H). Images in C, D, G and H are lateral views with anterior to the left. A, B, E and F show dorsal views with anterior to the top  or left . (I to O) Lateral views of control and hoxd4a-MO-injected larvae at 72 hpf.  Staining of hemoglobin with o-dianisidine reveals areas of hemorrhage such as in the head  and trunk  in some hoxd4a morphants.  Control larvae (K) but not hoxd4a morphants (L) show abundant RBCs passing through the heart (arrowheads). (M–O) Unlike control larvae (M), hoxd4a morphants display pericardial edema and edema over the adjacent yolk . Scale bars equal 100 µm. (P) The heart rate in morphants at 26–28 and 48 hpf was mildly reduced, but in a statistically significant manner as determined by unpaired Student’s t test (p<0.0001). Error bars give standard deviation.(TIF)Click here for additional data file.Figure S2Reduced expression of markers of primitive hematopoiesis gata1 and β embryonic globin (hbbe1) in hoxd4a morphants. WISH in control and morphant embryos at 26–28 hpf showing the expression of gata1  and hbbe1  in the ICM and PBI (white arrowheads). Normal expression of gata1 and hbbe1  is severely reduced in hoxd4a morphants  and rescued by co-injection with capped mRNA for hoxd4a . All images are lateral views with anterior to the left. ctrl, embryos injected with a non-specific morpholino. MO, embryos injected with the anti-hoxd4a morpholino. hoxd4a mRNA, embryos simultaneously injected with the anti-hoxd4a MO plus capped mRNA for hoxd4a. Scale bars equal 100 µm. All images are at the same magnification.(TIF)Click here for additional data file.Figure S3Reduced expression of markers of angiogenesis and venous specification in hoxd4a morphants. WISH in control and morphant embryos at 26–28 hpf showing the expression of fli1  and flk1 . Normal expression of fli1 and flk1  is severely reduced in hoxd4a morphants  and rescued by co-injection with capped mRNA for hoxd4a . White or black dots denote the tips of dorsally sprouting ISVs. Relative to controls , the expression of the arterial marker efnb2a is reduced in morphants at 26–28 hpf (I), while the venous marker ephb4a in morphants has recovered (J). Scale bars equal 100 µm.(TIF)Click here for additional data file.Figure S4Reduced expression of scl and lmo2 in hoxd4a morphants at 26–28 hpf. (A–J) Expression analysis of scl  and lmo2  at 26–28 hpf. Normal expression of scl and lmo2  is severely reduced in hoxd4a morphants  and rescued by co-injection with capped mRNA for hoxd4a  All images present lateral views with anterior to the left and dorsal on top. Scale bars equal 100 µm. All images are at the same magnification.(TIF)Click here for additional data file.Figure S5scl1 and fli1 act downstream of hoxd4a to direct formation of the hemangioblast. All images are of hoxd4a morphants at 26–28 hpf previously injected with capped mRNAs for either scl1 or fli1 as indicated on the left. WISH was performed to detect expression of scl1 and lmo2 (A–D), gata1 and hbbe1 (E–H) and fli1 and flk1 (I–L). Scale bars equal 100 µm. All images are at the same magnification.(TIF)Click here for additional data file.Figure S6Knockdown of hoxd4a results in decreased expression of meis1.1 but not cdx4 at 13 hpf (∼8 somites). (A–F) Expression of cdx4 , hoxd4a  and meis1.1  in control  and hoxd4a morphants  at the shield stage. The white arrowheads in C and D denote the hoxd4a anterior expression boundary in the hindbrain. Scale bars equal 100 µm. All images are at the same magnification.(TIF)Click here for additional data file.Figure S7The expression of multiple hox genes is reduced at 26–28 hpf in hoxd4a morphants. Images are dorsal views (A–H) and lateral views (I–P) of embryos taken through in situ hybridization for the indicated hox genes. Relative to control embryos , hox gene expression is reduced in hoxd4a morphants . All embryos were simultaneously probed for krox20a expression in r3 and r5 as in cdx4 expression is unchanged in control (Q) and hoxd4a morphants (R) at 26–28 hpf. Scale bars equal 100 µm. All images are at the same magnification.(TIF)Click here for additional data file.Table S1Primers used for cDNA cloning.(TIF)Click here for additional data file.Table S2Primers for quantitative RT-PCR.(TIF)Click here for additional data file.Video S1Anterior blood flow in control and hoxd4a morphant embryos. Lateral view focusing on the anterior half of a 48 hpf embryo including the region of the heart, future branchial arches and yolk of control embryos and hoxd4a morphants. In particular, note robust streaming of blood cells through the ducts of Cuvier over the yolk in the control, but an almost complete absence of circulation in the hoxd4a morphant.(7Z)Click here for additional data file.Video S2Trunk blood flow in control, hoxd4a morphant and rescued embryos. Lateral view of the trunk at 48 hpf in a control, hoxd4a morphant and rescuant previously injected with capped mRNA for hoxd4a. Circulation is vigorous in control and rescuant embryos, with abundant RBCs flowing caudally along the DA, streaming dorsally through the ISVs, caudally through the DLAV, and rostrally through the caudal vein and PCV. By contrast, the number of blood cells is greatly reduced in morphants and blood cells are unable to transit the truncated ISVs.(7Z)Click here for additional data file.Video S3Tail blood flow in control and hoxd4a morphant. Lateral view of the tail region in control and hoxd4a morphant embryos at 48 hpf. Control embryos display vigorous blood flow through the DA, ISVs, DLAV and caudal vein plexus. By contrast, morphants show a highly reduced blood cell count with individual blood cells moving slowly caudally through the DA and returning sporadically and haltingly through the caudal vein plexus. Blood cells do not transit from the DA to the DLAV along the ISVs, unlike control embryos. The reddish appearance of the tissue in the caudal vein plexus (white arrows) appears to be due to the accumulation of RBCs.(7Z)Click here for additional data file.Video S4Rescue by meis1.1 mRNA of trunk blood flow in hoxd4a morphant embryos. Video first showing vigorous circulation through the blood vessels of a control embryo at 48 hpf, followed by the absence of blood and weak circulation in a hoxd4a morphant. The last clip demonstrates significant rescue of blood cell count and vasculature in hoxd4a morphants rescued by co-injection of meis1.1 mRNA, including the ability of blood cells to traverse from the DA to the DLAV along intact ISVs.(7Z)Click here for additional data file.


