Nail Gun Injury and Endovascular Repair of Cervical Internal Carotid Artery.

A male patient aged 49 years presented to the emergency room after sustaining a nail-gun injury to the left neck (Zone III). Computed tomography (CT) angiogram demonstrated retained nail traversing in close proximity to the left internal carotid artery. Catheter angiogram with three-dimensional (3D) reconstruction revealed partial left internal carotid injury without active extravasation and with preserved flow through the vessel. The nail was removed by gentle traction with the simultaneous deployment of stent-graft across the injured segment. Balloon angioplasty of the stent was performed secondary to endoleak and active extravasation. Complete vessel reconstruction with maintained blood flow was achieved. The patient was extubated the following day and was discharged home on hospital day five without neurological deficits. This case report demonstrates the usefulness of endovascular repair of high cervical arterial injuries with special attention to the unique nature of nail gun injuries.

A high content, small molecule screen identifies candidate molecular pathways that regulate rod photoreceptor outer segment renewal.

The outer segment of the vertebrate rod photoreceptor is a highly modified cilium composed of many discrete membranous discs that are filled with the protein machinery necessary for phototransduction. The unique outer segment structure is renewed daily with growth at the base of the outer segment where new discs are formed and shedding at the distal end where old discs are phagocytized by the retinal pigment epithelium. In order to understand how outer segment renewal is regulated to maintain outer segment length and function, we used a small molecule screening approach with the transgenic (hsp70:HA-mCherryTM) zebrafish, which expresses a genetically-encoded marker of outer segment renewal. We identified compounds with known bioactivity that affect five content areas: outer segment growth, outer segment shedding, clearance of shed outer segment tips, Rhodopsin mislocalization, and differentiation at the ciliary marginal zone. Signaling pathways that are targeted by the identified compounds include cyclooxygenase in outer segment growth, γ-Secretase in outer segment shedding, and mTor in RPE phagocytosis. The data generated by this screen provides a foundation for further investigation of the signaling pathways that regulate photoreceptor outer segment renewal.

Phosphodiesterase Inhibitors Sildenafil and Vardenafil Reduce Zebrafish Rod Photoreceptor Outer Segment Shedding.

Purpose: The vertebrate rod photoreceptor undergoes daily growth and shedding to renew the rod outer segment (ROS), a modified cilium that contains the phototransduction machinery. It has been demonstrated that ROS shedding is regulated by the light-dark cycle; however, we do not yet have a satisfactory understanding of the molecular mechanisms that underlie this regulation. Given that phototransduction relies on the hydrolysis of cGMP via phosphodiesterase 6 (PDE6), we examined ROS growth and shedding in zebrafish treated with cGMP-specific PDE inhibitors. Methods: We used transgenic zebrafish that express an inducible, transmembrane-bound mCherry protein, which forms a stripe in the ROS following a heat shock pulse and serves as a marker of ROS renewal. Zebrafish were reared in constant darkness or treated with PDE inhibitors following heat shock. Measurements of growth and shedding were analyzed in confocal z-stacks collected from treated retinas. Results: As in dark-reared zebrafish, shedding was reduced in larvae and adults treated with the PDE5/6 inhibitors sildenafil and vardenafil but not with the PDE5 inhibitor tadalafil. In addition, vardenafil noticeably affected rod inner segment morphology. The inhibitory effect of sildenafil on shedding was reversible with drug removal. Finally, cones were more sensitive than rods to the toxic effects of sildenafil and vardenafil. Conclusions: We show that pharmacologic inhibition of PDE6 mimics the inhibition of shedding by prolonged constant darkness. The data show that the influence of the light-dark cycle on ROS renewal is regulated, in part, by initiating the shedding process through activation of the phototransduction machinery.

Characterizing information decay in patient handoffs.

