PBX1 and PBX3 transcription factors regulate SHH expression in the Frontonasal Ectodermal Zone through complementary mechanisms.

Sonic hedgehog (SHH) signaling from the frontonasal ectodermal zone (FEZ) is a key regulator of craniofacial morphogenesis. Along with SHH, pre-B-cell leukemia homeobox (PBX) transcription factors regulate midfacial development. PBXs act in the epithelium during fusion of facial primordia, but their specific interactions with SHH have not been fully investigated. We hypothesized that PBX1/3 regulate SHH expression in the FEZ by activating or repressing transcription. The hypothesis was tested by manipulating PBX1/3 expression in chick embryos and profiling epigenomic landscapes at early developmental stages. PBX1/3 expression was perturbed in the chick face beginning at stage 10 (HH10) using RCAS viruses, and the resulting SHH expression was assessed at HH22. Overexpressing PBX1 expanded SHH expression, while overexpressing PBX3 decreased SHH expression. Conversely, reducing PBX1 expression decreased SHH expression, but reducing PBX3 induced ectopic SHH expression. We performed ATAC-seq and mapped binding of PBX1 and PBX3 with ChIP-seq on the FEZ at HH22 to assess direct interactions of PBX1/3 with the SHH locus. These multi-omics approaches uncovered a 400 bp PBX1-enriched element within intron 1 of SHH (chr2:8,173,222-8,173,621). Enhancer activity of this element was demonstrated by electroporation of reporter constructs in ovo and luciferase reporter assays in vitro . When bound by PBX1, this element upregulates transcription, while it downregulates transcription when bound by PBX3. The present study identifies a cis- regulatory element, named SFE1, that interacts with PBX1/3 to modulate SHH expression in the FEZ and establishes that PBX1 and PBX3 play complementary roles in SHH regulation during embryonic development.

ESCRT-dependent control of craniofacial morphogenesis with concomitant perturbation of NOTCH signaling.

Craniofacial development is orchestrated by transcription factor-driven regulatory networks, epigenetic modifications, and signaling pathways. Signaling molecules and their receptors rely on endo-lysosomal trafficking to prevent accumulation on the plasma membrane. ESCRT (Endosomal Sorting Complexes Required for Transport) machinery is recruited to endosomal membranes enabling degradation of such endosomal cargoes. Studies in vitro and in invertebrate models established the requirements of the ESCRT machinery in membrane remodeling, endosomal trafficking, and lysosomal degradation of activated membrane receptors. However, investigations during vertebrate development have been scarce. By ENU-induced mutagenesis, we isolated a mouse line, Vps25ENU/ENU, carrying a hypomorphic allele of the ESCRT-II component Vps25, with craniofacial anomalies resembling features of human congenital syndromes. Here, we assessed the spatiotemporal dynamics of Vps25 and additional ESCRT-encoding genes during murine development. We show that these genes are ubiquitously expressed although enriched in discrete domains of the craniofacial complex, heart, and limbs. ESCRT-encoding genes, including Vps25, are expressed in both cranial neural crest-derived mesenchyme and epithelium. Unlike constitutive ESCRT mutants, Vps25ENU/ENU embryos display late lethality. They exhibit hypoplastic lower jaw, stunted snout, dysmorphic ear pinnae, and secondary palate clefting. Thus, we provide the first evidence for critical roles of ESCRT-II in craniofacial morphogenesis and report perturbation of NOTCH signaling in craniofacial domains of Vps25ENU/ENU embryos. Given the known roles of NOTCH signaling in the developing cranium, and notably the lower jaw, we propose that the NOTCH pathway partly mediates the craniofacial defects of Vps25ENU/ENU mouse embryos.

A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2.

A lingering question in developmental biology has centered on how transcription factors with widespread distribution in vertebrate embryos can perform tissue-specific functions. Here, using the murine hindlimb as a model, we investigate the elusive mechanisms whereby PBX TALE homeoproteins, viewed primarily as HOX cofactors, attain context-specific developmental roles despite ubiquitous presence in the embryo. We first demonstrate that mesenchymal-specific loss of PBX1/2 or the transcriptional regulator HAND2 generates similar limb phenotypes. By combining tissue-specific and temporally controlled mutagenesis with multi-omics approaches, we reconstruct a gene regulatory network (GRN) at organismal-level resolution that is collaboratively directed by PBX1/2 and HAND2 interactions in subsets of posterior hindlimb mesenchymal cells. Genome-wide profiling of PBX1 binding across multiple embryonic tissues further reveals that HAND2 interacts with subsets of PBX-bound regions to regulate limb-specific GRNs. Our research elucidates fundamental principles by which promiscuous transcription factors cooperate with cofactors that display domain-restricted localization to instruct tissue-specific developmental programs.