BRCA1 isoforms. Their proportion varies during the cell cycle, between tissues and in cancer suggesting functional importance of BRCA1 splicing regulation around this exon. Although the regulatory elements driving exon 11 splicing have never been identified, a selective constraint against synonymous substitutions  in a critical region of BRCA1 exon 11 has been reported to be associated with the necessity to maintain regulatory sequences.Alternative splicing across exon 11 produces several BRCA1 alternative splicing program. We report that in-frame deletions and translationally silent nucleotide substitutions in the critical region affect splicing regulation of BRCA1 exon 11.Here we have designed a specific minigene to investigate the possibility that this bias in synonymous codon usage reflects the need to preserve the BRCA1 exon 11. Identification of the trans-acting factors involved in regulating exon 11 alternative splicing will be important in understanding BRCA1-associated tumorigenesis.Using a hybrid minigene approach, we have experimentally validated the hypothesis that the need to maintain correct alternative splicing is a selective pressure against translationally silent sequence variations in the critical region of  BRCA1 gene are associated with a high risk of breast and ovarian cancer. Women heterozygous for such mutations have a lifetime risk of up to around 80% of developing breast cancer and up to 40% of developing ovarian cancer Pathogenic mutations in the BRCA1 is known to undergo alternative splicing of a number of its exons, including the large and functionally important exon 11 BRCA1 open reading frame and allow functional protein production.trans-acting splicing factors that recognise and bind specific pre-mRNA sequences. Their expression changes in cancer as well as in different tissues BRCA1 isoforms vary in quantity during the cell cycle and within different tissues The control of the ratio of splicing isoforms produced within a cell requires regulation by BRCA1 spanning the 3′ end of exon 10 and the 5′ end of exon 11 A bias towards the usage of particular codons, rather than their synonymous counterparts, has previously been reported in a critical region of BRCA1 splicing involving exon 11, we developed a minigene incorporating the whole of BRCA1 exons 8 to 11 and part of exon 12. Using this hybrid minigene approach, we have experimentally validated the hypothesis that maintenance of correct alternative splicing is the cause of selection against silent sequence variations in the BRCA1 critical region spanning exon 11.In order to investigate BRCA1 splicing, we constructed a pB1 wild-type minigene that incorporated sequence from BRCA1 exon 8 up to the first 89 nucleotides of exon 12  and D(11q) isoforms  and D(11q); showing comparable outcomes with that of endogenous BRCA1 in non-transfected MCF7 cells  and breast cancer cells MDA-MB-231 (ATCC). All cell lines tested resembled the increase in D11 isoform observed in MCF7 cells (data not shown).Furthermore, a minigene construct containing the genomic sequence from exons 9–12 had the same pattern of splicing Another version of the minigene behaved in exactly the same manner when we inserted the entire genomic sequence from exons 9 to 12 (data not shown).BRCA1 splicing. 13 synonymous substitutions were introduced by site-directed mutagenesis  were assayed for their effect on The resulting mutated minigenes were transiently transfected into MCF7 cell lines and splicing analysed as described . MutatioAll three synonymous substitutions at codon 231 appear to alter splicing. These changes lie 23 nucleotides distal to the start of exon 11 and significantly reduce the levels of FL and D(11q) in favour of increased levels of D(11) isoform. This suggests that sequence changes at this site alter the splicing of exon 11, through reduced recognition of the intron 10 acceptor site, resulting in its exclusion from the final mRNA. This finding points towards an important sequence element, such as an exonic splicing enhancer, located around codon 231.Codon 244 lies 60 nucleotides downstream from the start of exon 11 and is 55 nucleotides proximal to the 11q splice donor site. The T>C synonymous substitution at the third position of this codon seems to enhance levels of the D(11q) isoform. A similar effect was also observed in normal mammary epithelial cells (HMEpC supplied by ECACC) (data not shown).Codon 313 is situated 150 nucleotides 3′ (downstream) of the 11q splice donor site. Synonymous mutations in this codon do not appear to alter exon 11 splicing. Codons 195 and 196 are 9 and 6 nucleotides away from the 3′ end of exon 9 respectively and synonymous mutations at these sites would therefore most likely affect splicing of exon 9. Codon 215 lies 23 nucleotides from the 3′ end of exon 10 and so any splicing effects may be expected to affect this exon. Our assay does not show any significant alteration in relative exon 11 splice isoform abundance with synonymous mutations at these sites. We therefore evaluated their effect on skipping of exon 9 and 10. Splicing products revealed presence of four isoforms {FL, D(9), D(10) and D(9&10)} showing comparable outcomes with that of the pB1WT minigene  isoform . The data show several conserved sequences along the ‘critical region’ (supporting http://sfmap.technion.ac.il/) In order to identify putative splicing regulatory elements in the ‘critical region’ of BRCA1 exon 11 splicing is unchanged with deletion D1 and D4. Deletion D3 has a weak effect in reducing levels of the D(11q) isoform in favour of D(11). Transfection with the hybrid minigene carrying deletion D2 has the strongest effect, inducing almost complete skipping of exon 11.We then performed a pB1 minigene deletion analysis of the most conserved regions containing putative binding sites for splicing regulatory proteins. The D1, D2, D3 and D4 deletions are highlighted in http://sfmap.technion.ac.il/) and SpliceAid2 (www.introni.it/spliceaid.html) are shown in supporting The putative splicing factors predicted to bind in D2 and D3 by SFmap  isoform to the detriment of the full and D(11)isoforms. However, it is difficult to speculate upon the relevance of this aberrant splicing effect. This is for two main reasons. Firstly, the effect on the splicing ratio is only relatively weak. Secondly, the D(11q) isoform has been shown to play a role in apoptosis Of the 6 codons analysed, only synonymous substitutions at codon 231 and codon 244 affected splicing of Substitutions at codon 231 caused skipping of exon 11 with a marked increase in amounts of the D(11) isoform. Overexpression of this isoform in mouse epithelial mammary cells has been shown to cause atypical duct hyperplasia Substitutions at codons 195, 196, 215 and 313 did not have any discernible effect on isoform levels. However, there may be other ways by which synonymous changes can exert an effect Nevertheless, the possibility that substitutions at these codon positions affect regulatory elements of splicing should not be excluded. In fact protective variants might have occurred during evolution that compensate for aberrant splicing BRCA1 exon 11 variants. We therefore tested three further synonymous variants found in breast cancer patients to investigate their effect on BRCA1 splicing. Our findings show that the mutations c.795T>C and c.825C>T decrease and increase levels of the D(11q) isoform respectively. However, for the same reasons mentioned above it is not possible to classify these variants as pathogenic mutations causing aberrant splicing since the role of D(11q) is not clear. In fact, the D(11q) isoform has been described as both causing apoptosis and causing cancer The c.693G>A substitution at codon 231 has previously been reported to cause exon 11 skipping in a patient with breast cancer Both artificial and natural variants analysed in this study cause multiple splicing effects . Deletion 3, just next to the D(11q) donor site, decreased D(11q) isoform levels, probably due to the loss of a putative binding site for TIA1 (a donor site modulator) predicted by the SpliceAid analysis. NOVA 1 binding was predicted to bind both regions corresponding to deletion 2 and 3. Binding of NOVA1 at the end of an exon and beginning of an intron was proposed to induce exon inclusion We did not observe strong correlation on the splicing effect of nucleotide changes occurring inside the region of deletion 2 (c.732T>C) and deletion 3 (c.825C>T). This suggests that composite regulatory elements of splicing might be present in this region of BRCA1 exon 11 as has been previously suggested for CFTR exon 12 BRCA1-related cancer, it will be necessary to know the roles of each isoform individually and to understand the combined roles of different isoforms both in health and in tumorigenesis. Part of this understanding will require confirmation of which splicing factors are involved in regulating alternative splicing of BRCA1, including those suggested by this study that may be involved in the splicing of exon 11. Knowledge of these mechanisms would provide a framework for the development of new therapeutic agents capable of manipulating BRCA1 splicing and treating BRCA1-related cancers (eg. breast ovarian and prostate cancer). At some level it may be that cancer predisposition is not so much down to whether individual isoforms are simply present or absent but rather that subtle alterations to a complex and nuanced isoform profile are particularly relevant. Such an isoform environment may interact with other cellular pathways to make conditions favourable or otherwise towards tumour development. If this is the case, it will provide an added challenge of complexity to our understanding of tumorigenesis. However, it may also allow novel and innovative approaches to manipulating the cellular environment in order to prevent and treat cancer.In order to fully understand the effects of altered splice isoform ratios in BRCA1 genomic region from exon 8 to exon 12. Introns have been shortened by PCR amplification with oligonucleotides carrying a non complementary tail for specific restriction digestion and subsequent cloning in pCDNA3+.The pB1 WT minigene is shown in Exon 1 of the α-globin gene is used as the first exon of the minigene as it provides a strong splice donor site at its 3′ end as well as an ATG start codon at its 5′ end.Using specific oligonucleotides and a two-step PCR mutagenesis method Human breast cancer cell lines, MCF7 (ATCC number: HTB-22™), were grown in DMEM medium with 4500 mg/L glucose, pyruvate and L-glutamine supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. Cells were incubated at 37°C in a 5% CO2 atmosphere.Minigene plasmid vector was transfected into MCF7 cell lines using the FuGENE 6 transfection reagent from Roche. 100 µl of DMEM serum-free medium containing 2 µg of vector DNA and 3 µl of FuGENE reagent was incubated for 15 minutes at room temperature before the mixture was added in 6 cm-well cell cultures (50% confluent) in the presence of 10% fetal bovine serum.BRCA1 exon 11 in the pB1 minigene, RT-PCR was performed with 1.5 µg of total RNA using the pCSrev primer [5′ GCAACTAGAAGGCACAGTCGAGG 3′] to exclusively target only RNA products from the pB1 minigene. Given the large size of exon 11 (3426 nucleotides), the QIAGEN LongRange RT PCR kit was used following the manufacturer's instructions. 