Fibronectin signals through integrin α5β1 to regulate cardiovascular development in a cell type-specific manner.

Fibronectin (Fn1) is an evolutionarily conserved extracellular matrix glycoprotein essential for embryonic development. Global deletion of Fn1 leads to mid-gestation lethality from cardiovascular defects. However, severe morphogenetic defects that occur early in embryogenesis in these embryos precluded assigning a direct role for Fn1 in cardiovascular development. We noticed that Fn1 is expressed in strikingly non-uniform patterns during mouse embryogenesis, and that its expression is particularly enriched in the pharyngeal region corresponding with the pharyngeal arches 3, 4, and 6. This region bears a special importance for the developing cardiovascular system, and we hypothesized that the localized enrichment of Fn1 in the pharyngeal region may be essential for cardiovascular morphogenesis. To test this hypothesis, we ablated Fn1 using the Isl1(Cre) knock-in strain of mice. Deletion of Fn1 using the Isl1(Cre) strain resulted in defective formation of the 4th pharyngeal arch arteries (PAAs), aberrant development of the cardiac outflow tract (OFT), and ventricular septum defects. To determine the cell types responding to Fn1 signaling during cardiovascular development, we deleted a major Fn1 receptor, integrin α5 using the Isl1(Cre) strain, and observed the same spectrum of abnormalities seen in the Fn1 conditional mutants. Additional conditional mutagenesis studies designed to ablate integrin α5 in distinct cell types within the Isl1(+) tissues and their derivatives, suggested that the expression of integrin α5 in the pharyngeal arch mesoderm, endothelium, surface ectoderm and the neural crest were not required for PAA formation. Our studies suggest that an (as yet unknown) integrin α5-dependent signal extrinsic to the pharyngeal endothelium mediates the formation of the 4th PAAs

Making giant unilamellar vesicles via hydration of a lipid film

This unit describes protocols for making giant unilamellar vesicles (GUVs) based on rehydration of dried lipid films. These model membranes are useful for determining the impact of membrane and membrane-binding components on lipid bilayer stiffness and phase behavior. Due to their large size, they are especially amenable to studies using fluorescence and light microscopy, and may also be manipulated for mechanical measurements with optical traps or micropipets. In addition to their use in encapsulation, GUVs have proven to be useful model systems for studying many cellular processes, including tubulation, budding, and fusion, as well as peptide insertion. The introduction of enzymes or proteins can result in reorganization, leading to such diverse behavior as vesicle aggregation, fusion, and fission

Abnormalities of caudal pharyngeal pouch development in Pbx1 knockout mice mimic loss of Hox3 paralogs

AbstractPbx1 is a TALE-class homeodomain protein that functions in part as a cofactor for Hox class homeodomain proteins. Previous analysis of the in vivo functions of Pbx1 by targeted mutagenesis in mice has revealed roles for this gene in skeletal patterning and development and in the organogenesis of multiple systems. Both RNA expression and protein localization studies have suggested a possible role for Pbx1 in pharyngeal region development. As several Hox mutants have distinct phenotypes in this region, we investigated the potential requirement for Pbx1 in the development of the pharyngeal arches and pouches and their organ derivatives. Pbx1 homozygous mutants exhibited delayed or absent formation of the caudal pharyngeal pouches, and disorganized patterning of the third pharyngeal pouch. Formation of the third pouch-derived thymus/parathyroid primordia was also affected, with absent or hypoplastic primordia, delayed expression of organ-specific differentiation markers, and reduced proliferation of thymic epithelium. The fourth pouch and the fourth pouch-derived ultimobranchial bodies were usually absent. These phenotypes are similar to those previously reported in Hoxa3−/− single mutants and Hoxa1−/−;Hoxb1−/− or Hoxa3+/−;Hoxb3−/−;Hoxd3−/− compound mutants, suggesting that Pbx1 acts together with multiple Hox proteins in the development of the caudal pharyngeal region. However, some aspects of the Pbx1 mutant phenotype included specific defects that were less severe than those found in known Hox mutant mice, suggesting that some functions of Hox proteins in this region are Pbx1-independent

Thymus-Associated Parathyroid Hormone Has Two Cellular Origins with Distinct Endocrine and Immunological Functions

