Recent strategies targeting Embryonic Ectoderm Development (EED) for cancer therapy: Allosteric inhibitors, PPI inhibitors, and PROTACs.

The polycomb repressive complex 2 (PRC2), which comprised of the core subunits: Enhancer of Zeste Homolog 2 (EZH2), Suppressor of Zeste 12 (SUZ12), and Embryonic Ectoderm Development (EED), is an essential epigenetic gene silencer responsible for depositing repressive histone H3 lysine 27 trimethylation (H3K27me3) marks on chromatin. The aberrant activity of PRC2 is closely involved in tumorigenesis and progression, making its inhibition a viable strategy for epigenetic cancer therapy. Although the clinical development of small PRC2 inhibitors has made impressive progress, with one EZH2 inhibitor approved for cancer therapy and several other candidates in clinical trials, current EZH2 inhibitors are limited to treating certain hematological malignancies and have acquired drug resistance. EED is essential for PRC2 stabilization and allosterically stimulating PRC2 activity because it functions as a scaffold protein and an H3K27me3-recognizing protein. Thus, due to its novel mechanism of action, targeting EED provides a promising new strategy for inhibiting PRC2 function and exhibits the potential to overcome the issues encountered by EZH2 inhibitors. This review provides a comprehensive overview of available cancer therapy strategies that target EED, including allosteric inhibitors, protein-protein interaction (PPI) inhibitors, and proteolysis-targeting chimeras (PROTACs).

An intrafamilial phenotypic variability in Ellis-Van Creveld syndrome due to a novel 27 bps deletion mutation.

Ellis-van Creveld (EvC) syndrome is an autosomal recessive disease, characterized by ectodermal, skeletal, and cardiac anomalies. We report intrafamilial phenotypic variability in three new EvC syndrome cases. Affected males in this study showed only ectodermal abnormalities, whereas an affected female showed the classical presentation of EvC Syndrome, including bilateral postaxial polydactyly of hands and feet, and congenital heart defects. Whole exome sequencing was performed to identify the causative variant, followed by validation and segregation analysis using Sanger sequencing. A homozygous deletion variant (c.731_757del) was identified in exon 6 of the EVC gene (NM_153717.2). The identified variant is considered to be the most likely candidate variant for the EvC syndrome in the family based on previous reports validating the role of EVC variants in the EvC syndrome. The disease correctly segregated in the family members, as all affected members were homozygous, and obligate carriers were heterozygous. Our family is remarkable in highlighting the variable expressivity of the EvC phenotype within the same family, due to a homozygous deletion mutation in the EVC gene. The variable expressivity might be due to the hypomorphic nature of mutation, or the presence of additional variants in modifier genes or in the regulatory regions of the EVC/EVC2 genes.

Trophectoderm biopsy protocols may impact the rate of mosaic blastocysts in cycles with pre-implantation genetic testing for aneuploidy.

PURPOSE: This study aimed to analyze the impact of different biopsy protocols on the rate of mosaic blastocysts. METHODS: This is a retrospective cohort study which included 115 cycles with pre-implantation genetic testing for aneuploidy (PGT-A). Two groups were allocated based on the biopsy protocols: method 1 group, the zona pellucida (ZP) was drilled on day 3 embryos followed by trophectoderm (TE) biopsy; and method 2 group, the ZP was opened on day 5 or 6 blastocysts followed by TE biopsy. All biopsy samples were assessed using next-generation sequencing (NGS) at a single reference laboratory. The euploid, aneuploid, and mosaic blastocyst rates and clinical outcomes were compared. RESULTS: The mosaicism rate in the method 1 group was 19.58%, significantly higher than the method 2 group (8.12%; P < 0.05). No statistically significant difference was observed in euploid, aneuploid blastocyst rates, and clinical pregnancy rates between the two groups. Logistic regression analysis indicated that the biopsy protocols were independently associated with the mosaicism rates among all the variables. CONCLUSIONS: The present study showed that different biopsy protocols may have an impact on the mosaic blastocyst rate. ZP opening on day 3 combined with TE biopsy might increase the incidence of mosaic blastocysts.

