Nonsynonymous Mutations in Intellectual Disability and Autism Spectrum Disorder Gene PTCHD1 Disrupt N-Glycosylation and Reduce Protein Stability.

PTCHD1 has been implicated in Autism Spectrum Disorders (ASDs) and/or intellectual disability, where copy-number-variant losses or loss-of-function coding mutations segregate with disease in an X-linked recessive fashion. Missense variants of PTCHD1 have also been reported in patients. However, the significance of these mutations remains undetermined since the activities, subcellular localization, and regulation of the PTCHD1 protein are currently unknown. This paucity of data concerning PTCHD1 prevents the effective evaluation of sequence variants identified during diagnostic screening. Here, we characterize PTCHD1 protein binding partners, extending previously reported interactions with postsynaptic scaffolding protein, SAP102. Six rare missense variants of PTCHD1 were also identified from patients with neurodevelopmental disorders. After modelling these variants on a hypothetical three-dimensional structure of PTCHD1, based on the solved structure of NPC1, PTCHD1 variants harboring these mutations were assessed for protein stability, post-translational processing, and protein trafficking. We show here that the wild-type PTCHD1 post-translational modification includes complex N-glycosylation and that specific mutant proteins disrupt normal N-link glycosylation processing. However, regardless of their processing, these mutants still localized to PSD95-containing dendritic processes and remained competent for complexing SAP102.

Neuronal transcription of autism gene PTCHD1 is regulated by a conserved downstream enhancer sequence.

Patched domain-containing 1 (PTCHD1) is a well-established susceptibility gene for autism spectrum disorder (ASD) and intellectual disability (ID). Previous studies have suggested that alterations in the dosage of PTCHD1 may contribute to the etiology of both ASD and ID. However, there has not yet been a thorough investigation regarding mechanisms that regulate PTCHD1 expression. We sought to characterize the Ptchd1 promoter in a mouse neuronal model, as well as to identify and validate cis regulatory elements. We defined specific regions of the Ptchd1 promoter essential for robust expression in P19-induced neurons. Evolutionarily-conserved putative transcription factor binding sites within these regions were subsequently identified. Using a pairwise comparison of chromatin accessibility between mouse forebrain and liver tissues, a candidate regulatory region, ~ 9.1 kbp downstream of the Ptchd1 stop codon was defined. This region harbours two ENCODE-predicted enhancer cis-regulatory elements. Further, using DNase footprint analysis, a putative YY1-binding motif was also identified. Genomic deletion of the entire 8 kbp downstream open chromatin region attenuated Ptchd1 transcription by over 60% in our neuronal model, corroborating its predicted regulatory function. This study provides mechanistic insights related to the expression of PTCHD1, and provides important context to interpret genetic and genomic variation at this locus which may influence neurodevelopment.

PTCHD1: Identification and Neurodevelopmental Contributions of an Autism Spectrum Disorder and Intellectual Disability Susceptibility Gene.

Over the last one and a half decades, copy number variation and whole-genome sequencing studies have illuminated the considerable genetic heterogeneity that underlies the etiologies of autism spectrum disorder (ASD) and intellectual disability (ID). These investigations support the idea that ASD may result from complex interactions between susceptibility-related genetic variants (single nucleotide variants or copy number variants) and the environment. This review outlines the identification and neurobiological characterization of two such genes located in Xp22.11, Patched domain-containing 1 (PTCHD1), and its antisense lncRNA PTCHD1-AS. Animal models of Ptchd1 disruption have recapitulated a subset of clinical symptoms related to ASD as well as to ID. Furthermore, these Ptchd1 mouse knockout studies implicate the expression of Ptchd1 in both the thalamic and the hippocampal brain regions as being crucial for proper neurodevelopment and cognitive function. Altered kynurenine metabolic signalling has been postulated as a disease mechanism in one of these animal studies. Additionally, ASD patient-derived induced pluripotent stem cells (iPSCs) carrying a copy number loss impacting the antisense non-coding RNA PTCHD1-AS have been used to generate 2D neuronal cultures. While copy number loss of PTCHD1-AS does not affect the transcription of PTCHD1, the neurons exhibit diminished miniature excitatory postsynaptic current frequency, supporting its role in ASD etiology. A more thorough understanding of risk factor genes, such as PTCHD1 and PTCHD1-AS, will help to clarify the intricate genetic and biological mechanisms that underlie ASD and ID, providing a foundation for meaningful therapeutic interventions to enhance the quality of life of individuals who experience these conditions.