1/4 of the resulting cDNA was amplified in a PCR reaction using primers specific for the desired splice isoforms and PCR products were analysed by gel electrophoresis in 1.5% agarose. The forward primer [9–10F: 5′ ACTTATTGCAGTGTGGGAGA 3′] hybridises to the junction between exons 9 and 10. Reverse primers are a mixture of one specific for FL [11FLR: 5′ GGAGTCCGCCTATCATTACATG 3′] and one specific for D(11q) and D(11) [12R: 5′ CCAGATGCTGCTTCACCCT 3′]. 11FLR hybridises within exon 11 distal to the 11q splice site. 12R hybridises to proximal exon 12 and overlaps the exon 10/12 junction and also the exon 11q/12 junction.40 hours after transfection, RNA was extracted from cells using the RNeasy-plus kit from QIAGEN following the manufacturer's instructions. To analyse alternative splicing of BRCA1 alternative splicing of exons 9 and 10, RT-PCR was performed on 1 µg total RNA using random primers with the Promega kit. BRCA1-minigene specific PCR was performed on resulting cDNA using the forward specific primer alpha-8F [5′ GAGGCCCTGGAGAGGAcAA 3′] and the reverse primer 11+81Rev [5′ TCTCAGTGGTGTTCAAATCA 3′]. Alpha-8F hybridises to the junction between exon 1 of the α-globin gene and exon 8 of BRCA1. The c nucleotide in lowercase represents the insertion made in exon 8 of the pB1 minigene. 11+81Rev hybridises to exon 11 upstream of the donor site producing D(11q).For the analysis of th of PCR products were subjected to ‘reconditioning PCR’ for 6 cycles In order to eliminate heteroduplexes from mixed-template 1/10BRCA1 exon 11 were predicted using the computational tools SFmap Putative splicing regulatory sequences in The following calculation parameters were chosen for SFmap:Scoring function: COS(WR);Medium stringency ;Window size: 50.Figure S1Minigene splicing assay of BRCA1 exon 9 and 10.A. The pB1 wild type (WT) version of the minigene is shown. PCMV = promoter of the pCDNA3 vector. ATG = start codon. TAG = stop codon. +3C = insertion of cytosine as the third nucleotide in exon 8. pA = poly A signal. 1 = exon 1 of the alfa globin gene. BRCA1 exons from 8 to 12 are numbered. The black solid line represents introns. Dotted lines show alternative splicing of exon 9 and 10. B. Detection of BRCA1 splicing isoforms FL (inclusion of exon 9 and 10); D9 (skipping of exon 9); D10 (skipping of exon 10); D9,10 (skipping of exon 9 and 10).(TIF)Click here for additional data file.Figure S2BRCA1 alignments between nine eutherian mammals. The sequence of exon 11 critical region is in blue. Black characters represent the last 6 nucleotides of intron 10. The alternative donor site (5′ss) in exon 11 which gives rise to the Δ(11q) isoform is in white characters. Red are the nucleotide variations with respect to the human sequence. Ca. = Canis_familiaris; Eq. = Equus_caballus; Bo. = Bos_taurus; Ho. = Homo_sapiens; Pa. = Pan_troglodytes; Po. = Pongo_pygmaeus; Ma. = Macaca_mulatta; Mu. = Mus_musculus; Ra. = Rattus_norvegicus. Splicing regulatory proteins (predicted with SFmap) putatively binding to the human sequence are shown at the top of each sequence. The one corresponding to splicing regulatory motifs that are most conserved are highlighted. The list of putative sequences for splicing regulatory proteins and relative scores as reported in SFmap are listed.(DOC)Click here for additional data file.Figure S3Splicing factors predicted to bind. The sequence of deletion 2 and deletion 3 region are shown. Splicing factors predicted (by SFmap and/or SpliceAid) to bind these regions are listed.(XLS)Click here for additional data file.Table S1Information about all synonymous variants tested. The table includes the amino acid number, the nucleotide change, the dbSNP rs number, genomic location in hg19 coordinates (Chr 17 position),.PMID (for existing PubMed records) and our interpretation of the effects caused by the variations on different splicing events tested (isoform regulation).(XLS)Click here for additional data file.


CYP3A locus have been identified, genetic association studies have so far failed to explain a major part of the phenotypic variability. The term “missing heritability” has been used to denominate the gap between expected and known genetic contribution, e.g., for complex diseases, and is also used here in analogy. In this review we summarize CYP3A4 pharmacogenetics/genomics from the early inheritance estimations up to the most recent genetic and clinical studies, including new findings about SNPs in CYP3A4 (*22) and other genes (P450 oxidoreductase (POR), peroxisome proliferator-activated receptor alpha (PPARA)) with possible contribution to CYP3A4 variable expression.CYP3A4 is the most important drug metabolizing enzyme in adult humans because of its prominent expression in liver and gut and because of its broad substrate specificity, which includes drugs from most therapeutic categories and many endogenous substances. Expression and function of CYP3A4 vary extensively both intra- and interindividually thus contributing to unpredictable drug response and toxicity. A multitude of environmental, genetic, and physiological factors are known to influence CYP3A4 expression and activity. Among the best predictable sources of variation are drug–drug interactions, which are either caused by pregnane X-receptor (PXR), constitutive androstane receptor (CAR) mediated gene induction, or by inhibition through coadministered drugs or other chemicals, including also plant and food ingredients. Among physiological and pathophysiological factors are hormonal status, age, and gender, the latter of which was shown to result in higher levels in females compared to males, as well as inflammatory processes that downregulate CYP3A4 transcription. Despite the influence