Trim-Away mediated knock down uncovers a new function for Lbh during gastrulation of Xenopus laevis.

We previously identified the protein Lbh as necessary for cranial neural crest (CNC) cell migration in Xenopus through the use of morpholinos. However, Lbh is a maternally deposited protein and morpholinos achieve knockdowns through prevention of translation. In order to investigate the role of Lbh in earlier embryonic events, we employed the new technique "Trim-Away" to degrade this maternally deposited protein. Trim-Away utilizes the E3 ubiquitin ligase trim21 to degrade proteins targeted with an antibody and was developed in mammalian systems. Our results show that Xenopus is amenable to the Trim-Away technique. We also show that early knockdown of Lbh in Xenopus results in defects in gastrulation that present with a decrease in fibronectin matrix assembly, an increased in mesodermal cell migration and decrease in endodermal cell cohesion. We further show that the technique is also effective on a second abundant maternal protein PACSIN2. We discuss potential advantages and limit of the technique in Xenopus embryos as well as the mechanism of gastrulation inhibition.

Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications.

OBJECTIVE: To study whether embryonic cell-free DNA (cfDNA) in spent blastocyst media is representative of the chromosomal constitution of a blastocyst. DESIGN: Pilot prospective blinded study. SETTING: In vitro fertilization center and genetics laboratory. PATIENT(S): A total of 115 trophectoderm (TE) biopsies and spent blastocyst media (SBM) from 46 patients with ages ranging from 32 to 46 years, whose indications for preimplantation genetic testing of aneuploidy (PGT-A) were advanced maternal age, recurrent miscarriage, or recurrent implantation failure. INTERVENTIONS(S): Spent blastocyst media collection and TE biopsy. MAIN OUTCOME MEASURE(S): Concordance rates, sensitivity, and specificity between TE biopsies and SBM. Clinical outcomes in cases with euploid TE biopsies and euploid SBM compared with cases with euploid TE and aneuploid SBM. RESULT(S): In general, the total concordance rate for ploidy and sex was 78.7%, and sensitivity and specificity were 94.5% and 71.7%, respectively. A significant increase for all parameters was observed for day 6/7 samples compared with day 5 samples, with day 6/7 samples showing total concordance for ploidy and sex of 84%, and sensitivity and specificity of 95.2% and 82.1%, respectively. Ongoing implantation rates in euploid TE/euploid SBM showed a threefold increase compared with euploid TE/aneuploid SBM (52.9% vs. 16.7%, respectively), without reaching significant differences. Interestingly, no miscarriages were observed when TE and SBM were euploidy concordant. CONCLUSION(S): These results offer a better understanding of the dynamics of cfDNA during embryo development and despite more basic research being needed, they are reassuring to consider in the future this noninvasive approach as an alternative to TE biopsy for PGT-A.

Maternal and neonatal outcomes associated with trophectoderm biopsy.

OBJECTIVE: To assess whether pregnancies achieved with trophectoderm biopsy for preimplantation genetic testing (PGT) have different risks of adverse obstetric and neonatal outcomes compared with pregnancies achieved with IVF without biopsy. DESIGN: Observational cohort. SETTING: University-affiliated fertility center. PATIENT(S): Pregnancies achieved via IVF with PGT (n = 177) and IVF without PGT (n = 180) that resulted in a live birth. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Maternal outcomes including preeclampsia and placenta previa and neonatal outcomes including birth weight and birth defects. RESULT(S): There was a statistically significant increase in the risk of preeclampsia among IVF+PGT pregnancies compared with IVF without PGT pregnancies, with an incidence of 10.5% versus 4.1% (adjusted odds ratio [aOR] = 3.02; 95% confidence interval [95% CI], 1.10, 8.29). The incidence of placenta previa was 5.8% in IVF+PGT pregnancies versus 1.4% in IVF without PGT pregnancies (aOR = 4.56; 95% CI, 0.93, 22.44). Similar incidences of gestational diabetes, preterm premature rupture of membranes, and postpartum hemorrhage were observed. IVF+PGT and IVF without PGT neonates did not have a significantly different gestational age at delivery or rate of preterm birth, low birth weight, neonatal intensive care unit admission, neonatal morbidities, or birth defects. All trends, including the significantly increased risk of preeclampsia in IVF+PGT pregnancies, persisted upon stratification of analysis to only singleton live births. CONCLUSION(S): To date, this is the largest and most extensively controlled study examining maternal and neonatal outcomes after trophectoderm biopsy. There was a statistically significant three-fold increase in the odds of preeclampsia associated with trophectoderm biopsy. Given the rise in PGT use, further investigation is warranted.

