Rapid degradation of lindane (��-hexachlorocyclohexane) at low temperature by Sphingobium strains

Bioremediation of anthropogenic organic pollutants in cold climates is often limited by lower microbial or enzyme activity induced by low temperature. The present study addressed this issue through the degradation of ??-hexachlorocyclohexane (??-HCH) by three Sphingobium strains (S. indicum B90A, S. japonicum UT26 and S. francense Sp+) under low temperature (4 °C). After 5 days incubation at 4 °C, 79.7% and 43.8% of 5 and 25 mg L⁻¹ of ??-HCH added were degraded, respectively by the inoculation of 1.75 × 10⁷ cells mL⁻¹ of S. indicum B90A. An increase in inoculum concentration to 1.72 × 10⁸ cells mL⁻¹ significantly increased the degradation to 98.1 ± 1.7% of 5 mg L⁻¹ within 24 h. Further, S. indicum B90A and S. japonicum UT26 can rapidly degrade ??-HCH at 4 °C, while the degradation capability of S. francense Sp+ is relatively low. At 4 °C, ??-HCH is transformed to extremely low amounts of 1,2,4-trichlorobenzene (1,2,4-TCB) and 2,5-dichlorophenol (2,5-DCP) by S. indicum B90A, but most of ??-HCH were transformed to 2,5-Dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) by S. japonicum UT26. These results revealed that haloalkane dehalogenases in some Sphingobium species are very active at temperature as low as 4 °C and S. indicum B90A might be a good candidate for developing novel bioremediation techniques for cold regions to decontaminate ??-HCH from soils/waters.     

Expression of insect α6-like nicotinic acetylcholine receptors in Drosophila melanogaster highlights a high level of conservation of the receptor:spinosyn interaction

Insecticide research has often relied on model species for elucidating the resistance mechanisms present in the targeted pests. The accuracy and applicability of extrapolations of these laboratory findings to field conditions varies but, for target site resistance, conserved mechanisms are generally the rule rather than the exception (Perry et al., 2011). The spinosyn class of insecticides appear to fit this paradigm and are a pest control option with many uses in both crop and animal protection. Resistance to spinosyns has been identified in both laboratory-selected and field-collected pest insects.Studies using the model insect, Drosophila melanogaster, have identified the nicotinic acetylcholine receptor subunit, Dα6 as an important target of the insecticide spinosad (Perry et al., 2007, Watson et al., 2010). Field-isolated resistant strains of several agricultural pest insects provide evidence that resistance cases are often associated with mutations in orthologues to Dα6 (Baxter et al., 2010, Puinean et al., 2013).The expression of these receptors is difficult in heterologous systems. In order to examine the biology of the Dα6 receptor subunit further, we used Drosophila as a model and developed an in vivo rescue system. This allowed us to express four different isoforms of Dα6 and show that each is able to rescue the response to spinosad. Regulatory sequences upstream of the Dα6 gene able to rescue the resistance phenotype were identified. Expression of other D. melanogaster subunits revealed that the rescue phenotype appears to be Dα6 specific. We also demonstrate that expression of pest insect orthologues of Dα6 from a variety of species are capable of rescuing the spinosad response phenotype, verifying the relevance of this receptor to resistance monitoring in the field. In the absence of a robust heterologous expression system, this study presents an in vivo model that will be useful in analysing many other aspects of these receptors and their biology.     

Autopodial development is selectively impaired by misexpression of chordin-like 1 in the chick limb

Chordin-like 1 (CHRDL1) is a secreted bone morphogenetic protein (BMP) antagonist expressed in mesenchymal tissues whose function in development of the skeleton has not been examined in detail. Here we show Chrdl1 is dynamically expressed in the early distal limb bud mesenchyme, with expression becoming downregulated as development proceeds. Chrdl1 expression is largely excluded from the critical signaling center of the posterior limb bud, the Zone of Polarizing Activity (ZPA), as has been described for the BMP antagonist Gremlin (GREM1) ( Scherz et al., 2004, Science, 305, 396–399). Unlike Grem1, Chrdl1 is expressed in the hindlimb by a small subset of ZPA cells and their descendants suggesting divergent regulation and function between the various BMP antagonists. Ectopic expression of Chrdl1 throughout the avian limb bud using viral misexpression resulted in an oligodactyly phenotype with loss of digits from the anterior limb, although the development of more proximal elements of the zeugopod and stylopod were unaffected. Overgrowths of soft tissue and syndactyly were also observed, resulting from impaired apoptosis and failure of the anterior mesenchyme to undergo SOX9-dependent chondrogenesis, instead persisting as an interdigital-like soft tissue phenotype. Sonic hedgehog (SHH) and fibroblast growth factor (FGF) signaling were upregulated and persisted later in development, however these changes were only detected late in limb development at timepoints when endogenous Grem1 would normally be downregulated and increasing BMP signaling would cause termination of Shh and Fgf expression. Our results suggest that the early stages of the GREM1–SHH–FGF signaling network are resistant to Chrdl1-overexpression, leading to normal formation of proximal limb structures, but that later Bmp expression, impaired by ectopic CHRDL1, is essential for formation of the correct complement of digits.     

Pelagostrobilidium liui n. sp. (Ciliophora,Choreotrichida) from the Coastal Waters of Northeastern Taiwan and an Improved Description of Pelagostrobilidium minutum Liu et al., 2012

The number of somatic kineties in Pelagostrobilidium ranges from 4 to 6 according to the present state of knowledge. This study investigates Pelagostrobilidium liui n. sp. using live observation, protargol stain, and small subunit rDNA data sequencing. Pelagostrobilidium liui n. sp. is characterized by having a spherical‐shaped body, four somatic kineties, with kinety 2 spiraled around the left side of body, about six elongated external membranelles, and invariably no buccal membranelle. It differs from its most similar congener, Pelagostrobilidium minutum Liu et al., 2012 , in (i) cell shape; (ii) macronucleus width; (iii) oral apparatus; (iv) anterior orientation of kinety 2; (v) location where kinety 2 commences; (vi) arrangement of kinety 1; (vii) distance between the anterior cell end and the locations where kineties commence; and (viii) the presence of 12 different bases (including two deletions) in the small subunit rDNA sequences. The diagnosis of P. minutum Liu et al., 2012 is also improved to include the following new characteristics: invariably four somatic kineties; kineties 2 and 4 alone commence at the same level; kinety 2 originates from right anterior cell half on ventral side, extends sinistrally posteriorly, over kinety 1, around left posterior region, terminates near posterior cell end on dorsal side; kinety 1 commences below anterior third of kinety 2.     

Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ∼62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)–deficient enhanced green fluorescent protein (EGFP)–occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin^ΔOCEL. Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1–binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK–binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.Tight junctions seal the paracellular space in simple epithelia, such as those lining the lungs, intestines, and kidneys (Anderson et al., 2004 ; Fanning and Anderson, 2009 ; Shen et al., 2011 ). In the intestine, reduced paracellular barrier function is associated with disorders in which increased paracellular flux of ions and molecules contributes to symptoms such as diarrhea, malabsorption, and intestinal protein loss. Recombinant tumor necrosis factor (TNF) can be used to model this barrier loss in vitro or in vivo (Taylor et al., 1998 ; Clayburgh et al., 2006 ), and TNF neutralization is associated with restoration of intestinal barrier function in Crohn''s disease (Suenaert et al., 2002 ). Further, in vivo and in vitro studies of intestinal epithelia show that TNF-induced barrier loss requires myosin light chain kinase (MLCK) activation (Zolotarevsky et al., 2002 ; Clayburgh et al., 2005 , 2006 ; Ma et al., 2005 ; Wang et al., 2005 ). The resulting myosin II regulatory light chain (MLC) phosphorylation drives occludin internalization, which is required for cytokine-induced intestinal epithelial barrier loss (Clayburgh et al., 2005 , 2006 ; Schwarz et al., 2007 ; Marchiando et al., 2010 ). In addition, transgenic EGFP-occludin expression in vivo limits TNF-induced depletion of tight junction–associated occludin, barrier loss, and diarrhea (Marchiando et al., 2010 ). Conversely, in vitro studies show that occludin knockdown limits TNF-induced barrier regulation (Van Itallie et al., 2010 ). The basis for this discrepancy is not understood.One challenge is that, despite being identified 20 yr ago (Furuse et al., 1993 ), the contribution of occludin to tight junction regulation remains incompletely defined. The observation that occludin-knockout mice are able to form paracellular barriers and do not have obvious defects in epidermal, respiratory, or bladder tight junction function (Saitou et al., 2000 ; Schulzke et al., 2005 ) led many to conclude that occludin is not essential for tight junction barrier function. It is important to note, however, that barrier regulation in response to stress has not been studied in occludin-deficient animals.We recently showed that dephosphorylation of occludin serine-408 promotes assembly of a complex composed of occludin, ZO-1, and claudin-2 that inhibits flux across size- and charge-selective channels termed the pore pathway (Anderson and Van Itallie, 2009 ; Turner, 2009 ; Raleigh et al., 2011 ; Shen et al., 2011 ). Although this demonstrates that occludin can serve a regulatory role, it does not explain the role of occludin in TNF-induced barrier loss, which increases flux across the size- and charge-nonselective leak pathway (Wang et al., 2005 ; Weber et al., 2010 ). In vitro studies, however, do suggest that occludin contributes to leak pathway regulation, as occludin knockdown in either Madin–Darby canine kidney (MDCK) or human intestinal (Caco-2) epithelial monolayers enhances leak pathway permeability (Yu et al., 2005 ; Al-Sadi et al., 2011 ; Ye et al., 2011 ). Taken as a whole, these data suggest that occludin organizes the tight junction to limit leak pathway flux, whereas occludin removal, either by knockdown or endocytosis, enhances leak pathway flux.To define the mechanisms by which occludin regulates the leak pathway, we analyzed the contributions of occludin, as well as specific occludin domains, to basal and TNF-induced barrier regulation. The data indicate that TNF destabilizes tight junction–associated occludin via interactions mediated by the C-terminal coiled-coil occludin/ELL domain (OCEL). Further, these OCEL-mediated events are required for TNF-induced barrier regulation. Thus these data provide new insight into the structural elements and mechanisms by which occludin regulates leak pathway paracellular flux.     

TLR2 Directing PD-L2 Expression Inhibit T Cells Response in Schistosoma japonicum Infection

Toll-like receptor 2 (TLR2) was shown to be an important immune receptor involved in the recognition of schistosome antigens, especially soluble egg antigen (SEA). In mice models with Schistosoma japonicum acute infection, we observed enhanced T cell-mediated immune responses in TLR2 knock out (TLR2^−/−) mice compared with B6 mice. In Schistosoma japonicum chronic infection models, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) expression as well as TLR2 expression gradually increased in B6 mice, while only PD-L2 expression significantly decreased in TLR2^−/− mice. Meanwhile, Programmed Death 1(PD-1) expression on CD4⁺T cells was down-regulated in TLR2^−/− mice after a large number of egg appeared. We also found that stimulation with schistosome antigens, especially SEA, could up-regulate PD-L2 expression on BMDCs in a TLR2-dependent manner in vitro. Schistosome antigens primed-BMDCs with impaired expression of TLR2 or PD-L2 could induce CD4⁺T cells to produce low level of IL-10 or high level of IFN-γ. Our results indicated that TLR2 signaling can direct PD-L2 expression on DCs, which binds to PD-1 mainly on CD4⁺T cells, to help inhibit T cells response in Schistosoma japonicum infection.     

Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin

Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that ⁷⁷⁰DYWL⁷⁷³ of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around ⁷⁷⁰DYWL⁷⁷³ of ECL 4 in the ABCC2.     

Rohon-Beard sensory neurons are induced by BMP4 expressing non-neural ectoderm in Xenopus laevis

Rohon-Beard mechanosensory neurons (RBs), neural crest cells, and neurogenic placodes arise at the border of the neural- and non-neural ectoderm during anamniote vertebrate development. Neural crest cells require BMP expressing non-neural ectoderm for their induction. To determine if epidermal ectoderm-derived BMP signaling is also involved in the induction of RB sensory neurons, the medial region of the neural plate from donor Xenopus laevis embryos was transplanted into the non-neural ventral ectoderm of host embryos at the same developmental stage. The neural plate border and RBs were induced at the transplant sites, as shown by expression of Xblimp1, and XHox11L2 and XN-tubulin, respectively. Transplantation studies between pigmented donors and albino hosts showed that neurons are induced both in donor neural and host epidermal tissue. Because an intermediate level of BMP4 signaling is required to induce neural plate border fates, we directly tested BMP4′s ability to induce RBs; beads soaked in either 1 or 10 ng/ml were able to induce RBs in cultured neural plate tissue. Conversely, RBs fail to form when neural plate tissue from embryos with decreased BMP activity, either from injection of noggin or a dominant negative BMP receptor, was transplanted into the non-neural ectoderm of un-manipulated hosts. We conclude that contact between neural and non-neural ectoderm is capable of inducing RBs, that BMP4 can induce RB markers, and that BMP activity is required for induction of ectopic RB sensory neurons.     