HOX paralogs selectively convert binding of ubiquitous transcription factors into tissue-specific patterns of enhancer activation.

Gene expression programs determine cell fate in embryonic development and their dysregulation results in disease. Transcription factors (TFs) control gene expression by binding to enhancers, but how TFs select and activate their target enhancers is still unclear. HOX TFs share conserved homeodomains with highly similar sequence recognition properties, yet they impart the identity of different animal body parts. To understand how HOX TFs control their specific transcriptional programs in vivo, we compared HOXA2 and HOXA3 binding profiles in the mouse embryo. HOXA2 and HOXA3 directly cooperate with TALE TFs and selectively target different subsets of a broad TALE chromatin platform. Binding of HOX and tissue-specific TFs convert low affinity TALE binding into high confidence, tissue-specific binding events, which bear the mark of active enhancers. We propose that HOX paralogs, alone and in combination with tissue-specific TFs, generate tissue-specific transcriptional outputs by modulating the activity of TALE TFs at selected enhancers.

Uncovering tissue-specific binding features from differential deep learning.

Transcription factors (TFs) can bind DNA in a cooperative manner, enabling a mutual increase in occupancy. Through this type of interaction, alternative binding sites can be preferentially bound in different tissues to regulate tissue-specific expression programmes. Recently, deep learning models have become state-of-the-art in various pattern analysis tasks, including applications in the field of genomics. We therefore investigate the application of convolutional neural network (CNN) models to the discovery of sequence features determining cooperative and differential TF binding across tissues. We analyse ChIP-seq data from MEIS, TFs which are broadly expressed across mouse branchial arches, and HOXA2, which is expressed in the second and more posterior branchial arches. By developing models predictive of MEIS differential binding in all three tissues, we are able to accurately predict HOXA2 co-binding sites. We evaluate transfer-like and multitask approaches to regularizing the high-dimensional classification task with a larger regression dataset, allowing for the creation of deeper and more accurate models. We test the performance of perturbation and gradient-based attribution methods in identifying the HOXA2 sites from differential MEIS data. Our results show that deep regularized models significantly outperform shallow CNNs as well as k-mer methods in the discovery of tissue-specific sites bound in vivo.

Face morphogenesis is promoted by Pbx-dependent EMT via regulation of Snail1 during frontonasal prominence fusion.

Human cleft lip with or without cleft palate (CL/P) is a common craniofacial abnormality caused by impaired fusion of the facial prominences. We have previously reported that, in the mouse embryo, epithelial apoptosis mediates fusion at the seam where the prominences coalesce. Here, we show that apoptosis alone is not sufficient to remove the epithelial layers. We observed morphological changes in the seam epithelia, intermingling of cells of epithelial descent into the mesenchyme and molecular signatures of epithelial-mesenchymal transition (EMT). Utilizing mouse lines with cephalic epithelium-specific Pbx loss exhibiting CL/P, we demonstrate that these cellular behaviors are Pbx dependent, as is the transcriptional regulation of the EMT driver Snail1. Furthermore, in the embryo, the majority of epithelial cells expressing high levels of Snail1 do not undergo apoptosis. Pbx1 loss- and gain-of-function in a tractable epithelial culture system revealed that Pbx1 is both necessary and sufficient for EMT induction. This study establishes that Pbx-dependent EMT programs mediate murine upper lip/primary palate morphogenesis and fusion via regulation of Snail1. Of note, the EMT signatures observed in the embryo are mirrored in the epithelial culture system.

De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects.

We present eight patients with de novo, deleterious sequence variants in the PBX1 gene. PBX1 encodes a three amino acid loop extension (TALE) homeodomain transcription factor that forms multimeric complexes with TALE and HOX proteins to regulate target gene transcription during development. As previously reported, Pbx1 homozygous mutant mice (Pbx1-/-) develop malformations and hypoplasia or aplasia of multiple organs, including the craniofacial skeleton, ear, branchial arches, heart, lungs, diaphragm, gut, kidneys, and gonads. Clinical findings similar to those in Pbx mutant mice were observed in all patients with varying expressivity and severity, including external ear anomalies, abnormal branchial arch derivatives, heart malformations, diaphragmatic hernia, renal hypoplasia and ambiguous genitalia. All patients but one had developmental delays. Previously reported patients with congenital anomalies affecting the kidney and urinary tract exhibited deletions and loss of function variants in PBX1. The sequence variants in our cases included missense substitutions adjacent to the PBX1 homeodomain (p.Arg184Pro, p.Met224Lys, and p.Arg227Pro) or within the homeodomain (p.Arg234Pro, and p.Arg235Gln), whereas p.Ser262Glnfs*2, and p.Arg288* yielded truncated PBX1 proteins. Functional studies on five PBX1 sequence variants revealed perturbation of intrinsic, PBX-dependent transactivation ability and altered nuclear translocation, suggesting abnormal interactions between mutant PBX1 proteins and wild-type TALE or HOX cofactors. It is likely that the mutations directly affect the transcription of PBX1 target genes to impact embryonic development. We conclude that deleterious sequence variants in PBX1 cause intellectual disability and pleiotropic malformations resembling those in Pbx1 mutant mice, arguing for strong conservation of gene function between these two species.