Clinical delineation of GTPBP2-associated neuro-ectodermal syndrome: Report of two new families and review of the literature.

The GTPBP2 gene encodes a guanosine triphosphate (GTP)-binding protein of unknown function. Biallelic loss-of-function variants in the GTPBP2 gene have been previously reported in association with a neuro-ectodermal clinical presentation in six individuals from four unrelated families. Here, we provide detailed descriptions of three additional individuals from two unrelated families in the context of the previous literature. Both families carry nonsense variants in GTPBP2: homozygous p.(Arg470*) and compound heterozygous p.(Arg432*)/p.(Arg131*). Key features of this clinically recognizable condition include prenatal onset microcephaly, tone abnormalities, and movement disorders, epilepsy, dysmorphic features, retinal dysfunction, ectodermal dysplasia, and brain iron accumulation. Our findings suggest that some aspects of the clinical presentation appear to be age-related; brain iron accumulation may appear only after childhood, and the ectodermal findings and peripheral neuropathy are most prominent in older individuals. In addition, we present prenatal and neonatal findings as well as the first Caucasian and black African families with GTPBP2 biallelic variants. The individuals described herein provide valuable additional phenotypic information about this rare, novel, and progressive neuroectodermal condition.

Pregnancy rates after pre-implantation genetic screening for aneuploidy are only superior when trophectoderm biopsy is performed on hatching embryos.

PURPOSE: In vitro fertilization with trophectoderm embryo biopsy and pre-implantation genetic screening with comprehensive chromosomal screening (PGS-CCS) for aneuploidy is becoming increasingly more popular. Embryos are cryopreserved and implanted in a subsequent frozen thawed embryo transfer cycle (FET). No studies have investigated differences in pregnancy outcomes by timing of trophectoderm biopsy relative to stages of blastocyst development. METHODS: Retrospective study of all patients (n = 363) at a single IVF center between January 1, 2013 and December 31, 2016 undergoing single embryo transfer with PGS-CCS where embryos were cryopreserved with subsequent FET. Embryo expansion and grading was assessed both at the time of biopsy and transfer. Pregnancy rates were analyzed by embryo expansion and embryo grading. RESULTS: Implantation, clinical pregnancy, and live birth rates improved significantly with increased embryo expansion at the time of embryo biopsy (P < 0.001). Pregnancy loss decreased with increases in embryo expansion prior to biopsy (P < 0.001). Superior live birth rates with PGS-CCS were seen when embryos were hatching at the time of biopsy (p < 0.001). For fresh and frozen embryo transfers without PGS-CCS, embryo expansion did not affect pregnancy outcomes. CONCLUSIONS: PGS-CCS significantly increases implantation and live birth rates only if embryos are hatching at the time of biopsy. The embryo biopsy itself on a non-hatching embryo significantly damages the embryo in ways which are not reflected in future embryo expansion. IVF labs should wait until embryos hatch before performing trophectoderm biopsy.

Congenital Dermal Sinus and Limited Dorsal Myeloschisis: "Spectrum Disorders" of Incomplete Dysjuction Between Cutaneous and Neural Ectoderms.