The Role of Hepatocyte Hemojuvelin in the Regulation of Bone Morphogenic Protein-6 and Hepcidin Expression in Vivo

Both hemojuvelin (HJV) and bone morphogenic protein-6 (BMP6) are essential for hepcidin expression. Hepcidin is the key peptide hormone in iron homeostasis, and is secreted predominantly by hepatocytes. HJV expression is detected in hepatocytes, as well as in skeletal and heart muscle. HJV binds BMP6 and increases hepcidin expression presumably by acting as a BMP co-receptor. We characterized the role of hepatocyte HJV in the regulation of BMP6 and hepcidin expression. In HJV-null (Hjv^−/−) mice that have severe iron overload and marked suppression of hepcidin expression, we detected 4-fold higher hepatic BMP6 mRNA than in wild-type counterparts. These results indicate that Hjv^−/− mice do not lack BMP6. Furthermore, iron depletion in Hjv^−/− mice decreased hepatic BMP6 mRNA. Expression of HJV in hepatocytes of Hjv^−/− mice using an AAV2/8 vector, increased hepatic hepcidin mRNA by 65-fold and phosphorylated Smad1/5/8 in the liver by about 2.5-fold. However, no significant change in BMP6 mRNA was detected in either the liver or the small intestine of these animals. Our results revealed a close correlation of hepatic BMP6 mRNA expression with hepatic iron-loading. Together, our data indicate that the regulation of hepatic BMP6 expression by iron is independent of HJV, and that expression of HJV in hepatocytes plays an essential role in hepcidin expression by potentiating the BMP6-mediated signaling.     

How a first research experience had an impact on my scientific journey

As I look back to my scientific trajectory on the occasion of being the recipient of the E. B. Wilson Medal of the American Society for Cell Biology, I realize how much an early scientific experience had an impact on my research many years later. The major influence that the first scientific encounters can have in defining a scientist’s path makes the choice of the training environment so important for a future career.