Distinct epithelial-to-mesenchymal transitions induced by PIK3CAH1047R and PIK3CB.

The most common PIK3CA mutation, producing the H1047R mutant of p110α, arises in myriad malignancies and is typically observed in low-grade breast tumours. In contrast, amplification is observed for wild-type PIK3CB, encoding p110ß, and occurs at low frequency but in aggressive, high-grade metastatic tumours. We hypothesized that mutant p110αH1047R and wild-type p110ß give rise to distinct transformed phenotypes. We show that p110αH1047R and wild-type p110ß, but not wild-type p110α, transform MCF-10A cells and constitutively stimulate phosphoinositide 3-kinase (PI3K)-AKT pathway signalling. However, their resultant morphological transformed phenotypes are distinct. p110αH1047R induced an epithelial-to-mesenchymal transition (EMT) commensurate with SNAIL (also known as SNAI1) induction and loss of E-cadherin. Upon p110ß expression, however, E-cadherin expression was maintained despite cells readily delaminating from epithelial sheets. Distinct from the prominent filopodia in p110αH1047R-expressing cells, p110ß induced formation of lamellipodia, and these cells migrated with significantly greater velocity and decreased directionality. p110ß-induced phenotypic alterations were accompanied by hyperactivation of RAC1; the dependency of transformation of p110ß-binding to Rac1 revealed using a Rac1-binding mutant of p110ß. Thus, PIK3CB amplification induces a transformed phenotype that is dependent upon a p110ß-Rac1 signalling loop and is distinct from the transformed phenotype induced by p110αH1047R.

The protein-specific activities of the transmembrane modules of Ptch1 and Ptch2 are determined by their adjacent protein domains.

Signaling through the Hedgehog (Hh) pathway is mediated by the Patched (Ptch) family of proteins. Although the vertebrate Ptch proteins Ptch1 and Ptch2 harbor two closely related transmembrane modules related to sterol-sensing domains (SSDs), the role of these closely related receptors in the Hh pathway are not equivalent. Ptch1 is essential for development and appears to be the principal receptor mediating responses to Hh ligands, whereas Ptch2 is nonessential, and its role in Hh-signaling remains ambiguous. We hypothesized that the SSDs of the Ptch proteins function as generic modules whose protein-specific activities are determined by the adjacent cytoplasmic and luminal domains. We first showed that individual N-terminal and C-terminal halves of Ptch1 associated noncovalently to mediate ligand-dependent regulation of Hh signaling. The analogous regions of Ptch2 also interacted noncovalently but did not repress the Hh pathway. However, the SSD of Ptch2 were capable of repressing Hh signaling, as determined using chimeric proteins where the SSDs of Ptch1 were replaced by those from Ptch2. Replacement of the SSDs of Ptch1 with the analogous regions from the cholesterol transporter NPC1 failed to produce a chimeric protein capable of Hh repression. Further refinement of the specific regions in Ptch1 and Ptch2 revealed that specific cytoplasmic domains of Ptch1 were necessary but not sufficient for repression of Hh signaling and that the two principal luminal domains of Ptch1 and Ptch2 were interchangeable. These data support a model where the SSDs of the Ptch family proteins exhibit generic activities and that the adjacent cytoplasmic and luminal domains determine their protein-specific activities.

Activities of the Cytoplasmic Domains of Patched-1 Modulate but Are Not Essential for the Regulation of Canonical Hedgehog Signaling.