of these non-genetic factors, the genetic influence on CYP3A4 activity was estimated in previous twin studies and using information on repeated drug administration to account for 66% up to 88% of the interindividual variation. Although many single nucleotide polymorphisms (SNPs) within the  CYP3A5*1 allele and with low CYP3A4 expression . Average microsomal content has been estimated between ∼60 pmol/mg of microsomal protein  which occurs in white populations at ∼2–9% but at higher frequencies in Africans. This single nucleotide polymorphism (SNP) was initially found to be associated with higher tumor grade and stage in prostate cancer and showed higher nifedipine oxidase activity in human livers  was identified by a systematic screen for SNPs showing allelic mRNA expression imbalance in human liver CYP3A4*1 another 21 alleles are included, comprising at least 21 amino acid changes and 2 frame shift variants. Although this collection is a useful resource for functional data related to the alleles, it only provides limited information on genetic variability. Further valuable information on CYP3A4 SNPs and clinical pharmacogenetics is available on the homepage of The Pharmacogenomics Knowledgebase PharmGKBCYPallele database or PharmGKB they lack cross reference to documented functional impact  to large structural variations like copy number variants and large deletions and insertions, as well as frequency information and haplotype context . While in the HapMap data  contains in total 471 SNPs assigned to the major transcript (ENST00000336411), including NCBI dbSNP database content (build 134). The 1000 Genomes Project to date (accessed September 2012) contributed 129 new SNPs to this collection. Besides 115 intronic variant positions 7 non-synonymous amino acid changes were previously unknown, of which four variants were reported on the CYPallele homepage  and only one was predicted to be deleterious by phenotype prediction tools SIFT and PolyPhen  genes including CYP3A4 in diverse populations will profit enormously from 1000 Genomes and new candidate SNPs influencing CYP3A4 expression and activity will certainly be discovered, although prediction or experimental testing of functional impact of the many novel variants pose a challenging task.For the CYP3A4 expression have been studied only in recent years by single gene or pathway-directed approaches.Regulation of CYP3A4 phenotype expression is enormously complex including influences from networks of nuclear receptors and other transcription factors  is a microsomal flavoprotein and an obligatory electron donator in the microsomal P450 monooxygenase reaction. In contrast to the multiplicity of CYPs, mammals have only a single POR should thus be expected to influence CYP activity. In recent years rare POR missense mutations in humans were discovered that impair POR function and cause disordered steroidogenesis, ambiguous genitalia, and Antley–Bixler syndrome  is common with frequencies ranging from 19 to 37% in all major ethnicities. In recombinant systems the variant retained >50% of the wild type activity toward several CYPs  and constitutive androstane receptor  are important transcription factors in the regulatory network of CYP3A4 constitutive and inducible expression. Both genes are polymorphic and genetic variants have been studied for impact on CYP3A4 transcription  gene] display largely overlapping substrate selectivity  gene to be associated with higher expression of FoxA2 mRNA and its target genes PXR and CYP3A4. Polymorphisms in FoxA2, HNF4α, FoxA3 (HNF3Γ), PXR, MDR1, and the CYP3A4 promoter together with sex explained 24.6% of the variation in hepatic CYP3A4 mRNA expression. However the study lacked information on the relevance of these variations for CYP3A4 protein and activity.In search for additional influential genes, Lamba et al.  phenotypARNT), glucocorticoid receptor (GR), progesterone receptor membrane component 2 (PGRMC2), and peroxisome proliferator-activated receptor alpha (PPARA) to be consistently associated with CYP3A4 phenotype in human liver in the multivariate analysis. Validation in an atorvastatin-treated volunteer cohort confirmed decreased atorvastatin-2-hydroxylation in carriers of PPARA SNP rs4253728. Moreover, homozygous carriers of the variant had reduced PPARα protein expression in liver, and shRNA-mediated PPARA gene knock-down in primary human hepatocytes decreased mRNA expression levels of CYP3A4 by more than 50%. Multivariate analysis revealed that two linked PPARA SNPs alone explained ∼8–9% of the atorvastatin hydroxylase activity variation, whereas all genetic and non-genetic factors together accounted for ∼33% of atorvastatin 2-hydroxylase variation in this liver cohort , whereas all remaining 24 SNPs were described as trans-acting elements, and only one of these (rs12041966 located on chromosome 1) influenced testosterone hydroxylation  for numerous liver-expressed genes The in vivo phenotype is still far away from the expected 60–80% of genetic determination. The use of next generation sequencing approaches for the identification of causal variants in CYP3A4 as well as the numerous genes of its many influential pathways may lead to the identification of many more rare and common DNA variants that together account for a sizeable fraction of this variability. The examples of POR and PPARA demonstrate that CYP3A4 variability is at least in part determined by polymorphisms in genes outside the CYP3A4 locus. However, investigating and validating trans-acting factors is much more difficult due to the more indirect nature of interaction which is more likely to be masked by covariates, thus necessitating larger studies, in vitro or in vivo. In addition, mathematical algorithms are needed to combine many genetic variants, some of which contribute only small fractions to the total variability, into practically useful signatures