OBJECTIVE: The purpose of this study was to analyze the decay of information with multiple sequential patient handoff reports given by third-year medical students who have not had standardized patient handoff training. METHODS: We examine the information decay of quantitative parameters included in 2 different simulated patient history and physical handoffs conducted among third-year medical students. Both student self-surveys and third party observer surveys tracked accuracy of information. A total of 93 students were surveyed for the first patient scenario and 103 students were surveyed for second patient scenario. Survey data were aggregated into 2 separate spreadsheets, one for each patient scenario tested. A total of 16 data points pertaining to the checklist were analyzed for common trends in handoff accuracy and information decay. RESULTS: Quantitative analysis of information passed between handoffs showed that between the 2 case scenarios, there was a consistent loss of information between one presenter to the next. Overall, 33% of information was lost between the first and third handoffs. Within the progression of individual handoffs, a narrative decay was demonstrated. There was a regression in handoff accuracy, trending down to an average of only 45% of information being passed on successfully by the time each presenter reached the last piece of information in their patient presentation. When examining the survey data points that had greater than an 80% success rate of being included in the handoffs, there appeared to be no correlation between their inherent qualities. CONCLUSIONS: This study showed there is a significant decrease in accuracy of information during sequential patient handoff exercises. The information decay may be a result of time, memory, or relevance of the information to the student. Future studies incorporating teaching effective handoffs early in the clinical curriculum would be an area of future research.

Two types of transgenic lines for doxycycline-inducible, cell-specific gene expression in zebrafish ultraviolet cone photoreceptors.

Temporal and spatial control of gene expression is important for studying the molecular and cellular mechanisms of development, physiology, and disease. We used the doxycycline (Dox)-inducible, Tet-On system to develop transgenic zebrafish for inducible, cell specific control of gene expression in the ultraviolet (UV) cone photoreceptors. Two constructs containing the reverse tetracycline-controlled transcriptional transactivator (rtTA) gene driven by the UV opsin-specific promoter (opn1sw1) were used to generate stable transgenic zebrafish lines using the Tol2-based transgenesis method. One construct included a self-reporting GFP (opn1sw1:rtTA, TRE:GFP) and the other incorporated an epitope tag on the rtTA protein (opn1sw1:rtTA(flag)). UV cone-specific expression of TRE-controlled transgenes was induced by Dox treatment in larvae and adults. Induction of gene expression was observed in 96% of all larval UV cones within 16 h of Dox treatment. UV cone-specific expression of two genes from a bidirectional TRE construct injected into one-cell Tg(opn1sw1:rtTA(flag)) embryos were also induced by Dox treatment. In addition, UV cone-specific expression of Crb2a(IntraWT) was induced by Dox treatment in progeny from crosses of the TRE-response transgenic line, Tg(TRE:HA-Crb2a(IntraWT)), to the Tg(opn1sw1:rtTA, TRE:GFP) line and the Tg(opn1sw1:rtTA(flag)) line. These lines can be used in addition to the inducible, rod-specific gene expression system from the Tet-On Toolkit to elucidate the photoreceptor-specific effects of genes of interest in photoreceptor cell biology and retinal disease.

Two types of Tet-On transgenic lines for doxycycline-inducible gene expression in zebrafish rod photoreceptors and a gateway-based tet-on toolkit.

The ability to control transgene expression within specific tissues is an important tool for studying the molecular and cellular mechanisms of development, physiology, and disease. We developed a Tet-On system for spatial and temporal control of transgene expression in zebrafish rod photoreceptors. We generated two transgenic lines using the Xenopus rhodopsin promoter to drive the reverse tetracycline-controlled transcriptional transactivator (rtTA), one with self-reporting GFP activity and one with an epitope tagged rtTA. The self-reporting line includes a tetracycline response element (TRE)-driven GFP and, in the presence of doxycycline, expresses GFP in larval and adult rods. A time-course of doxycycline treatment demonstrates that maximal induction of GFP expression, as determined by the number of GFP-positive rods, is reached within approximately 24 hours of drug treatment. The epitope-tagged transgenic line eliminates the need for the self-reporting GFP activity by expressing a FLAG-tagged rtTA protein. Both lines demonstrate strong induction of TRE-driven transgenes from plasmids microinjected into one-cell embryos. These results show that spatial and temporal control of transgene expression can be achieved in rod photoreceptors. Additionally, system components are constructed in Gateway compatible vectors for the rapid cloning of doxycycline-inducible transgenes and use in other areas of zebrafish research.