The Hedgehog (Hh) pathway is a highly conserved signaling cascade crucial for cell fate determination during embryogenesis. Response to the Hh ligands is mediated by the receptor Patched-1 (Ptch1), a 12-pass transmembrane glycoprotein. Despite its essential role in Hh signaling and its activity as a tumor suppressor, Ptch1 remains largely uncharacterized. We demonstrate here that Ptch1 binds to itself to form oligomeric structures. Oligomerization is mediated by two distinct, structurally disordered, intracellular domains spanning amino acids 584-734 ("middle loop") and 1162-1432 (C terminus). However, oligomerization is not required for Ptch1-dependent regulation of the canonical Hh pathway operating through Smo. Expression of a mutant protein that deletes both regions represses the Hh pathway and responds to the addition of Hh ligand independent of its inability to bind other factors such as Smurf2. Additionally, deletion of the cytoplasmic middle loop domain generates a Ptch1 mutant that, despite binding to Hh ligand, constitutively suppresses Hh signaling and increases the length of primary cilia. Constitutive activity because of deletion of this region is reversed by further deletion of specific sequences in the cytoplasmic C-terminal domain. These data reveal an interaction between the cytoplasmic domains of Ptch1 and that these domains modulate Ptch1 activity but are not essential for regulation of the Hh pathway.

Oestrogen receptor-alpha regulates non-canonical Hedgehog-signalling in the mammary gland.

Mesenchymal dysplasia (mes) mice harbour a truncation in the C-terminal region of the Hh-ligand receptor, Patched-1 (mPtch1). While the mes variant of mPtch1 binds to Hh-ligands with an affinity similar to that of wild type mPtch1 and appears to normally regulate canonical Hh-signalling via smoothened, the mes mutation causes, among other non-lethal defects, a block to mammary ductal elongation at puberty. We demonstrated previously Hh-signalling induces the activation of Erk1/2 and c-src independently of its control of smo activity. Furthermore, mammary epithelial cell-directed expression of an activated allele of c-src rescued the block to ductal elongation in mes mice, albeit with delayed kinetics. Given that this rescue was accompanied by an induction in estrogen receptor-alpha (ERα) expression and that complex regulatory interactions between ERα and c-src are required for normal mammary gland development, it was hypothesized that expression of ERα would also overcome the block to mammary ductal elongation at puberty in the mes mouse. We demonstrate here that conditional expression of ERα in luminal mammary epithelial cells on the mes background facilitates ductal morphogenesis with kinetics similar to that of the MMTV-c-src(Act) mice. We demonstrate further that Erk1/2 is activated in primary mammary epithelial cells by Shh-ligand and that this activation is blocked by the inhibitor of c-src, PP2, is partially blocked by the ERα inhibitor, ICI 182780 but is not blocked by the smo-inhibitor, SANT-1. These data reveal an apparent Hh-signalling cascade operating through c-src and ERα that is required for mammary gland morphogenesis at puberty.

Distinct effects of the mesenchymal dysplasia gene variant of murine Patched-1 protein on canonical and non-canonical Hedgehog signaling pathways.

Hedgehog (Hh) signaling requires regulation of the receptor Patched-1 (Ptch1), which, in turn, regulates Smoothened activity (canonical Hh signaling) as well as other non-canonical signaling pathways. The mutant Ptch1 allele mesenchymal dysplasia (mes), which truncates the Ptch1 C terminus, produces a limited spectrum of developmental defects in mice as well as deregulation of canonical Hh signaling in some, but not all, affected tissues. Paradoxically, mes suppresses canonical Hh signaling and binds to Hh ligands with an affinity similar to wild-type mouse Ptch1 (mPtch1). We characterized the distinct activities of the mes variant of mPtch1 mediating Hh signaling through both canonical and non-canonical pathways. We demonstrated that mPtch1 bound c-src in an Hh-regulated manner. Stimulation with Sonic Hedgehog (Shh) of primary mammary mesenchymal cells from wild-type and mes animals activated Erk1/2. Although Shh activated c-src in wild-type cells, c-src was constitutively activated in mes mesenchymal cells. Transient assays showed that wild-type mPtch1, mes, or mPtch1 lacking the C terminus repressed Hh signaling in Ptch1-deficient mouse embryo fibroblasts and that repression was reversed by Shh, revealing that the C terminus was dispensable for mPtch1-dependent regulation of canonical Hh signaling. In contrast to these transient assays, constitutively high levels of mGli1 but not mPtch1 were present in primary mammary mesenchymal cells from mes mice, whereas the expression of mPtch1 was similarly induced in both mes and wild-type cells. These data define a novel signal transduction pathway involving c-src that is activated by the Hh ligands and reveals the requirement for the C terminus of Ptch in regulation of canonical and non-canonical Hh signaling pathways.