During my scientific career I have worked on different topics. However, my scientific journey represents a progression of steps that build onto each other. Each experience, no matter how successful, had an impact on how I interpreted subsequent results, on how I assessed their implications, and on how I made subsequent programmatic decisions. My research at the interface of cell biology and neuroscience has spanned from membrane traffic and membrane remodeling, to phosphoinositide signaling, and more recently also to the role of membrane contact sites in the control of membrane lipid homeostasis in physiology and disease. While only partially related, all these topics are interconnected by an intellectual thread. As an example—which shows how defining our training years can be—I will summarize here how an early scientific experience in medical school contributed to shape in unexpected ways my research directions many years later.From the day I enrolled in medical school at the University of Milano I knew I wanted to be a scientist. While a medical student, I explored several research environments that would expose me to leading-edge science. Eventually I encountered a young scientist, Jacopo Meldolesi, who had just returned to Italy after studying the secretory pathway as a postdoc with Jim Jamieson and George Palade at Rockefeller University, and I decided to join his lab to carry out the required short medical school thesis project. Jacopo was interested in the regulation of secretion and proposed that I explore mechanisms underlying the so-called “PI (Phosphatidyl Inositol) effect” described by Hokin and Hokin in the 1950s (Hokin and Hokin, 1953). These investigators had found that stimulation of secretory cells resulted in the rapid cleavage of PI immediately followed by its resynthesis. It remained unclear whether these changes reflected a signaling reaction elicited by secretagogues or metabolic changes associated with membrane traffic reactions triggered by the stimulus. So, the idea was to determine in which membrane compartment this turnover occurred: selectively at the plasma membrane (thus supporting a role in signaling) or in membranes of the secretory pathway. We were puzzled by finding that at least under the rather poor time resolution of our experiments—incubation of pancreatic lobules for tens of minutes with radioactive inositol following stimulation with acetylcholine—newly synthesized PI was found at similar concentrations in all membranes. But then we became aware of the recently described proteins that transport lipids between membranes through the cytosol and independent of membrane traffic (Wirtz and Zilversmit, 1969). We thought that perhaps these proteins could contribute to rapidly equilibrating PI pools in different compartments, questioning the validity of our experimental approach to identify the site of PI resynthesis. I moved on to other projects, and I thought I would never again work with lipids.In the following years the work of several labs established that the PI effect discovered by Hokin and Hokin mainly reflected agonist-stimulated phospholipase C (PLC)-dependent cleavage of the phosphoinositide PI(4,5)P₂ at the plasma membrane to generate the second messengers diacylglycerol (DAG) and IP3 (phosphoinositides are the phosphorylated products of PI) (Lapetina and Michell, 1973; Inoue et al., 1977; Berridge et al., 1983). These findings became textbook knowledge, and the idea that PI(4,5)P₂ had primarily a function in signal transduction at the cell surface became mainstream. The additional discovery of the PI(3,4)P₂ and PI(3,4,5)P₃ as a mediators of growth factor actions added a new twist to the field by showing that not only metabolites generated by PI(4,5)P₂ cleavage, but also phosphoinositides themselves, could have a signaling role (Traynor-Kaplan et al., 1988; Whitman et al., 1988; Auger et al., 1989). However, once again, this signaling appeared to be implicated in classical signal transduction, not in membrane traffic.In the meantime, I had moved to Yale, first as a postdoc with Paul Greengard and then as a faculty in the Section of Cell Biology (which subsequently became the Department of Cell Biology). Following my postdoc with Greengard, I had become interested in the cell biology of synapses and in the molecular machinery underlying the exo-endocytic recycling of synaptic vesicles. In what turned out to be a seminal finding, in the mid-1990s Peter McPherson, a postdoc in my laboratory, identified a nerve terminal–enriched protein that shared some interactions with the GTPase dynamin, another abundant presynaptic protein studied in our lab (McPherson et al., 1994). As dynamin mediates the fission reaction of endocytosis by cutting the neck of endocytic buds to generate free vesicles (Takei et al., 1995; Roux et al., 2006; Ferguson and De Camilli, 2012), we speculated that this new protein would also have a role in endocytic traffic. Peter set out to clone it, and I vividly recall my surprise when, upon my returning from a summer vacation in Italy he reported to me that a domain of this protein, which we called synaptojanin, had homology to an inositol 5-phosphatase that dephosphorylates PI(4,5)P₂ (McPherson et al., 1996). A second domain of this protein was subsequently found to have inositol 4-phosphatase activity (Guo et al., 1999), so that synaptojanin can dephosphorylate PI(4,5)P₂ all the way to PI. Thus, I was back to lipids, specifically to inositol phospholipids, and to a potential role of these lipids not in the transduction of extracellular signals, but in membrane traffic. With a mind primed by my project in medical school, I delved with enthusiasm into this topic. PI(4,5)P₂ had been shown to interact with clathrin adaptors (Beck and Keen, 1991), but the physiological significance of these findings had remained unclear. Our studies led to a model in which the selective concentration of PI(4,5)P₂ in the plasma membrane is required for the recruitment of clathrin adaptors and other endocytic factors to this membrane, while the tight coupling of PI(4,5)P₂ dephosphorylation by synaptojanin to the dynamin-dependent fission reaction of endocytosis is required to allow the shedding of such factors once the vesicle has undergone separation from the plasma membrane (Cremona et al., 1999; Cremona and De Camilli, 2001; Wenk et al., 2001). We also discovered curvature-generating/sensing proteins (BAR domain–containing proteins, primarily endophilin) that are responsible for achieving this coupling by coordinating the formation of the narrow neck of endocytic buds with the recruitment of synaptojanin (Takei et al., 1999; Farsad et al., 2001; Frost et al., 2009; Wu et al., 2010; Milosevic et al., 2011). This model, which is encapsulated in Figure 1 and which is supported by genetic studies in mice and other model organisms, posits that a cycle of PI phosphorylation and dephosphorylation is nested within the synaptic vesicle cycle. The implication of a phosphoinositide phosphatase in membrane transport converged with the identification of a PI kinase (the PI 3-kinase Vps34) involved in “vacuolar protein sorting” in yeast (Schu et al., 1993). This raised the possibility that the phosphorylation and dephosphorylation of inositol phospholipids could have a general significance in membrane traffic (De Camilli et al., 1996).Open in a separate windowFIGURE 1:Schematic cartoon depicting the occurrence of a cycle of phosphatidylinositol phosphorylation–dephosphorylation nested within the exo-endocytic recycling of synaptic vesicles at synapses. PI(4,5)P₂ in the plasma membrane helps define this membrane as the acceptor for the fusion of synaptic vesicles and functions as a coreceptor for endocytic factors responsible for their reinternalization. PI(4,5)P₂ dephosphorylation by synaptojanin allows the shedding of endocytic factors and the reutilization of vesicles for a new cycle of secretion. Recruitment of synaptojanin is coupled to dynamin-dependent fission via its binding to the BAR domain–containing protein endophilin, a curvature-generating/sensing protein that also binds dynamin.The field grew very rapidly. It was found that reversible phosphorylation of phosphatidylinositol at the 3,4 and 5 positions of the inositol ring by a multiplicity of enzymes generates seven phosphoinositide species whose differential protein-binding specificities help control, often via a dual key mechanism (Wenk and De Camilli, 2004), membrane cytosol interfaces (Di Paolo and De Camilli, 2006; Vicinanza et al., 2008; Balla, 2013). Along with small GTPases (Behnia and Munro, 2005), phosphoinositides are key determinants of the identity of membranes or membrane subdomains and thus control the multiplicity of reactions that occur at membrane–cytosol interfaces, such as the recruitment and shedding of trafficking proteins, the function of integral membrane proteins, and the assembly and disassembly of signaling and cytoskeleton complexes. Importantly, it became clear that, as we had found for synaptojanin, interconversion of phosphoinositide species along the secretory and endocytic pathways functions as a switch to release membrane-associated factors that define one compartment and to recruit factors that define the next compartment (Odorizzi et al., 2000; Di Paolo and De Camilli, 2006; Zoncu et al., 2009). I think I would not have immediately grasped the significance of the identification of synaptojanin and then invested much of our work in this field had it not been for my early research experience in medical school.The roles of phosphoinositides in membrane identity imply the existence of mechanisms to tightly control their localizations and also to ensure availability of PI in membranes—primarily the plasma membrane—where the backbone of this lipid is consumed by PLC. Surprisingly, genetic evidence suggested that one mechanism to control PI4P levels in membranes is via their direct contacts with the endoplasmic reticulum (ER), where a main PI4P phosphatase is localized (Foti et al., 2001; Stefan et al., 2011; Mesmin et al., 2013). Concerning availability of PI, DAG generated by PLC and its phosphorylated downstream product phosphatidic acid must be returned to the ER for metabolic recycling by the ER-localized phosphatidylinositol synthase, and newly synthesized PI then needs to be rapidly transported to the plasma membrane to replace its depleted pool. Strong evidence indicates that these reactions are mediated at least in part by lipid transfer proteins, and we have now learned that much of this protein-mediated lipid transport occurs at membrane contact sites (Mesmin et al., 2013; Wong et al., 2017; Cockcroft and Raghu, 2018; Pemberton et al., 2020). This is what motivated my lab to enter the young field of membrane contact sites. Here, once again, my early appreciation of the importance of lipid transfer proteins during medical school had an impact on my decision to invest in this rapidly developing area of cell biology.Not only has it been rewarding to help expand the inventory of proteins known to function both as membrane tethers and as lipid transporters, and to elucidate their function and regulation (Giordano et al., 2013; Schauder et al., 2014; Chung et al., 2015; Dong et al., 2016; Saheki et al., 2016), but this has been an opportunity, in collaboration with my Yale colleague Karin Reinisch, to participate in the discovery of something unexpected and new in the biology of eukaryotic cells. Until recently, it was thought that nonvesicular lipid transport, including the transport occurring at membrane contact sites, occurred only via protein modules that function as shuttles between two bilayers, often acting as countertransporters, delivering different cargoes as they move back and forth between two closely apposed membranes. However, our investigations of VPS13, a protein originally identified in yeast for its function in membrane transport and subsequently linked to lipid dynamics (Lang et al., 2015; Park et al., 2016), raised the possibility that this protein, and thus also the closely related autophagy factor ATG2, may be the founding members of a protein superfamily with bulk lipid transport properties, that is, proteins that could facilitate the net fluxes of lipids between adjacent membranes, and thus in membrane expansion (Kumar et al., 2018). As was subsequently shown by structural studies (Valverde et al., 2019; Li et al., 2020), these rod-like proteins harbor a hydrophobic groove that spans their length, thus supporting a bridge-like model of lipid transport to support bulk lipid flow (Li et al., 2020; Guillen-Samander et al., 2021; Leonzino et al., 2021). Inspection of protein sequence and structure databases suggests the existence of several other proteins with these properties, implying that this mode of lipid transport (a bridge-like mechanism), previously described for the transport of lipids from the inner to the outer membrane of Gram-negative bacteria (Bishop, 2019), may be of broad relevance in cell biology. As mutations in VPS13 family proteins result in neurodegenerative diseases, including a Huntington-like disease and Parkinson’s disease (Ugur et al., 2020), this is a field rich in medical implications. In fact, my curiosity about disease mechanisms stemming from my medical school training contributed to making me specially interested in studying these proteins.It has been a wonderful journey, rich with twists and turns, exploring new territories and continuously discovering that there are untapped frontiers whose existence we still do not know about. During the course of my career, I found especially rewarding bringing together different fields and finding new connections between fundamental cell biology and medicine, more so in recent years, where genetic studies of diseases often provide the first clues to molecular and cellular mechanisms. As I have emphasized in this essay, each of our experiences, no matter how successful, becomes part of the body of knowledge that defines us and has an impact on our research trajectory. I typically encourage students to search for training opportunities in different settings rather than follow a linear path, as this will enable them to bring together in their independent careers the expertise and knowledge of different worlds and thus to carry out original science. Our specific and unique paths are what make our research innovative and special.     