In mammals, parathyroid hormone (PTH) is a key regulator of extracellular calcium and inorganic phosphorus homeostasis. Although the parathyroid glands were thought to be the only source of PTH, extra-parathyroid PTH production in the thymus, which shares a common origin with parathyroids during organogenesis, has been proposed to provide an auxiliary source of PTH, resulting in a higher than expected survival rate for aparathyroid Gcm2−/− mutants. However, the developmental ontogeny and cellular identity of these “thymic” PTH–expressing cells is unknown. We found that the lethality of aparathyroid Gcm2−/− mutants was affected by genetic background without relation to serum PTH levels, suggesting a need to reconsider the physiological function of thymic PTH. We identified two sources of extra-parathyroid PTH in wild-type mice. Incomplete separation of the parathyroid and thymus organs during organogenesis resulted in misplaced, isolated parathyroid cells that were often attached to the thymus; this was the major source of thymic PTH in normal mice. Analysis of thymus and parathyroid organogenesis in human embryos showed a broadly similar result, indicating that these results may provide insight into human parathyroid development. In addition, medullary thymic epithelial cells (mTECs) express PTH in a Gcm2-independent manner that requires TEC differentiation and is consistent with expression as a self-antigen for negative selection. Genetic or surgical removal of the thymus indicated that thymus-derived PTH in Gcm2−/− mutants did not provide auxiliary endocrine function. Our data show conclusively that the thymus does not serve as an auxiliary source of either serum PTH or parathyroid function. We further show that the normal process of parathyroid organogenesis in both mice and humans leads to the generation of multiple small parathyroid clusters in addition to the main parathyroid glands, that are the likely source of physiologically relevant “thymic PTH.

GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET

The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [^3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [^3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ~30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst

Uncovering precision phenotype-biomarker associations in traumatic brain injury using topological data analysis

Background: Traumatic brain injury (TBI) is a complex disorder that is traditionally stratified based on clinical signs and symptoms. Recent imaging and molecular biomarker innovations provide unprecedented opportunities for improved TBI precision medicine, incorporating patho-anatomical and molecular mechanisms. Complete integration of these diverse data for TBI diagnosis and patient stratification remains an unmet challenge. Methods and findings: The Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) Pilot multicenter study enrolled 586 acute TBI patients and collected diverse common data elements (TBI-CDEs) across the study population, including imaging, genetics, and clinical outcomes. We then applied topology-based data-driven discovery to identify natural subgroups of patients, based on the TBI-CDEs collected. Our hypothesis was two-fold: 1) A machine learning tool known as topological data analysis (TDA) would reveal data-driven patterns in patient outcomes to identify candidate biomarkers of recovery, and 2) TDA-identified biomarkers would significantly predict patient outcome recovery after TBI using more traditional methods of univariate statistical tests. TDA algorithms organized and mapped the data of TBI patients in multidimensional space, identifying a subset of mild TBI patients with a specific multivariate phenotype associated with unfavorable outcome at 3 and 6 months after injury. Further analyses revealed that this patient subset had high rates of post-traumatic stress disorder (PTSD), and enrichment in several distinct genetic polymorphisms associated with cellular responses to stress and DNA damage (PARP1), and in striatal dopamine processing (ANKK1, COMT, DRD2). Conclusions: TDA identified a unique diagnostic subgroup of patients with unfavorable outcome after mild TBI that were significantly predicted by the presence of specific genetic polymorphisms. Machine learning methods such as TDA may provide a robust method for patient stratification and treatment planning targeting identified biomarkers in future clinical trials in TBI patients

Canonical notch signaling controls the early thymic epithelial progenitor cell state and emergence of the medullary epithelial lineage in fetal thymus development

Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T cell repertoire. During thymus organogenesis, these functionally distinct sub-lineages are thought to arise from a common thymic epithelial progenitor cell (TEPC). However, the mechanisms controlling cTEC and mTEC production from the common TEPC are not understood. Here, we show that emergence of the earliest mTEC lineage-restricted progenitors requires active NOTCH signaling in progenitor TEC and that, once specified, further mTEC development is NOTCH independent. In addition, we demonstrate that persistent NOTCH activity favors maintenance of undifferentiated TEPCs at the expense of cTEC differentiation. Finally, we uncover a cross-regulatory relationship between NOTCH and FOXN1, a master regulator of TEC differentiation. These data establish NOTCH as a potent regulator of TEPC and mTEC fate during fetal thymus development, and are thus of high relevance to strategies aimed at generating/regenerating functional thymic tissue in vitro and in vivo