Wnt-signalling in the embryonic mammary gland.

The first member of the Wnt-family ligands was identified 30 years ago as a factor in mouse mammary tumours whose expression was deregulated due to the promoter activity emanating from the proximal integration of the Mouse Mammary Tumour Virus genome (Nusse and Varmus, Embo J 31:2670-84, 2012). The Wnt-ligands invoke a number of molecular-genetic signalling cascades fundamental to the patterning of developing tissues and organs during embryogenesis as well as during postnatal development. The Wnt-signalling cascade that controls the activities of ß-catenin and the T-cell Factor (Tcf)/Lympoid enhancer factor (Lef1) plays a fundamental role in control of all stages of embryonic mammary gland development. We provide here a brief overview of the known aspects of Wnt-signalling activities in the embryonic mammary gland and its interactions with other signalling cascades in this developing tissue.

Mammary epithelial-restricted expression of activated c-src rescues the block to mammary gland morphogenesis due to the deletion of the C-terminus of Patched-1.

Mesenchymal dysplasia (mes) mice expressing a C-terminally truncated version of the Hedgehog (Hh)-ligand receptor, Patched-1 (Ptch1), exhibit a limited spectrum of developmental defects including blocked ductal morphogenesis of the mammary gland during puberty. Given that the Hh-ligands can stimulate signalling cascades distinct from the canonical pathway involving Smo and the Gli-family proteins and that Ptch1 binds to factors harbouring SH3-domains, we determined whether the mes mammary gland defect could be rescued by activating non-canonical signalling pathways downstream of Ptch1. We demonstrate here that expression of constitutively active c-src (c-src(Act)) in mammary epithelial cells overcomes the block to mammary epithelial morphogenesis in mes mice. Specifically, MMTV-directed expression of c-src(Act) rescued blocked ductal morphogenesis in mes mice, albeit only after animals were more than 15 weeks of age. The overall morphology resembled wild type mice expressing c-src(Act) although 40% of mes/MMTV-c-src(Act) mice exhibited terminal end buds at 24 weeks of age. C-src(Act) restored the proliferative capacity of mes epithelial cells, self-renewal capacity of mammary progenitor cells and increased the expression of Esr1, Ptch1 and Gli1. These data reveal the cooperative interactions between signalling cascades involving c-src and Ptch1 and suggest that Hh-signalling may be permissive for c-src/Esr1-dependent mammary gland morphogenesis.

Activation of Erk by sonic hedgehog independent of canonical hedgehog signalling.

Hedgehog (Hh) signalling is mediated through the Patched-1 (Ptch1) receptor. Hh-binding to Ptch1 blocks the inhibitory effects of Ptch1 on the activity of the transmembrane protein, Smoothened (Smo), resulting induction of target genes by the Gli-family of transcription factors. We demonstrate here that Hh-binding to Ptch1 stimulates activation of Erk1/2. This activation is insensitive to the small molecule Smo antagonists and occurs in a cell line that does not express Smo. Specifically, the C-terminus of Ptch1 harbours motifs encoding Class I and II SH3-binding sites. SH3-domain binding activity was verified using GST-c-src(SH3), -Grb2(SH3) and -p85beta(SH3) fusion-proteins. Ectopically expressed Grb2 or p85beta could also be co-immunoprecipitated with the Ptch1 C-terminus. Addition of Shh to serum-starved human mammary epithelial cells and Shh Light II fibroblasts stimulated phosphorylation of Erk1/2. Erk1/2 activation was observed in cells where Smo activity had been inhibited using cyclopamine and in the breast epithelial cell line, MCF10A, that does not express Smo. These data reveal novel binding activities for the C-terminal region of Ptch1 and define a signalling pathway stimulated by the Hh-ligands operating independently of pathways requiring Smo.