Different doses of bone morphogenetic protein 4 promote the expression of early germ cell-specific gene in bone marrow mesenchymal stem cells

Bone morphogenetic protein (BMP)-4 has a crucial role on primordial germ cells (PGCs) development in vivo which can promote stem cell differentiation to PG-like cells. In this study, we investigated the expression of Mvh as one of the specific genes in primordial germ cells after treatment with different doses of BMP4 on bone mesenchymal stem cells (BMSCs)-derived PGCs. Following isolation of BMSCs from male mouse femur and tibia, cells were cultured in medium for 72 h. Passage 4 murine BMSCs were characterized by CD90, CD105, CD34, and CD45 markers and osteo-adipogenic differentiation. Different doses of BMP4 (0, 0.01, 0.1, 1, 5, 25, 50, and 100 ng/ml) were added to BMSCs for PGCs differentiation during 4-days culture. Viability percent, proliferation rates, and expression of Mvh gene were analyzed by RT-qPCR. Data analysis was done with ANOVA test. CD90⁺, CD105⁺, CD34⁻, and CD45⁻ BMSCs were able to differentiate to osteo-adipogenic lineages. The results revealed that proliferation rate and viability percent were raised significantly (p ≤ 0.05) by adding 1, 5, 25 ng/ml of BMP4 and there were decreased to the lowest rate after adding 100 ng/ml BMP4 (p ≤ 0.05). There were significant up regulation (p ≤ 0.05) in Mvh expression between 25, 50, and 100 ng/ml BMP4 with other doses. So the selective dose of BMP-4 for treatment during 4-day culture was 25 ng/ml. The results suggest that addition of 25 ng/ml BMP4 had the best effects based on gene-specific marker expression.     

A Computational Analysis of Localized Ca<Superscript>2+</Superscript>-Dynamics Generated by Heterogeneous Release Sites

We investigate the role of heterogeneous expression of IP₃R and RyR in generating diverse elementary Ca²⁺ signals. It has been shown empirically (Wojcikiewicz and Luo in Mol. Pharmacol. 53(4):656–662, 1998; Newton et al. in J. Biol. Chem. 269(46):28613–28619, 1994; Smedt et al. in Biochem. J. 322(Pt. 2):575–583, 1997) that tissues express various proportions of IP₃ and RyR isoforms and this expression is dynamically regulated (Parrington et al. in Dev. Biol. 203(2):451–461, 1998; Fissore et al. in Biol. Reprod. 60(1):49–57, 1999; Tovey et al. in J. Cell Sci. 114(Pt. 22):3979–3989, 2001). Although many previous theoretical studies have investigated the dynamics of localized calcium release sites (Swillens et al. in Proc. Natl. Acad. Sci. U.S.A. 96(24):13750–13755, 1999; Shuai and Jung in Proc. Natl. Acad. Sci. U.S.A. 100(2):506–510, 2003a; Shuai and Jung in Phys. Rev. E, Stat. Nonlinear Soft Matter Phys. 67(3 Pt. 1):031905, 2003b; Thul and Falcke in Biophys. J. 86(5):2660–2673, 2004; DeRemigio and Smith in Cell Calcium 38(2):73–86, 2005; Nguyen et al. in Bull. Math. Biol. 67(3):393–432, 2005), so far all such studies focused on release sites consisting of identical channel types. We have extended an existing mathematical model (Nguyen et al. in Bull. Math. Biol. 67(3):393–432, 2005) to release sites with two (or more) receptor types, each with its distinct channel kinetics. Mathematically, the release site is represented by a transition probability matrix for a collection of nonidentical stochastically gating channels coupled through a shared Ca²⁺ domain. We demonstrate that under certain conditions a previously defined mean-field approximation of the coupling strength does not accurately reproduce the release site dynamics. We develop a novel approximation and establish that its performance in these instances is superior. We use this mathematical framework to study the effect of heterogeneity in the Ca²⁺-regulation of two colocalized channel types on the release site dynamics. We consider release sites consisting of channels with both Ca²⁺-activation and inactivation (“four-state channels”) and channels with Ca²⁺-activation only (“two-state channels”) and show that for the appropriate parameter values, synchronous channel openings within a release site with any proportion of two-state to four-state channels are possible, however, the larger the proportion of two-state channels, the more sensitive the dynamics are to the exact spatial positioning of the channels and the distance between channels. Specifically, the clustering of even a small number of two-state channels interferes with puff/spark termination and increases puff durations or leads to a tonic response.     