for application on clinical studies and individualized medicine.In conclusion, the predictive power of currently known genetic polymorphisms with relevance for CYP3A4 The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea; and, although multiple genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. In this study, we investigated the roles of Tbx4 and Tbx5 in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in Tbx4 and Tbx5 show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. Lung branching undergoes arrest ex vivo when Tbx4 and Tbx5 are both completely lacking. Lung-specific Tbx4 heterozygous;Tbx5 conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings. Sox9, a master regulator of cartilage formation, is expressed in the trachea; but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. Tbx4;Tbx5 double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction. Finally, we show that Tbx4 and Tbx5 interact with Fgf10 during the process of lung growth and branching but not during tracheal/bronchial cartilage development.Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Both  Tbx4 and Tbx5, in three processes essential to the development of the respiratory system: the specification of the lung and trachea primordia, the growth and branching of the airways to form the lung, and formation of cartilage rings of the trachea and bronchi. In the absence of Tbx5, only one lung is specified, and no trachea. Both Tbx4 and Tbx5 regulate the process of lung branching by controlling the expression of the secreted growth factor Fgf10 and activation of Fgf10 signaling. In the trachea, both Tbx4 and Tbx5 are important for condensation of cells to form cartilage rings, although this is regulated by a distinct pathway that does not involve Fgf10.Defective development of the mammalian respiratory system can lead to tracheal, bronchial, or pulmonary malformations causing severe consequences at birth or during postnatal life. Studies using mouse genetics have begun to reveal complex regulatory mechanisms that guide the development of the respiratory system, but understanding how disruption of these mechanisms leads to malformations is far from complete. In this study, we analyze the role of two T-box transcription factors,  The development of the respiratory system represents an evolutionary hallmark that allowed vertebrates to survive on land utilizing air as a source of oxygen. Because the respiratory system is dispensable for embryonic survival in mammals, defects in development of the respiratory system manifest at or after birth. Indeed, abnormal development of the respiratory system in humans is associated with multiple disorders such as tracheal/bronchial atresia, tracheoesophageal fistula, bronchogenic cysts, pulmonary/lobar atresia and pulmonary hypoplasia Nkx2.1 has been identified as the earliest marker of lung endoderm specification. At E9.25 (25–28 somites), the primary lung buds appear as ventro-lateral outpouchings of the foregut connected ventrally by the tracheal primordium. The lung buds grow in a ventro-posterior direction and continue to elongate until E11.5. The point of connection of the lung buds is thought to be the origin of the tracheal tube, which separates from the esophagus in a caudal to cranial direction by E11.5 In the mouse embryo, the endodermal foregut tube is patterned by signals from the lateral plate mesoderm leading to specification of the lung and trachea at embryonic day (E) 9.0 (19–24 somites). Wnt2, Fgf10, Bmp4, Shh and retinoic acid synthesis genes, have been shown to play important roles in lung specification and branch formation. Complete absence of both Wnt2 and Wnt2b in mesenchyme surrounding the anterior foregut or absence of β-catenin in the foregut epithelium leads to a loss of specification of lung primordia as seen by the absence of Nkx2.1 expression Fgf10, which is normally expressed in mesenchyme surrounding the epithelial branching tips, form a short trachea but have no lungs Bmp4 signaling by overexpression of Xnoggin leads to a decrease in lung size and irregularly shaped lung lobes Shh null mutant mice have only a rudimentary lung sac due to branching severe branching defect Shh in lung epithelial cells leads to the formation of hypoplastic lungs with reduced branching of the peripheral tubules Genes involved in different signaling pathways, including Sox9 has been implicated as an important regulator of mesenchymal condensation and chondrocyte differentiation Sox9, FGF2, Igf1, Tgfβ2 and Bmp2 enhance chondrocyte formation Shh, Sox2, retinoic acid synthesis genes and Fgf signaling pathway genes have been shown to affect cartilage ring formation Fgf10 mutants form a partial tracheal tube in spite of the failure of lung formation Fgf10 leads to defects in tracheal ring formation and that overexpression of Fgf10 between E11.5 and E13.5 disrupts tracheal rings by altering the periodic expression of Shh in the trachea After E11.5, mesenchyme surrounding the dorsal aspect of the trachea differentiates into the trachealis smooth muscle. Mesenchyme surrounding the ventral aspect of the trachea and lateral aspect of the main stem bronchi segments and differentiates into C shaped rings composed of chondrocytes. Ventral tracheal cartilage is formed by migration of cells that undergo mesenchymal condensation Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing chick lung buds and trachea between stages 15–21 Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later Tbx1 homozygous null mutants die at birth due to severe heart defects; the lungs are never fully inflated Tbx4 homozygous mutants, lung buds form but the embryos die at E10.5 due to failure of allantois development and the subsequent lack of chorio-allantoic fusion leading to placental insufficiency Tbx5 mutants die around E10 due to defects in heart development Tbx4 and Tbx5, but not Tbx2 and Tbx3, in lung