BACKGROUND: The existence of tethering tracts in spinal dysraphism, other than congenital dermal sinus (CDS), has been recognized and has been summated into an integrated concept of limited dorsal myeloschisis (LDM). OBJECTIVE: To elucidate the underlying embryology of LDM in relation to CDS by focusing on the pathological features of special cases of tethering tracts. METHODS: Out of 389 spinal dysraphism patients who were operated on from 2010 to 2016, 5 patients who had tethering tracts composed of both CDS and LDM (or "probable LDM" if only fibrous tissue was found) were identified. Their clinical presentation, radiological images, operative findings, and pathology were thoroughly reviewed. RESULTS: Three nonsaccular-type patients harbored stalks in which the squamous epithelial lined sinus (CDS) was found in the distal portion, and fibroneural (LDM) or fibrous (probable LDM) tissue in the proximal part. Two patients had saccular lesions, and a stalk was found inside the sac, connecting a small pit on the skin to the spinal cord. The tracts were pathologically identical to a CDS. CONCLUSION: This study reports the coexistence of CDS and LDM (or probable LDM) components. These unique cases support the hypothesis that the CDS and LDM are among a spectrum of an anomaly that is caused by failure of complete dysjunction between cutaneous and neural ectoderms. Neurosurgeons should be aware of the possibility of coexisting "CDS" components in cases suggestive of LDM. In such cases, not only untethering but also meticulous removal of the squamous epithelium is critical.

Karyotype of the blastocoel fluid demonstrates low concordance with both trophectoderm and inner cell mass.

OBJECTIVE: To compare the genomic profiles of blastocoel fluid (BF), inner cell mass (ICM), and trophectoderm (TE) cells derived from the same blastocyst. DESIGN: Prospective study. SETTING: Academic and in vitro fertilization units. PATIENT(S): Sixteen donated cryopreserved embryos at blastocyst stage. INTERVENTION(S): BF, TE, and ICM cells were retrieved from each blastocyst for chromosome analysis by means of next-generation sequencing (NGS). MAIN OUTCOME MEASURE(S): Aneuploidy screening and assessment of mosaicism in BF, TE and ICM samples with subsequent comparison of genomic profiles between the three blastocyst compartments. RESULT(S): Out of 16 blastocysts, 10 BF samples and 14 TE and ICM samples provided reliable NGS data for comprehensive chromosome analysis. Only 40.0% of BF-DNA karyotypes were fully concordant with TE or ICM, compared with 85.7% concordance between TE and ICM. In addition, BF-DNA was burdened with mosaic aneuploidies and the total number of affected chromosomes in BF was significantly higher compared with the TE and ICM. CONCLUSION(S): BF-DNA can be successfully amplified and subjected to NGS, but owing to increased discordance with ICM and TE, BF does not adequately represent the status of the rest of the embryo. To overcome biologic and technical challenges associated with BF sampling and processing, blastocentesis would require improvement in both laboratory protocols and aneuploidy calling algorithms. Therefore, TE biopsy remains the most effective way to predict embryonic karyotype, and the use of BF as a single source of DNA for preimplantation genetic screening is not yet advised.

Frequencies of chromosome-specific mosaicisms in trophoectoderm biopsies detected by next-generation sequencing.