BMP and BMP receptor expression during murine organogenesis

Cell–cell communication is critical for regulating embryonic organ growth and differentiation. The Bone Morphogenetic Protein (BMP) family of transforming growth factor β (TGFβ) molecules represents one class of such cell–cell signaling molecules that regulate the morphogenesis of several organs. Due to high redundancy between the myriad BMP ligands and receptors in certain tissues, it has been challenging to address the role of BMP signaling using targeting of single Bmp genes in mouse models. Here, we present a detailed study of the developmental expression profiles of three BMP ligands (Bmp2, Bmp4, Bmp7) and three BMP receptors (Bmpr1a, Bmpr1b, and BmprII), as well as their molecular antagonist (noggin), in the early embryo during the initial steps of murine organogenesis. In particular, we focus on the expression of Bmp family members in the first organs and tissues that take shape during embryogenesis, such as the heart, vascular system, lungs, liver, stomach, nervous system, somites and limbs. Using in situ hybridization, we identify domains where ligand(s) and receptor(s) are either singly or co-expressed in specific tissues. In addition, we identify a previously unnoticed asymmetric expression of Bmp4 in the gut mesogastrium, which initiates just prior to gut turning and the establishment of organ asymmetry in the gastrointestinal tract. Our studies will aid in the future design and/or interpretation of targeted deletion of individual Bmp or Bmpr genes, since this study identifies organs and tissues where redundant BMP signaling pathways are likely to occur.     

The promising effects of BMP2 transfected mesenchymal stem cells on human osteosarcoma

Selective targeting of transfected mesenchymal stem cells (MSCs) carrying specific antioncogenes to the tumor was suggested as a treatment option. Bone morphogenetic protein-2 (BMP2) was shown to inhibit the proliferation and aggressiveness of osteosarcoma (OS) cells. Here, we aimed to assess the homing efficiency of intraperitoneally administered hMSCs transfected with BMP2 to the tumoral site and their effects on OS using an orthotopic xenograft murine model. Orthotopic xenograft murine model of OS in six-week-old female NOD/SCID mice using 143B cells was established. hMSCs transfected with BMP2 (BMP2⁺hMSC) were used. In vivo experiments performed on four groups of mice that received no treatment, or intraperitoneally administered BMP2, hMSCs, and BMP2⁺hMSCs. Histopathological and immunohistochemical studies were used to evaluate the pathological identification and to assess the dimensions and necrotic foci of the tumor, the features of lung metastases, and immunostaining against p27, Ki-67, and caspase-3 antibodies. The osteogenic differentiation markers BMP2, BMP4, COL1A1, OPN, OCN and PF4 evaluated using RT-PCR. The tumor dimensions in the hMSCs group were significantly higher than those of the remaining groups (p < 0.01). The number of metastatic foci in the BMP2⁺hMSCs group was significantly lower than those of the other groups (p < 0.01). The current results showed that the intraperitoneal route could be efficiently used for targeting hMSCs to the tumoral tissues for effective BMP2 delivery. In this study, the effects of BMP2 transfected hMSCs on human OS and metastasis were promising for achieving osteogenic differentiation and reduced metastatic process.     

Discovery of keratin function and role in genetic diseases: the year that 1991 was