Lymphoid enhancer factor-1 mediates loading of Pax3 to a promoter harbouring lymphoid enhancer factor-1 binding sites resulting in enhancement of transcription.

The transcription factor, Pax3, alters transcription by binding directly to promoter regions harbouring sequences recognized by either its paired domain or its homeodomain. We demonstrated previously that the promoter regions of many of the genes whose expression was altered during a Pax3-induced mesenchymal-to-epithelial transition harboured sequences recognized by lymphoid enhancer factor-1 (Lef1). Given the apparent lack of DNA-binding consensus sequences for Pax3 in these promoters, it was hypothesized that Pax3 might alter transcriptional activity of promoters harbouring Lef1-binding sites independent of Pax3 binding to DNA. We describe here a novel mode of Pax3-dependent regulation of transcription that is mediated through DNA-independent binding to Lef1. Specifically, we demonstrate that Pax3 binds to Lef1, determined in binding assays and co-immunoprecipitation of endogenous Pax3 and Lef1. Binding assays employing deletion mutants of Pax3 and Lef1 determined that association was mediated through the homeodomain of Pax3 and the first half of the Lef1 DNA-binding domain. The significance of this association was demonstrated in transcriptional assays using a luciferase reporter gene downstream of a model promoter harbouring Lef1 DNA-binding consensus sites. Pax3 augmented Lef1-dependent transactivation from this promoter. This increase in transcriptional activity occurred in the absence and presence of added beta-catenin. Chromatin immunoprecipitation assays demonstrated further that Pax3-association to complexes bound to DNA harbouring Lef1 consensus sequences was dependent on Lef1. These data reveal a novel mode of transcriptional regulation by Pax3. This mode of transcriptional regulation suggests further that Pax3 activity may directly effect the expression of factors regulated by signal transduction pathways dependent on Lef1.

Ptch1 is required locally for mammary gland morphogenesis and systemically for ductal elongation.

Systemic hormones and local growth factor-mediated tissue interactions are essential for mammary gland development. Using phenotypic and transplantation analyses of mice carrying the mesenchymal dysplasia (mes) allele of patched 1 (Ptch1(mes)), we found that Ptch1(mes) homozygosity led to either complete failure of gland development, failure of post-pubertal ductal elongation, or delayed growth with ductal dysplasia. All ductal phenotypes could be present in the same animal. Whole gland and epithelial fragment transplantation each yielded unique morphological defects indicating both epithelial and stromal functions for Ptch1. However, ductal elongation was rescued in all cases, suggesting an additional systemic function. Epithelial function was confirmed using a conditional null Ptch1 allele via MMTV-Cre-mediated disruption. In Ptch1(mes) homozygotes, failure of ductal elongation correlated with diminished estrogen and progesterone receptor expression, but could not be rescued by exogenous ovarian hormone treatment. By contrast, pituitary isografts were able to rescue the ductal elongation phenotype. Thus, Ptch1 functions in the mammary epithelium and stroma to regulate ductal morphogenesis, and in the pituitary to regulate ductal elongation and ovarian hormone responsiveness.

Genetic interaction between Lef1 and Alx4 is required for early embryonic development.