OBJECTIVE: To examine the chromosome-specific frequencies of mosaicism detected by next-generation sequencing (NGS) compared with constitutional aneuploidy. DESIGN: Retrospective cross-sectional review of NGS results from trophectoderm biopsies analyzed by per-chromosome prevalence of mosaicism and constitutional aneuploidy. SETTING: Private fertility clinic. PATIENT(S): A total of 378 patients who underwent preimplantation genetic screening by NGS for routine clinical indications from February 2016 to April 2017. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Aneuploidies and mosaicisms were tabulated per chromosome, and whole-chromosome and segmental mosaicisms were also analyzed. RESULT(S): NGS results were analyzed from 1,547 blastocysts. Mosaicism was detected as the sole abnormality in 17.5% (n = 270) of samples but were also found in 196/634 aneuploid embryos, so the overall incidence of mosaicism per biopsy was 30.1%. Mosaicism did not statistically vary when stratified by maternal age. The mean rate of overall mosaicism per chromosome was 2.46%. When whole chromosome and segmental mosaicisms were compared, unequal frequencies were found in several chromosomes. Trisomy was more frequently detected as whole-chromosome mosaicism, although monosomy was more frequently seen in segmental mosaicism. Aneuploidy and mosaicism displayed different patterns of distribution in various chromosomes. CONCLUSION(S): Mosaicism is unequally detected in various chromosomes and appears distinct from the distribution pattern of constitutional aneuploidy. Whole chromosome and segmental mosaicisms are also differentially detected. These results contribute to the study of mosaicism, illuminating a differential pattern of detection across the genome.

Diagnosis of parental balanced reciprocal translocations by trophectoderm biopsy and comprehensive chromosomal screening.

PURPOSE: This study investigates a case series of eight couples who underwent trophectoderm (TE) biopsy and comprehensive chromosomal screening (CCS) for routine aneuploidy screening and were found to have CCS results concerning for previously undetected parental balanced reciprocal translocations. METHODS: In each case, controlled ovarian hyperstimulation and in vitro fertilization (IVF) yielded multiple blastocysts that each underwent CCS with high-density oligonucleotide microarray comparative genomic hybridization (aCGH). RESULTS: Parental translocations were suspected based on the finding of identical break point mutations in multiple embryos from each couple. Confirmation of these suspected translocations within blastocysts was performed with next-generation sequencing (NGS). Subsequent parental karyotypic evaluation resulted in a diagnosis of parental balanced reciprocal translocation in each case. CONCLUSIONS: We demonstrated that high-resolution aCGH and NGS on TE biopsies can accurately detect parental reciprocal translocations when previously unrecognized.

Mosaicism in Cutaneous Disorders.

Genetic mosaicism arises when a zygote harbors two or more distinct genotypes, typically due to de novo, somatic mutation during embryogenesis. The clinical manifestations largely depend on the differentiation status of the mutated cell; earlier mutations target pluripotent cells and generate more widespread disease affecting multiple organ systems. If gonadal tissue is spared-as in somatic genomic mosaicism-the mutation and its effects are limited to the proband, whereas mosaicism also affecting the gametes, such as germline or gonosomal mosaicism, is transmissible. Mosaicism is easily appreciated in cutaneous disorders, as phenotypically distinct mutant cells often give rise to lesions in patterns determined by the affected cell type. Genetic investigation of cutaneous mosaic disorders has identified pathways central to disease pathogenesis, revealing novel therapeutic targets. In this review, we discuss examples of cutaneous mosaicism, approaches to gene discovery in these disorders, and insights into molecular pathobiology that have potential for clinical translation.

Generation and characterization of human iPSC line from CD34+ cells isolated from umbilical cord blood belonging to Indian origin.

We describe here the reprogramming of CD34+ cells isolated from umbilical cord blood obtained after full term delivery of a healthy female child of Indian origin. The cells were nucleofected by episomal vectors expressing Oct4, Sox2, L-Myc, Klf4, Lin28 and p53DD (negative mutation in p53). Colonies were identified by alkaline phosphatase staining and characterized for expression of pluripotency markers at protein level by immunofluorescence, flow cytometry and at transcript level by PCR. Genomic stability of the cell line was checked by G-banded karyotype. The ability to differentiate to endoderm, mesoderm and ectoderm in vitro was confirmed by immunofluorescence staining.

A single trophectoderm biopsy at blastocyst stage is mathematically unable to determine embryo ploidy accurately enough for clinical use.