DRD2 C957T polymorphism is associated with improved 6-month verbal learning following traumatic brain injury

Traumatic brain injury (TBI) often leads to heterogeneous clinical outcomes, which may be influenced by genetic variation. A single-nucleotide polymorphism (SNP) in the dopamine D2 receptor (DRD2) may influence cognitive deficits following TBI. However, part of the association with DRD2 has been attributed to genetic variability within the adjacent ankyrin repeat and kinase domain containing 1 protein (ANKK1). Here, we utilize the Transforming Research and Clinical Knowledge in Traumatic Brain Injury Pilot (TRACK-TBI Pilot) study to investigate whether a novel DRD2 C957T polymorphism (rs6277) influences outcome on a cognitive battery at 6 months following TBI-California Verbal Learning Test (CVLT-II), Wechsler Adult Intelligence Test Processing Speed Index Composite Score (WAIS-PSI), and Trail Making Test (TMT). Results in 128 Caucasian subjects show that the rs6277 T-allele associates with better verbal learning and recall on CVLT-II Trials 1-5 (T-allele carrier 52.8 ± 1.3 points, C/C 47.9 ± 1.7 points; mean increase 4.9 points, 95% confidence interval [0.9 to 8.8]; p = 0.018), Short-Delay Free Recall (T-carrier 10.9 ± 0.4 points, C/C 9.7 ± 0.5 points; mean increase 1.2 points [0.1 to 2.5]; p = 0.046), and Long-Delay Free Recall (T-carrier 11.5 ± 0.4 points, C/C 10.2 ± 0.5 points; mean increase 1.3 points [0.1 to 2.5]; p = 0.041) after adjusting for age, education years, Glasgow Coma Scale, presence of acute intracranial pathology on head computed tomography scan, and genotype of the ANKK1 SNP rs1800497 using multivariable regression. No association was found between DRD2 C947T and non-verbal processing speed (WAIS-PSI) or mental flexibility (TMT) at 6 months. Hence, DRD2 C947T (rs6277) may be associated with better performance on select cognitive domains independent of ANKK1 following TBI

Clinical predictors of 3- and 6-month outcome for mild traumatic brain injury patients with a negative head CT scan in the emergency department: A TRACK-TBI pilot study

Aconsiderable subset of mild traumatic brain injury (mTBI) patients fail to return to baseline functional status at or beyond 3 months postinjury. Identifying at-risk patients for poor outcome in the emergency department (ED) may improve surveillance strategies and referral to care. Subjects with mTBI (Glasgow Coma Scale 13–15) and negative ED initial head CT < 24 h of injury, completing 3- or 6-month functional outcome (Glasgow Outcome Scale-Extended; GOSE), were extracted from the prospective, multicenter Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) Pilot study. Outcomes were dichotomized to full recovery (GOSE = 8) vs functional deficits (GOSE < 8). Univariate predictors with p < 0.10 were considered for multivariable regression. Adjusted odds ratios (AOR) were reported for outcome predictors. Significance was assessed at p < 0.05. Subjects who completed GOSE at 3- and 6-month were 211 (GOSE < 8: 60%) and 185 (GOSE < 8: 65%). Risk factors for 6-month GOSE < 8 included less education (AOR = 0.85 per-year increase, 95% CI: (0.74–0.98)), prior psychiatric history (AOR = 3.75 (1.73–8.12)), Asian/minority race (American Indian/Alaskan/Hawaiian/Pacific Islander) (AOR = 23.99 (2.93–196.84)), and Hispanic ethnicity (AOR = 3.48 (1.29–9.37)). Risk factors for 3-month GOSE < 8 were similar with the addition of injury by assault predicting poorer outcome (AOR = 3.53 (1.17–10.63)). In mTBI patients seen in urban trauma center EDs with negative CT, education, injury by assault, Asian/minority race, and prior psychiatric history emerged as risk factors for prolonged disability