In 1991, a set of transgenic mouse studies took the fields of cell biology and dermatology by storm in providing the first credible evidence that keratin intermediate filaments play a unique and essential role in the structural and mechanical support in keratinocytes of the epidermis. Moreover, these studies intimated that mutations altering the primary structure and function of keratin filaments underlie genetic diseases typified by cellular fragility. This Retrospective on how these studies came to be is offered as a means to highlight the 25th anniversary of these discoveries.Although intermediate filaments (IFs) have been characterized at some level for a longer period of time (Oshima, 2007 ), they were officially discovered as such as recently as 1968 by Howard Holtzer and colleagues while studying the developing skeletal muscle (Ishikawa et al., 1968 ). The advent of gene cloning methods and monospecific antibody production in the late 1970s and throughout the 1980s led to an explosion of data and knowledge about IFs that established them as a large family of genes and proteins that are individually regulated in a tight and evolutionarily conserved tissue- and differentiation-specific manner. Researchers also uncovered some of the remarkable properties of IFs as purified elements in vitro and in living systems and recognized that they occur in the nucleus as well as in the cytoplasm. In spite of the fast pace of progress during that period, however, it was not possible to produce evidence that spoke unequivocally about the functional importance of IFs in cells and tissues, let alone their role in disease.Beginning in the mid- to late 1980s, pioneering experimentation along two distinct lines was underway in the laboratory of Elaine Fuchs, then at the University of Chicago. The eventual merger of these approaches yielded the first formal insight into IF function in vivo, as well as into their direct involvement in human disease. In an effort to define structure–function relationships with regard to the assembly and network formation properties of IFs, one such approach was the application of systematic deletion mutagenesis to keratin 14 (K14), a type I IF that is expressed with its type II partner keratin 5 (K5) in the progenitor basal layer of the epidermis and related complex epithelia. These studies demonstrated that deleting sequences from either end of the central α-helical rod domain of the K14 protein was deleterious for filament formation in a dominant manner both in transfected cells (Albers and Fuchs, 1987 , 1989 ; Figure 1) and the setting of IF polymerization assays involving purified proteins in vitro (e.g., Coulombe et al., 1990 ). The second key effort in the Fuchs lab in the late 1980s resulted in the demonstration that the proximal 2.5 kb and distal 700 base pairs corresponding respectively to the 5′ upstream and 3′ downstream regions of the cloned human K14 gene were sufficient to confer tissue-specific, that is, K14-like, regulation in transgenic mice in vivo (Vassar et al., 1989 ; Figure 1). This tour de force paved the way for the production of a human K14 gene promoter–based cassette (e.g., Saitou et al., 1995 ) that could reliably direct the expression of any open reading frame in a K14-like manner in transgenic mice. As an aside, this tool has had a profound effect on epithelial and skin biology research.Open in a separate windowFIGURE 1:Schematic representation of the strategy and outcome of the experiments that led to the discovery of keratin function and role in genetic disease. Original figures are reproduced to give a realistic account of the data. (A) Examples of a disrupted keratin filament network in cultured epithelial cells transfected with and expressing a dominantly acting K14 deletion mutant (arrows). (Reproduced from Albers and Fuchs, 1987 , with permission.) (B) Preferential expression of a substance P-epitope–tagged transgenic human K14 protein in the basal layer of tail skin epidermis in mouse, conveying the tissue- and differentiation-specific behavior of the transgene. (Reproduced from Vassar et al., 1989 , with permission.) (C) The two experimental approaches described in A and B were combined to assess the consequences of tissue-specific expression of dominantly acting K14 mutants in skin tissue in vivo. (D) Newborn mouse littermates. The mouse at the top is transgenic (Tg) and expresses a mutated form of K14 in the epidermis. It is showing severe skin blistering (arrows), particularly in its front paws, which are heavily used by mouse newborns to feed from their mother. The bottom mouse is a nontransgenic control showing no such blistering. (E, F) Hematoxylin-eosin–stained skin tissue sections showing the location of subepidermal cleavage within the epidermis of a K14 mutant–expressing transgenic mouse (opposing arrows in E). Cleavage occurs at the level of the basal layer, where the mutant keratin is expressed. Again, this is never seen in control wild-type (Wt) skin (F). Bar, 100 μm (E, F). (D–F are from Coulombe et al., 1991b , with permission.) (G) Leg skin in a patient suffering from the Dowling–Meara form of epidermolysis bullosa simplex. Characteristic of this severe variant of this disease, several skin blisters are often grouped in a herpetiform manner (Fine et al., 1991 ).Subsequent use of the human K14 promoter–based cassette to direct the expression of epitope-tagged and selected deletion mutants of K14 gave rise to transgenic mouse pups that exhibited extensive blistering of the skin preferentially at sites of frictional trauma (Coulombe et al., 1991b ; Vassar et al., 1991 ; Figure 1). Electron microscopy showed that skin blistering occurred secondary to a loss of the integrity of keratinocytes located in the basal layer of the epidermis, that is, the precise site of mutant K14 protein accumulation. Such blistering did not occur in transgenic mice expressing a full-length version of human K14 modified to carry only an epitope tag at the C-terminus at similar or higher levels (Coulombe et al., 1991b ; Vassar et al., 1991 ). In addition, the severity of skin blistering in mutant K14–expressing transgenic pups could be directly related to the extent to which the mutant protein had been shown to disrupt filament assembly in transfected cell assays and in IF reconstitution assays in vitro. For instance, tissue-specific expression of a K14 mutant that could severely disrupt 10-nm filament assembly was associated with whole-body skin blistering and the untimely death of mouse pups and, from a pathology perspective, with “tonofilament clumping” and a paucity of visible keratin IFs in transgenic basal keratinocytes. By comparison, expression of another K14 mutant with a less deleterious effect on 10-nm IF assembly was compatible with the survival of transgenic mouse pups and resulted in skin blistering largely limited to the front paws in newborn mice together with altered organization of keratin IFs in basal keratinocytes of transgenic epidermis in situ, albeit without tonofilament clumping. This initial set of mouse strains thus revealed the existence of a direct link between the so-called “genotype” (i.e., mutant K14 characteristics) and the skin phenotype (Coulombe et al., 1991b ; Vassar et al., 1991 ; Fuchs and Coulombe, 1992 ). Electrophoretic analyses of protein samples confirmed that the K14 mutant proteins acted dominantly to produce such spectacular phenotypes in transgenic mouse skin. Finally, blistering also occurred in the mutant K14–expressing transgenic mice in other stratified epithelia known both to express K14 and experience trauma, notably in the oral mucosa (Coulombe et al., 1991b ; Vassar et al., 1991 ).It is worth celebrating the 25th anniversary of these pioneering experiments for the following two reasons. First, the study of these mice provided the first formal demonstration that keratin IFs play a fundamentally important role in structural support in surface epithelia such as the epidermis and oral mucosa. Without proper IF support, epidermal keratinocytes are rendered fragile and cannot sustain trivial frictional stress (Coulombe et al., 1991b ; Fuchs and Coulombe, 1992 ). The second reason is the observation that the phenotype of these K14 mutant–expressing mice proved eerily similar to those of individuals afflicted with the disease epidermolysis bullosa simplex (EBS), a rare, dominantly inherited and debilitating skin condition in which the epidermis and oral mucosa undergo blistering after exposure to trivial mechanical trauma. As observed in the mouse model, tissue cleavage had been shown to result from the loss of integrity of keratinocytes located in the basal layer (Fine et al., 1991 ). Further, other researchers had previously reported on anomalies in the organization of keratin IFs in the basal epidermal keratinocytes of EBS patients (Anton-Lamprecht, 1983 ; Ito et al., 1991 ) or in cultures of epidermal keratinocytes established from EBS patients (Kitajima et al., 1989 ). The Fuchs laboratory thus teamed up with Amy Paller, a physician-scientist and pediatric dermatologist with deep expertise in genodermatoses, and mutations were soon discovered in the K14 gene of two independent and sporadic cases of a severe variant of the disease known as Dowling–Meara EBS (Coulombe et al., 1991a ; Figure 1). The two mutations were heterozygous missense alleles that affected the very same codon in K14 (Arg-125) and were correctly predicted at the time to correspond to a mutational hot spot in type I keratin genes. The mutations were shown to dominantly disrupt 10-nm IF assembly in vitro and/or in transfected keratinocytes in culture (Coulombe et al., 1991a ). Soon thereafter, a team led by Ervin Epstein at University of California, San Francisco (San Francisco, CA), reported on the use of classical linkage analysis to uncover a missense mutation in the K14 gene of a small pedigree with Koebner-type EBS, a less severe variant of the disease (Bonifas et al., 1991 ). The next year, Birgit Lane and colleagues (Lane et al., 1992 ) reported on the occurrence of mutations in keratin 5 (K5), the formal type II keratin assembly partner for K14 in vivo, in another instance of Dowling–Meara EBS.In the years since 1991, a role in structural support has been formally demonstrated for all classes of IFs (Coulombe et al., 2009 ), including the nuclear-localized lamins (e.g., Lammerding et al., 2004 ). Moreover, we now know of several hundred independent instances of mutations in either K5 or K14 in the setting of the EBS disease, with the vast majority of those consisting of dominantly acting missense alleles (Szeverenyi et al., 2008 ; Human Intermediate Filament Database, www.interfil.org, maintained at the Centre for Molecular Medicine and Bioinformatics Institute, Singapore). We also learned that, as anticipated, EBS largely represents a loss-of-function phenotype, since K14-null mice (Lloyd et al., 1995 ), K14-null individuals (Chan et al., 1994 ; Rugg et al., 1994 ), and K5-null mice (Peters et al., 2001 ) all exhibit an EBS-like skin-blistering phenotype (Coulombe et al., 2009 ). Mutations such as Arg125Cys in K14 markedly compromise the remarkable mechanical properties of keratin filaments (Ma et al., 2001 ), as well as the steady-state dynamics of keratin filaments in transfected keratinocytes in culture (Werner et al., 2004 ). Finally, mutations affecting the coding sequence of IF genes have been shown to underlie >100 diseases affecting the human population (Omary et al., 2004 ; Szeverenyi et al., 2008 ; www.interfil.org). Consistent with the exquisite tissue- and cell type–specific regulation of IF genes, these diseases collectively affect a myriad of tissues and organs and are relevant to nearly all branches of medicine. These observations attest to the importance and profound effect that the generation and characterization of mutant K14–expressing transgenic mice has had for cell biology, epithelial physiology, dermatology, and medicine.Many thoughts spring to mind when reminiscing about my involvement with this body of work. First, this effort was prescient of the power of team science and, in particular, of the potential effect of close collaborations involving biologists and physician-scientists. I learned a great deal and benefited immensely from working closely with many colleagues on this project, including Bob Vassar, Kathryn Albers, Linda Degenstein, Liz Hutton, Anthony Letai, Amy Paller, and, last but not least, my postdoctoral mentor and the laboratory head, Elaine Fuchs. Second, there is no substitute for elements such as innovation, hard work, perseverance, boldness, accountability, and great leadership. Elaine had the vision and created the exceptional circumstances necessary to make this set of discoveries possible, and, of equal importance, she was an integral part of the day-to-day progress and maturation of the entire project. Finally, as we all know, there is an intangible element of luck involved in discovery research. In this instance, a strong argument can be made that the studies highlighted here may not have had such a deep and defining effect had the effort been devoted to any IF other than the K5–K14 keratin pairing.What are some of the lingering issues regarding this specific topic that preoccupy us still, 25 years later? Two challenges loom particularly large. First, we have yet to achieve a satisfactory understanding of how mutations in keratin proteins can cause disease. This is due in part to the lack of an atomic-level understanding of the core structure of IFs (which has been a tough nut to crack; Lee et al., 2012 ), along with the reality that, for any relevant IF gene, there is a broad variety of disease-associated (mostly missense) mutations that pepper their primary structure (www.interfil.org). Second, we have yet to achieve success toward the treatment of EBS or any IF-based disorder. Disease characteristics such as low incidence, a dominantly inherited character, genetic heterogeneity (e.g., broad mutational landscape), and, in the case of EBS and related conditions, an intrinsically high rate of cell turnover within the main target tissue significantly add to the challenge of devising safe and effective therapeutic strategies (Coulombe et al., 2009 ). Although efforts are still underway to foster progress on these two challenging issues, the field as a whole has made significant progress in uncovering a plethora of noncanonical functions of keratin IFs (Hobbs et al., 2016 ) in addition to understanding their regulation, dynamics, and many remarkable properties.     