organ cultures results in inhibition of branching and loss of Fgf10 expression Tbx4 function leads to a reduction in Fgf10 expression in lung mesenchyme and inhibits lung bud formation. Ectopic expression of Tbx4 leads to ectopic expression of Fgf10 and Nkx2.1 and lung bud formation in the esophagus. Additionally, ectopic expression of Tbx4 at the boundary between the trachea and the esophagus can lead to lack of separation of these two structures, resulting in a tracheoesophageal fistula Tbx5 mutations cause Holt Oram syndrome characterized by heart and forelimb abnormalities. A single de-novo mutation in TBX5 has been linked to right lung agenesis The T-box transcription factor genes are important during embryonic development. All members of this gene family contain a conserved DNA-binding T-box domain, which binds to a conserved sequence, the T-box binding element, to activate or repress transcription of specific target genes Tbx4 and Tbx5 in lung and trachea development in the mouse, we made use of conditional alleles to bypass early embryonic lethality. We studied three distinct processes, namely 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation. We show that during early stages of development, Tbx5 is important for specification of the lung buds and the trachea. After specification, Tbx4 and Tbx5 interact during lung growth and branching and the regulation of branching is dependent on Fgf10 signaling. Additionally, Tbx4 and Tbx5 interact in the formation of mesenchymal condensations, which ultimately form the tracheal/bronchial cartilage rings independent of Fgf10 signaling.To study the roles of Tbx5 expression is first detected using in situ hybridization (ISH) at E9.0 (24 somites) in the mesenchyme of the lung and trachea primordia, concurrent with Nkx2.1 expression in the ventral foregut epithelium (Tbx4 expression is detected in the lung buds when they first appear a few hours later at E9.25 (28 somites) in a pattern similar to Tbx5 , the foregut tube is devoid of Nkx2.1 expression and the lung buds are not present Nkx2.1 expression  and removal of Tbx4 in addition to Tbx5 did not exacerbate the Tbx5 phenotype , late enough to bypass lethality but well before lung branching begins. Embryos with CreER and different combinations of the flTbx4 and flTbx5 alleles were examined at E12.5 and E13.5. Conditional Tbx4 null lungs were similar to controls at E12.5 but had fewer branching tips at E13.5 . We hypothesize that the variability in phenotype is due to variable extent of recombination of the flTbx5 allele  became cyanotic at birth and died shortly thereafter due to respiratory distress. Unlike the variable lung size in the lung-specific Tbx5 null mutants, the lungs of these mutants were consistently smaller than controls at E13.5  nor Tbx4 single heterozygous lungs show a branching defect, double heterozygous lungs are smaller at E13.5 and E18.5 with a reduced number of branching tips at E13.5. Because these genes are closely related Tbx4 and Tbx5 could physically interact with each other as heterodimers to activate or repress transcription of downstream targets, as has been suggested for other T-box genes Tbx4;Tbx5 double heterozygous lungs are smaller than conditional Tbx4 nulls. Unequal and distinct functions could be explained by domains outside of the T-box. For instance, the C terminal domain of both Tbx4 and Tbx5 has transcription activating capability which has been correlated to the shared limb outgrowth promoting activity of these genes In lung branching morphogenesis,  another . AlthougTbx4 nulls;Tbx5 heterozygous and lung-specific Tbx4 heterozygous;Tbx5 null lungs, reduction of Tbx4 or Tbx5 leads to a severe decrease in branching, a defect in formation of the lobes and failure of lobe separation, while absence of Tbx4 and Tbx5 leads to branching arrest. These results are in line with the published observations that antisense RNAs used against both Tbx4 and Tbx5 inhibit lung branching in culture and that this affect is explained by loss of Fgf10 expression Fgf10 promoter in vitro suggesting that Fgf10 is a direct downstream target Fgf10 and Wnt2 in the lung mesenchyme. The Fgf10 targets Bmp4 and Spry2 show very low levels of expression in the epithelium of Tbx4 and Tbx5-deficient mutants and Etv5, another Fgf10 target, shows greatly reduced expression in the conditional double null lungs, leading to the hypothesis that the Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. Lungs that are triple heterozygous for Tbx4, Tbx5 and Fgf10 are reduced in size compared to Tbx4;Tbx5 double heterozygous lungs, which supports this hypothesis. Lung-specific Tbx4 heterozygote;Tbx5 null lungs are severely retarded but lung size is not affected by further loss of Fgf10, demonstrating epistasis and supporting the hypothesis that Fgf10 lies downstream of Tbx4 and Tbx5. Lack of rescue of branch formation and lack of activation of the Fgf10 signaling pathway by exogenously supplied Fgf10 in culture suggests that there are other factors downstream of Tbx4 and Tbx5 that affect Fgf10 signaling. Although, lungs deficient in Tbx4 and Tbx5 retain the ability to respond to Fgf10 coated beads . The extracellular matrix (ECM) molecules, heparan sulfate (HS) proteoglycans aid in Fgf10-Fgfr2 interactions during lung development Tbx4 and Tbx5-deficient mutants. Inhibition of heparanase decreases submandibular gland morphogenesis in culture due to deficiency of Fgf signaling Tbx4 regulates ECM molecules in the developing allantois, specifically the chondroitin sulfate proteoglycan versican (R. Arora and V. E. Papaioannou unpublished observations), suggesting that ECM might be one of the targets for T-box genes in regulating development of other organs as well.One possibility for such a factor is a mesenchymal signaling molecule that communicates with the epithelium and activates downstream target(s) required for the