BACKGROUND: It has become increasingly apparent that the trophectoderm (TE) at blastocyst stage is much more mosaic than has been appreciated. Whether preimplantation genetic screening (PGS), utilizing a single TE biopsy (TEB), can reliably determine embryo ploidy has, therefore, increasingly been questioned in parallel. METHODS: We for that reason here established 2 mathematical models to assess probabilities of false-negative and false-positive results of an on average 6-cell biopsy from an approximately 300-cell TE. This study was a collaborative effort between investigators at The Center for Human Reproduction in New York City and the Center for Studies in Physics and Biology and the Brivanlou Laboratory of Stem Cell Biology and Molecular Embryology, the latter two both at Rockefeller University in New York City. RESULTS: Both models revealed that even under best case scenario, assuming even distribution of mosaicism in TE (since mosaicism is usually clonal, a highly unlikely scenario), a biopsy of at least 27 TE cells would be required to reach minimal diagnostic predictability from a single TEB. CONCLUSIONS: As currently performed, a single TEB is, therefore, mathematically incapable of reliably determining whether an embryo can be transferred or should be discarded. Since a single TEB, as currently performed, apparently is not representative of the complete TE, this study, thus, raises additional concern about the clinical utilization of PGS.

Dental and extra-oral clinical features in 41 patients with WNT10A gene mutations: A multicentric genotype-phenotype study.

WNT10A gene encodes a canonical wingless pathway signaling molecule involved in cell fate specification as well as morphogenetic patterning of the developing ectoderm, nervous system, skeleton, and tooth. In patients, WNT10A mutations are responsible for ectodermal-derived pathologies including isolated hypo-oligodontia, tricho-odonto-onycho-dermal dysplasia and Schöpf-Schulz-Passarge syndrome (SSPS). Here we describe the dental, ectodermal, and extra-ectodermal phenotypic features of a cohort of 41 patients from 32 unrelated families. Correlations with WNT10A molecular status (heterozygous carrier, compound heterozygous, homozygous) and patient's phenotypes were performed. Mild to severe oligodontia was observed in all patients bearing biallelic WNT10A mutations. However, patients with compound heterozygous mutations presented no significant difference in phenotypes compared with homozygous individuals. Anomalies in tooth morphology were frequently observed with heterozygous patients displaying hypodontia. No signs of SSPS, especially eyelids cysts, were detected in our cohort. Interestingly, extra-ectodermal signs consisted of skeletal, neurological and vascular anomalies, the latter suggesting a wider phenotypic spectrum associated with WNT10A mutations. Indeed, the Wnt pathway plays a crucial role in skeletal development, lipid metabolism, and neurogenesis, potentially explaining patient's clinical manifestations.

CFTR-ß-catenin interaction regulates mouse embryonic stem cell differentiation and embryonic development.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated anion channel capable of conducting both Cl- and HCO3-, mutations of which cause cystic fibrosis (CF), a common autosomal recessive disease. Although CF patients are known to have varied degree of developmental problems, the biological role of CFTR in embryonic development remains elusive. Here, we show that CFTR is functionally expressed in mouse ESCs. CFTR-/- mESCs exhibit dramatic defect in mesendoderm differentiation. In addition, CFTR physically interacts with ß-catenin, defect of which leads to premature degradation of ß-catenin and suppressed activation of ß-catenin signaling. Furthermore, knockdown of CFTR retards the early development of Xenopus laevis with impaired mesoderm/endoderm differentiation and ß-catenin signaling. Our study reveals a previously undefined role of CFTR in controlling ESC differentiation and early embryonic development via its interaction with ß-catenin, and provides novel insights into the understanding of embryonic development.

The Gene Regulatory Network of Lens Induction Is Wired through Meis-Dependent Shadow Enhancers of Pax6.