Identification and expression of an encoding steroid receptor coactivator (SRA) in amphioxus (Branchiostoma japonicum)

Steroid receptor coactivator (SRA), a class of genes encoding both functional RNA and protein, has been shown to be present in vertebrates but little is known in invertebrates. Here we isolated a cDNA encoding a SRA homolog from amphioxus Branchiostoma japonicum, named AmphiSRA. The cDNA contained a 525 bp open reading frame corresponding to a deduced protein of 174 amino acids with a predicted molecular mass of ~21 kDa. Phylogenetic analysis showed that AmphiSRA was located at the base of its vertebrate counterparts, suggesting that it represents the archetype of vertebrate SRA. The genomic DNA sequence of AmphiSRA contained four exons and three introns, which was similar to B. floridae SRA exon/intron organization. The recombinant SRAP expressed in vitro shows a band with a molecular mass of 21 kDa and western blot confirmed it, which proved it is an encoding isoform. AmphiSRA is found to display a tissue specific expression pattern, with a predominant expression in gill, intestine, testis, neural tube and notochord. The whole-mount in situ hybridization demonstrated the expression of AmphiSRA in all the stages of development assayed. These implicated that SRA maybe play an important role during embryonic development of cephalochordate amphioxus.     

BMP4 loss-of-function mutations in developmental eye disorders including SHORT syndrome

BMP4 loss-of-function mutations and deletions have been shown to be associated with ocular, digital, and brain anomalies, but due to the paucity of these reports, the full phenotypic spectrum of human BMP4 mutations is not clear. We screened 133 patients with a variety of ocular disorders for BMP4 coding region mutations or genomic deletions. BMP4 deletions were detected in two patients: a patient affected with SHORT syndrome and a patient with anterior segment anomalies along with craniofacial dysmorphism and cognitive impairment. In addition to this, three intragenic BMP4 mutations were identified. A patient with anophthalmia, microphthalmia with sclerocornea, right-sided diaphragmatic hernia, and hydrocephalus was found to have a c.592C>T (p.R198X) nonsense mutation in BMP4. A frameshift mutation, c.171dupC (p.E58RfsX17), was identified in two half-siblings with anophthalmia/microphthalmia, discordant developmental delay/postaxial polydactyly, and poor growth as well as their unaffected mother; one affected sibling carried an additional BMP4 mutation in the second allele, c.362A>G (p.H121R). This is the first report indicating a role for BMP4 in SHORT syndrome, Axenfeld?CRieger malformation, growth delay, macrocephaly, and diaphragmatic hernia. These results significantly expand the number of reported loss-of-function mutations, further support the critical role of BMP4 in ocular development, and provide additional evidence of variable expression/non-penetrance of BMP4 mutations.