Lymphoid Enhancer Factor-1 (Lef1) facilitates the assembly of transcriptional regulatory complexes and mediates nuclear responses to Wnt signals. We determined previously that the mesenchymally restricted, paired-like homeodomain protein Aristaless-like 4 (Alx4) interacts with Lef1 and together alters promoter activity of candidate genes. In order to define their overlapping functions, mice deficient for both Lef1 and Alx4 activity (Lef1-/-/Alx4lstD/lstD) were produced. Whereas embryos lacking either Lef1 or Alx4 activity remain viable up to or after birth, early embryonic lethality results when both factors were absent. No viable Lef1-/-/Alx4lstD/lstD embryos were recovered beyond 9.5 dpc. Between E8.5 and E10, viable Lef1-/-/Alx4lstD/lstD embryos were developmentally delayed 0.5 days relative to littermates of all other genotypes. Principle among the alterations seen in Lef1-/-/Alx4lstD/lstD animals was defective vasculature in both embryonic and extra-embryonic tissues. In the yolk sac, while the vascular network is present, it were greatly diminished and large vitelline vessels were largely absent. Platelet/endothelial cell adhesion molecule (PECAM) staining revealed that the major vessels in the head of compound mutant embryos were absent, while the other vessels were finer than those seen in normal littermates. Pools of blood and pericardial effusion were also apparent in Lef1-/-/Alx4lstD/lstD animals, further indicative of a defective vasculature. These data confirm genetically the interaction between Lef1 and Alx4 and further reveal unknown, overlapping roles for these transcription factors in embryonic vasculogenesis.

Loss of Alx4, a stromally-restricted homeodomain protein, impairs mammary epithelial morphogenesis.

Postnatal development of the mammary gland is determined by reciprocal interactions between the ductal epithelia and adjacent stroma. Alx4 is a mesenchymally restricted homeodomain transcription factor expressed in a number of developing tissues, including skin appendages such as hair follicles, whiskers and teeth. We show here that Alx4 is expressed in a subset of ERalpha-expressing mammary stromal cells adjacent to terminal end buds and alveoli during puberty and pregnancy, respectively. Alx4 expression is induced in mammary stromal cells at the onset of puberty and can be induced in prepubescent mice by administration of 17beta-estradiol. In order to determine the role of Alx4 during mammary gland development, we characterized mammary gland development of mice homozygous for the null allele of Alx4, lst(D). Mammary glands from animals lacking Alx4 activity exhibit profound alterations in ductal morphogenesis. Overall development is delayed, ducts being grossly distorted in size and structure. Terminal end buds are also disoriented, displaying aberrant architecture during bifurcation. Despite the developmental delay, the ductal network typically reaches the limits of the fat pad. However, during puberty and in the adult virgin mice, the frequency and density of branch points is significantly reduced. We show further that the defective ductal morphogenesis is due to defects in stromal cells. Specifically, when injected into the cleared fat pad of wild-type recipients, mixed populations of wild-type epithelial cells and Alx4-deficient stromal cells give rise to retarded ductal morphogenesis. Wild-type stromal cells mixed with Alx4-deficient epithelial cells result in normal progression of ductal development. Defective branching morphogenesis in Alx4-deficient females is not due to a loss in expression of HGF, since the level of HGF message in mammary stromal cells is similar in mutant and wild-type littermates. MMP3 is similarly expressed while a 40% increase in MMP2 and a 50% decrease in MMP9 message levels in Alx4-deficient mice relative to their wild-type littermates is observed. Thus, the activity of the stromally restricted homeodomain factor, Alx4, is required for normal branching morphogenesis of the ductal epithelia during pubescent mammary gland development.

Lef1 is required for the transition of Wnt signaling from mesenchymal to epithelial cells in the mouse embryonic mammary gland.