A tissue-specific, Gata6-driven transcriptional program instructs remodeling of the mature arterial tree.

Connection of the heart to the systemic circulation is a critical developmental event that requires selective preservation of embryonic vessels (aortic arches). However, why some aortic arches regress while others are incorporated into the mature aortic tree remains unclear. By microdissection and deep sequencing in mouse, we find that neural crest (NC) only differentiates into vascular smooth muscle cells (SMCs) around those aortic arches destined for survival and reorganization, and identify the transcription factor Gata6 as a crucial regulator of this process. Gata6 is expressed in SMCs and its target genes activation control SMC differentiation. Furthermore, Gata6 is sufficient to promote SMCs differentiation in vivo, and drive preservation of aortic arches that ought to regress. These findings identify Gata6-directed differentiation of NC to SMCs as an essential mechanism that specifies the aortic tree, and provide a new framework for how mutations in GATA6 lead to congenital heart disorders in humans.

Hoxa2 selectively enhances Meis binding to change a branchial arch ground state.

Hox transcription factors (TFs) are essential for vertebrate development, but how these evolutionary conserved proteins function in vivo remains unclear. Because Hox proteins have notoriously low binding specificity, they are believed to bind with cofactors, mainly homeodomain TFs Pbx and Meis, to select their specific targets. We mapped binding of Meis, Pbx, and Hoxa2 in the branchial arches, a series of segments in the developing vertebrate head. Meis occupancy is largely similar in Hox-positive and -negative arches. Hoxa2, which specifies second arch (IIBA) identity, recognizes a subset of Meis prebound sites that contain Hox motifs. Importantly, at these sites Meis binding is strongly increased. This enhanced Meis binding coincides with active enhancers, which are linked to genes highly expressed in the IIBA and regulated by Hoxa2. These findings show that Hoxa2 operates as a tissue-specific cofactor, enhancing Meis binding to specific sites that provide the IIBA with its anatomical identity.

Living with life insurance: a qualitative analysis of the experience of male implantable defibrillator recipients in Spain.

BACKGROUND AND OBJECTIVES: The implantation of defibrillators should not be studied simply on the basis of clinical improvement or quality of life: it is also important to understand the significance, which the recipients attach to the defibrillator and their experiences with it. The aim of this work was, therefore, to determine the experience of Spanish implantable defibrillator recipients. DESIGN: A qualitative phenomenological study. METHODS: Purposeful sampling of male implantable defibrillator recipients older than 18 years of age attended at the defibrillator consultancy at the Hospital Fuenlabrada or belonging to the Heart Patients' Association (Asociación de Pacientes Coronarios, APACOR). A secondary, theoretical sampling was also carried out to gain a more in-depth understanding of certain aspects identified in the first sampling, such as living with the discharges and difficulties during sexual activity. Data were collected using unstructured and semi-structured questionnaires and applying a question guide, field notes and the recipients' personal diaries/letters. Data collection was terminated once theoretical saturation was reached. Data were analysed using the Giorgi method. Finally, the seven themes, which showed what it means to be an implantable cardioverter-defibrillator recipient, were described. RESULTS: The defibrillator is perceived positively and is considered to be a form of life insurance, whereas the discharges are a limiting factor. The recipient's outlook on life changes. Acceptance of the changes resulting from the implant leads to the development of strategies to facilitate everyday life. CONCLUSIONS: An understanding of the significance attached by recipients to their disease, diagnosis and treatment allows their behaviour and expectations to be understood. RELEVANCE TO CLINICAL PRACTICE: Provide the basis for nursing assessment after discharge, understand the effects of the device in the recipient and track the process of adapting the recipient to daily life.

[Surgical complications in salvage surgery of patients with head and neck carcinomas treated with concomitant chemoradiotherapy (CCR)]. / Complicaciones quirúrgicas en la cirugía de rescate de pacientes con carcinomas de cabeza y cuello tratados con quimioterapia y radioterapia concomitantes.

INTRODUCTION: One way of treating head and neck carcinomas is using concomitant chemoradiotherapy (CCR). In this study we will try to evaluate the incidence of complications in rescue surgery after CCR. MATERIAL AND METHOD: We have studied data from 103 patients diagnosed as having stage III or IV squamous head and neck carcinoma between 1997 and 2005. They were treated following two different CCR protocols. RESULTS: Of the 103 patients, 26 (25 %) required rescue surgery. Eight patients in this group (30.76 % of those operated on) presented complications. The average stay in our department was 52.8 days (7-197 days). CONCLUSIONS: Patients treated with CCR who have needed rescue surgery apparently have a higher rate of complications and a longer stay than those treated with surgery alone.