activation of the Fgf10 pathway. We examined expression of Tbx4 and Tbx5 causes defects in both formation of the tracheal cartilage rings and development of the trachealis smooth muscle indicating interactions between Tbx4 and Tbx5. Tbx4 and Tbx5 heterozygous tracheas have 10–11 cartilage rings but Tbx4;Tbx5 double heterozygous tracheas have 6–8 cartilage rings, some of which are incomplete. Additional reduction of Tbx4 and Tbx5 in the lung-specific Tbx4 heterozygous;Tbx5 null lungs leads to a complete disruption of cartilage ring formation supporting a genetic interaction between Tbx4 and Tbx5 in trachea formation.Appropriate dorsal smooth muscle development and ventral tracheal/bronchial cartilage development is important for the normal functioning of the trachea. Smooth muscle provides tracheal flexibility and the cartilaginous rings prevent tracheal collapse. Defects in trachea formation may result in tracheomalacia or tracheal stenosis. Reduction of Sox9, a master regulator of the process of chondrogenesis, is expressed normally at E12.5 throughout the ventral mesenchyme in lung-specific Tbx4 heterozygous;Tbx5 null tracheas but at E13.5 these tracheas show a lack of characteristic mesenchymal condensations and reduced Sox9 expression. Either Tbx4 and Tbx5 control of Sox9 expression becomes more sensitive to dosage at E13.5 or Tbx4 and Tbx5 control another factor, possibly an ECM molecule, which is important for formation of mesenchymal condensations and, in the absence of these condensations, there is a down regulation of Sox9 expression  at a concentration of 20 mg/ml in sunflower oil (Sigma) was administered to pregnant females by intraperitoneal injection between 15.30 and 19.30 hours on E8.5 or between 23.00 and 24.00 hours on E9.0.Whole-mount ISH, immunohistochemistry (IHC), immunofluorescence (IF) and ISH on cryosections was performed as described previously For histology embryos were removed from the uterine horns, dissected out of the decidua and fixed in Bouin's fixative (Sigma). After dehydration in ethanol, embryos were embedded in paraffin wax, sectioned at 8 µm thickness and stained with hematoxylin and eosin (H & E).Alcian blue staining was performed according to standard protocols 2 on Transwell-Col filters (Fisher Scientific) containing 1.5 ml BGJb media (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 0.2 mg/ml vitamin C (Sigma) and 2 µM 4-OH tamoxifen (Sigma). For lung bud culture, lung buds were dissected at E11.5 in phosphate buffered saline (PBS) with 0.1% bovine serum albumin (Sigma) and cultured in media containing DMEM (Invitrogen) with 10% fetal bovine serum, 1% penicillin/streptomycin (Invitrogen) and 1 µM 4-OH tamoxifen on 3.0 µm filters (Millipore) or 0.4 µm Transwell filters (Fisher Scientific). Similar results were obtained using both types of filter; results reported are for experiments with Millipore filters. Where specified, Fgf10 (R&D) was added after 1 day of culture at a concentration of 500 ng/ml. In some experiments heparin beads coated with Fgf10 (100 µg/ml) or BSA (100 µg/ml) were placed near the branching tips of the explants after 1 day of culture. Transwell filters were used for the bead experiments.Foregut culture was carried out as described previously E-cadherin RNA probe using ISH. In case of whole mount lungs, lobes were separated and photographed to count the number of branching tips. The cultured lungs were photographed and the branching tips were counted. For some experiments, after E-cadherin ISH, lungs were post fixed in 4% PFA, washed with PBT, dehydrated in 100% methanol, cleared in BABB and then photographed.Lungs were stained with either E-cadherin antibody using IHC or Figure S1flTbx4 and flTbx5. Females carrying embryos with flTbx4, flTbx5 and CreER alleles were injected with either 8 mg tamoxifen at E9.0 and dissected at E12.5  or 7 mg tamoxifen at E8.5 and dissected at E13.5 . fl/flTbx4 was completely excised to mutant −/−Tbx4 at both doses and times  whereas the single conditional allele of fl/+Tbx5 was incompletely excised by injection at E9.0 (E) but completely excised by injection at E8.5 (F). In addition when lungs with the genotype fl/flTbx4; fl/flTbx5; CreER were treated with 1 µm tamoxifen in culture, the Tbx4 locus was nearly completely excised at the end of 1 day of culture (C) but the Tbx5 locus was only partially excised (G). At the end of a 4 day culture both Tbx4 (D) and Tbx5 (H) loci achieved virtually complete excision. fl, floxed conditional PCR band; WT, wild type PCR band; Mut, excised PCR band.Analysis of excision efficiency of (TIF)Click here for additional data file.Figure S2Tbx4 and Tbx5 does not affect alveolar differentiation.  T1α IHC on cryosections of control and lung-specific Tbx4 heterozygous;Tbx5 null lungs at E18.5 shows comparable staining in the lung epithelium. Nuclear fast red was used as a counter stain. A′ and B′ are higher magnification views of boxed regions in A and B.  Prosurfactant protein C (Pro-SPC) IF on cryosections of control and lung-specific Tbx4 heterozygous;Tbx5 null lungs at E18.5 shows comparable staining in the lung epithelium. DAPI was used to stain the nuclei. Scale bars represent 100 µm.Loss of (TIF)Click here for additional data file.Figure S3Tbx4 and Tbx5 has multiple affects on branching morphogenesis.  H & E sections of lung-specific Tbx4 heterozygous;Tbx5 null lungs (B) show lack of separation of the cranial (cr), medial (m) and caudal (cd) lobes in the right lung as compared to control lungs (A). Black arrowheads point to the space created between the lobes in the control lungs (A) and to the corresponding regions of the mutant lungs (B).  Control lungs stained for E-cadherin at E13 show greater outgrowth (yellow parentheses) of lateral branches (C) than the lung-specific Tbx4 heterozygous;Tbx5 null lungs (D). Scale bars represent 100 µm.Loss of (TIF)Click here for additional data file.