Inductive reciprocal signaling between mesenchymal and adjacent epithelia gives rise to skin appendages such as hair follicles and mammary glands. Lef1-mediated canonical Wnt signaling is required for morphogenesis of these skin appendages during embryogenesis. In order to define the role of canonical Wnt signaling during early embryonic mammary gland development, we determined the temporal and spatial changes in Wnt signaling during embryogenesis in wild-type and Lef1-deficient embryos harboring a Tcf/Lef1-betagal reporter (TOPGAL) transgene. In contrast to previous studies using TOPGAL mice from a distinct founder, we observe that Wnt signaling acts initially on mesenchymal cells associated with the sequential appearance of mammary placodes. As placode development progresses between 12.5 and 15.5 dpc, Wnt signaling progressively accumulates in the mammary epithelial compartment. By 18.5 dpc, betagal activity is confined to mesenchymal and epithelial cells near the nipple region. In Lef1-deficient embryos, the transition of Wnt signaling from mesenchyme to the mammary epithelia is blocked for placodes #1, 4 and 5 despite the expression of Tcf1 in epithelial cells. These placodes ultimately disappear by 15.5 dpc, while placodes 2 and 3 typically did not form in the absence of Lef1. Progressive loss of placodes 1, 4, and 5 is accompanied by increased apoptosis in mesenchymal cells adjacent to the mammary epithelial placodes. While factors important for embryonic mammary gland development, such as FGF7, are expressed normally in Lef1-deficient animals, one mediator of the Hedgehog (Hh)-signaling pathway is aberrantly expressed. Specifically, Shh, Ihh, and Gli2 are expressed in mammary epithelial cells at levels in Lef1-deficient animals similar to wild-type littermates. However, the signal for Ptc-1 is strongly reduced in mesenchymal cells surrounding the mammary placode in Lef1 mutants relative to wild-type embryos. The loss of Ptc-1, both a receptor for and transcriptional target of Hh signaling, suggests that Hh signaling is blocked in Lef1-deficient embryos. Thus, these data reveal distinct requirements of different mammary placodes for Lef1-dependent Wnt signaling. They further define dynamic changes in which cells integrate Lef1-dependent Wnt signaling during progression of embryonic mammary gland development.

Cell-type-specific regulation of distinct sets of gene targets by Pax3 and Pax3/FKHR.

The oncogenic fusion protein, Pax3/FKHR, is a more potent transcription factor relative to its normal counterpart, Pax3. Since Pax3 induced a mesenchymal to epithelial transition (MET) in human SaOS-2 osteosarcomas, we hypothesized that Pax3/FKHR would also induce a morphological change in SaOS-2 cells. We demonstrate here that Pax3/FKHR more potently induces a MET in SaOS-2 cells than Pax3. This greater potency was further evident where Pax3/FKHR, but not Pax3, induced a morphological alteration in U2-OS osteosarcoma cells. By microarray analysis, we determined that Pax3/FKHR altered the expression of gene targets in a manner quantitatively and qualitatively distinct from Pax3. Three classes of genes were identified: (i) genes induced or repressed by Pax3 and Pax3/FKHR, (ii) genes induced or repressed by Pax3/FKHR but not Pax3 and (iii) genes induced by Pax3/FKHR but repressed by Pax3. Chromatin immunoprecipitations confirmed the direct binding of Pax3/FKHR to the promoter region of several factors including cannabinoid receptor-1, EPHA2 and EPHA4. Verification of the microarray data also revealed coordinate alteration in the expression of factors involved in BMP4 signalling. Regulation of gene expression by Pax3 and Pax3/FKHR is, however, cell-type specific. BMP4 expression, for example, was repressed by both Pax3 and Pax3/FKHR in SaOS-2 cells, while in the rhabdomyosarcoma, RD, Pax3/FKHR, but not Pax3, induced BMP4 expression. Thus, our data reveal that Pax3/FKHR regulates a distinct but overlapping set of genes relative to Pax3 and that the global set of Pax3 and Pax3/FKHR gene targets is cell-type specific.

Homeless people's trust and interactions with police and paramedics.