Lens induction is a classical developmental model allowing investigation of cell specification, spatiotemporal control of gene expression, as well as how transcription factors are integrated into highly complex gene regulatory networks (GRNs). Pax6 represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of Pax6 during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of Pax6 transcription start site. Despite this generally accepted regulatory pathway, Meis1-, Meis2- and EE-deficient mice have surprisingly mild eye phenotypes at placodal stage of lens development. Here, we show that simultaneous deletion of Meis1 and Meis2 in presumptive lens ectoderm results in arrested lens development in the pre-placodal stage, and neither lens placode nor lens is formed. We found that in the presumptive lens ectoderm of Meis1/Meis2 deficient embryos Pax6 expression is absent. We demonstrate using chromatin immunoprecipitation (ChIP) that in addition to EE, Meis homeoproteins bind to a remote, ultraconserved SIMO enhancer of Pax6. We further show, using in vivo gene reporter analyses, that the lens-specific activity of SIMO enhancer is dependent on the presence of three Meis binding sites, phylogenetically conserved from man to zebrafish. Genetic ablation of EE and SIMO enhancers demostrates their requirement for lens induction and uncovers an apparent redundancy at early stages of lens development. These findings identify a genetic requirement for Meis1 and Meis2 during the early steps of mammalian eye development. Moreover, they reveal an apparent robustness in the gene regulatory mechanism whereby two independent "shadow enhancers" maintain critical levels of a dosage-sensitive gene, Pax6, during lens induction.

Sneddon's syndrome: it is all in the ectoderm.

A 51-year-old man gave a 2-year history of worsening mobility, cognitive decline and headaches. He had a history of thromboembolic stroke, recurrent transient ischaemic attacks and a spontaneous intraventricular haemorrhage. On examination, he had livedo reticularis and perniosis and a systolic murmur. Catheter cerebral angiography showed peripheral small-vessel and medium-vessel vasculopathy resulting in pruning of the distal cortical vessels and tortuous irregular distal collaterals. Skin biopsy showed subtle vasculopathy with ectasia of capillaries and postcapillary venules but no frank vasculitis or arterial thrombosis. Repeated serum antiphospholipid antibody titres were negative. The clinical features, skin biopsy and angiogram findings strongly supported a diagnosis of Sneddon's syndrome. Clinicians should consider Sneddon's syndrome in patients with livedo reticularis and stroke. There are treatment dilemmas in this situation when ischaemic and haemorrhagic cerebral events coexist.

The apical ectodermal ridge of the mouse model of ectrodactyly Dlx5;Dlx6-/- shows altered stratification and cell polarity, which are restored by exogenous Wnt5a ligand.

The congenital malformation split hand/foot (SHFM) is characterized by missing central fingers and dysmorphology or fusion of the remaining ones. Type-1 SHFM is linked to deletions/rearrangements of the DLX5-DLX6 locus and point mutations in the DLX5 gene. The ectrodactyly phenotype is reproduced in mice by the double knockout (DKO) of Dlx5 and Dlx6. During limb development, the apical ectodermal ridge (AER) is a key-signaling center responsible for early proximal-distal growth and patterning. In Dlx5;6 DKO hindlimbs, the central wedge of the AER loses multilayered organization and shows down-regulation of FGF8 and Dlx2. In search for the mechanism, we examined the non-canonical Wnt signaling, considering that Dwnt-5 is a target of distalless in Drosophila and the knockout of Wnt5, Ryk, Ror2 and Vangl2 in the mouse causes severe limb malformations. We found that in Dlx5;6 DKO limbs, the AER expresses lower levels of Wnt5a, shows scattered ß-catenin responsive cells and altered basolateral and planar cell polarity (PCP). The addition of Wnt5a to cultured embryonic limbs restored the expression of AER markers and its stratification. Conversely, the inhibition of the PCP molecule c-jun N-terminal kinase caused a loss of AER marker expression. In vitro, the addition of Wnt5a on mixed primary cultures of embryonic ectoderm and mesenchyme was able to confer re-polarization. We conclude that the Dlx-related ectrodactyly defect is associated with the loss of basoapical and PCP, due to reduced Wnt5a expression and that the restoration of the Wnt5a level is sufficient to partially reverts AER misorganization and dysmorphology.