Complicaciones quirúrgicas en la cirugía de rescate de pacientes con carcinomas de cabeza y cuello tratados con quimioterapia y radioterapia concomitantes / Surgical complications in salvage surgery of patients with head and neck carcinomas treated with concomitant chemoradiotherapy (CCR)

Introducción: Una de las modalidades de tratamiento de los carcinomas epidermoides de cabeza y cuello es con radioterapia y quimioterapia concomitantes (RQC). En este estudio se intentará evaluar la incidencia de complicaciones que presenta la cirugía de rescate tras la realización de RQC. Material y método: Se ha estudiado los datos de 103 pacientes diagnosticados de carcinoma epidermoide de cabeza y cuello en estadios III y IV tratados entre 1997 y 2005. Se los trató siguiendo dos protocolos distintos de RQC. Resultados: De los 103 pacientes, requirieron cirugía de rescate 26 (25 %). Dentro de este grupo presentaron complicaciones 8 pacientes (el 30,76 % de los intervenidos). La estancia media en el servicio de otorrinolaringología fue de 52,8 (7-197) días. Conclusiones: Los pacientes que han recibido RQC y han necesitado cirugía de rescate posteriormente presentan una aparente mayor tasa de complicaciones, así como un ingreso más prolongado, que aquellos en quienes inicialmente se opta por la cirugía

Introduction: One way of treating head and neck carcinomas is using concomitant chemoradiotherapy (CCR). In this study we will try to evaluate the incidence of complications in rescue surgery after CCR. Material and method: We have studied data from 103 patients diagnosed as having stage III or IV squamous head and neck carcinoma between 1997 and 2005. They were treated following two different CCR protocols. Results: Of the 103 patients, 26 (25 %) required rescue surgery. Eight patients in this group (30.76 % of those operated on) presented complications. The average stay in our department was 52.8 days (7-197 days). Conclusions: Patients treated with CCR who have needed rescue surgery apparently have a higher rate of complications and a longer stay than those treated with surgery alone

Abordaje quirúrgico de la exostosis retrocalcánea / Surgical approach of the retrocalcaneal exotosis

El dolor posterior del talón representa una patología relativamente común que se asocia a la calcificación tanto del cuerpo del tendón como de su inserción. Clínicamente los pacientes presentan eritema, y edema en la inserción del tendón de Aquiles. La zona posterior del talón se presenta sensible a la presión, y el dolor puede ir acompañado de una bursitis localizada. Las alternativas al tratamiento conservador suelen ser limitadas y poco eficaces. El tratamiento quirúrgico está indicado cuando el tratamiento conservador fracasa y el paciente tiene dificultades para realizar sus actividades diarias. En el presente artículo presentamos la revisión de 2 técnicas quirúrgicas para el abordaje de la exostosis retrocalcánea y para la solución de la patología tendinosa asociada

The posterior heel pain represents a relatively common pathology that it is associated with the calcification of the body of the tendon as well as its insertion. Clinically, the patients show eritema, and edema in the insertion of the Aquilles tendon. The posterior aspect of the heel appears too tender to the digital pressure, and the pain can go accompanied of a located bursitis. The alternatives to the conservative treatment usually are limited and not to much effective. The surgical treatment is indicated when the conservative treatment fails and the patients have difficulties to make their daily activities. In the present article we present the review of 2 surgical techniques to approach the retrocalcaneal exostosis and for the solution of the associated tendinopathy

Los pies en distintas culturas y cosmovisiones. Los pies en el pensamiento chino / The feet in different cultures and world view. The feet in the chinese thought

En todas las sociedades el cuerpo y sus distintas partes se perciben de forma diferente, pero siempre en relación al pensamiento filosófico religioso de entender la vida y al ser humano, ello influirá también en la forma de comprender la salud y la enfermedad. En la tradición china, además, las huellas de los pies de Buda son sagradas y se veneran. Es frecuente utilizar reproducciones de los pies de Buda como amuletos (AU)

Los pies en distintas culturas y cosmovisiones: los pies en la Cábala / The feet in different cultures and world view. The feet in the cabala

La forma de percibir y experimentar el cuerpo humano es diferente según épocas y sociedades. El cuerpo humano, y sus partes, está repleto de metáforas y de simbolismo. Los pies situados en la parte inferior y en contacto con el suelo se han pensado de maneras distintas según cosmovisiones. Pretendemos estudiar cómo se interpretan los pies en la simbología de la Cábala (AU)