Although the health impact of patients' trust in physicians has been well documented, less is known about the possible health effects of trust in police or paramedics. Homeless people frequently interact with police officers and paramedics, and these experiences may affect their health and future willingness to seek emergency assistance. We examined homeless people's self-reported interactions with police and paramedics in Toronto, Canada, and their level of trust in these emergency service providers. In a sample of 160 shelter users, 61% had interacted with police in the last 12 months, and 37% had interacted with paramedics (P = .0001). The proportion of subjects who expressed willingness to call police in an emergency was significantly lower than those willing to call paramedics in an emergency (69% vs. 92%, P = .0001). On a Likert scale ranging from a minimum of 0 to a maximum of 5, trust levels were lower in police than in paramedics (median level 3 vs. 5, P = .0001). Among shelter users, 9% (95% confidence interval [CI], 5% to 14%) reported an assault by a police officer in the last year, and 0% (95% CI, 0% to 4%) reported an assault by a paramedic. These findings showed that homeless people have much lower levels of trust in police than paramedics. Reports of negative interactions with police are not uncommon, and homeless people's perceptions of the police may pose a barrier to seeking emergency assistance. Further research is needed for objective characterization of homeless people's interactions with police officers and the potential health implications of low levels of trust in the police.

The prevalence of psychological morbidity in West Bank Palestinian children.

OBJECTIVE: To determine the prevalence of psychological morbidity among Palestinian children living in the southern Bethlehem District of the West Bank during July 2000. METHODS: We undertook a descriptive study using the Rutter A2 (parent) Scale to determine psychological morbidity. This questionnaire comprises 31 questions that were answered by a parent of the 206 subject children (ages 6 to 13 years). We selected subjects based on a multistage, randomized selection of 8 Palestinian villages and their households in the southern region of Bethlehem, West Bank. We used the Gaza Socioeconomic Adversities Questionnaire to determine differences in economic status among families. RESULTS: For all families interviewed, the father was employed, none were receiving financial assistance, and all but 1 owned their own house. The results of the Rutter A2 Scale revealed a rate of psychological morbidity ("caseness") of 42.3% among Palestinian children. The rate for boys was 46.3% and for girls, 37.8%. CONCLUSIONS: The prevalence of psychological morbidity among Palestinian children in the West Bank was significantly higher (factor of 2; chi2 = 23.26, df 1, P < 0.001), relative to the level of psychological morbidity determined independently for children in Gaza during 2000. We predict that these rates will have increased substantially owing to the escalated violence that began in this region 2 months after we conducted our study. We further predict that children in Israeli settlements in the West Bank will also exhibit elevated levels of psychological morbidity, relative to their counterparts in Israel.

Shh expression is required for embryonic hair follicle but not mammary gland development.

The embryonic mammary gland and hair follicle are both derived from the ventral ectoderm, and their development depends on a number of common fundamental developmental pathways. While the Hedgehog (Hh) signaling pathway is required for hair follicle morphogenesis, the role of this pathway during embryonic mammary gland development remains undetermined. We demonstrate here that, unlike the hair follicle, both Shh and Ihh are expressed in the developing embryonic mouse mammary rudiment as early as E12.5. In Shh(-/-) embryos, hair follicle development becomes arrested at an early stage, while the mammary rudiment, which continues to express Ihh, develops in a manner indistinguishable from that of wild-type littermates. The five pairs of mammary buds in Shh(-/-) female embryos exhibit normal branching morphogenesis at E16.5, forming a rudimentary ductal structure identical to wild-type embryonic mammary glands. We further demonstrate that loss of Hh signaling causes altered cyclin D1 expression in the embryonic dermal mesenchyme. Specifically, cyclin D1 is expressed at E14.5 principally in the condensed mesenchymal cells of the presumptive hair follicles and in both mesenchymal and epithelial cells of the mammary rudiments in wild-type and Shh-deficient embryos. By E18.5, robust cyclin D1 expression is maintained in mammary rudiments of both wild-type and Shh-deficient embryos. In hair follicles of wild-type embryos by E18.5, cyclin D1 expression switches to follicular epithelial cells. In contrast, strong cyclin D1 expression is observed principally in the mesenchymal cells of arrested hair follicles in Shh(-/-) embryos at E18.5. These data reveal that, despite the common embryonic origin of hair follicles and mammary glands, distinct patterns of Hh-family expression occur in these two tissues. Furthermore, these data suggest that cyclin D1 expression in the embryonic hair follicle is mediated by both Hh-independent and Hh-dependent mechanisms.