Generation of a genetically encoded marker of rod photoreceptor outer segment growth and renewal.

Vertebrate photoreceptors are specialized light sensing neurons. The photoreceptor outer segment is a highly modified cilium where photons of light are transduced into a chemical and electrical signal. The outer segment has the typical cilary axoneme but, in addition, it has a large number of densely packed, stacked, intramembranous discs. The molecular and cellular mechanisms that contribute to vertebrate photoreceptor outer segment morphogenesis are still largely unknown. Unlike typical cilia, the outer segment is continuously regenerated or renewed throughout the life of the animal through the combined process of distal outer segment shedding and proximal outer segment growth. The process of outer segment renewal was discovered over forty years ago, but we still lack an understanding of how photoreceptors renew their outer segments and few, if any, molecular mechanisms that regulate outer segment growth or shedding have been described. Our lack of progress in understanding how photoreceptors renew their outer segments has been hampered by the difficulty in measuring rates of renewal. We have created a new method that uses heat-shock induction of a fluorescent protein that can be used to rapidly measure outer segment growth rates. We describe this method, the stable transgenic line we created, and the growth rates observed in larval and adult rod photoreceptors using this new method. This new method will allow us to begin to define the genetic and molecular mechanisms that regulate rod outer segment renewal, a crucial aspect of photoreceptor function and, possibly, viability.

Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size.

BACKGROUND: Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains. RESULTS: We examined the localization of Crb2a constructs and their effects on rod morphology. We found that only the full-length Crb2a protein approximated the normal localization of Crb2a protein apical to adherens junctions in the photoreceptor inner segment. Several Crb2a construct proteins localized abnormally to the outer segment and one construct localized abnormally to the cell body. Overexpression of full-length Crb2a greatly increased inner segment size while expression of several other constructs increased outer segment size. CONCLUSIONS: Our observations suggest that particular domains in Crb2a regulate its localization and thus may regulate its regionalized function. Our results also suggest that the PDZ-binding domain in Crb2a might bring a protein(s) into the Crb complex that alters the function of the FERM-binding domain.

Tissue-specific requirements for specific domains in the FERM protein Moe/Epb4.1l5 during early zebrafish development.

BACKGROUND: The FERM domain containing protein Mosaic Eyes (Moe) interacts with Crumbs proteins, which are important regulators of apical identity and size. In zebrafish, loss-of-function mutations in moe result in defects in brain ventricle formation, retinal pigmented epithelium and neural retinal development, pericardial edema, and tail curvature. In humans and mice, there are two major alternately spliced isoforms of the Moe orthologue, Erythrocyte Protein Band 4.1-Like 5 (Epb4.1l5), which we have named Epb4.1l5long and Epb4.1l5short, that differ after the FERM domain. Interestingly, Moe and both Epb4.1l5 isoforms have a putative C' terminal Type-I PDZ-Binding Domain (PBD). We previously showed that the N' terminal FERM domain in Moe directly mediates interactions with Crumbs proteins and Nagie oko (Nok) in zebrafish, but the function of the C'terminal half of Moe/Epb4.1l5 has not yet been examined. RESULTS: To define functionally important domains in zebrafish Moe and murine Epb4.1l5, we tested whether injection of mRNAs encoding these proteins could rescue defects in zebrafish moe- embryos. Injection of either moe or epb4.1l5long mRNA, but not epb4.1l5short mRNA, could rescue moe- embryonic defects. We also tested whether mRNA encoding C' terminal truncations of Epb4.1l5long or chimeric constructs with reciprocal swaps of the isoform-specific PBDs could rescue moe- defects. We found that injection of the Epb4.1l5short chimera (Epb4.1l5short+long_PBD), containing the PBD from Epb4.1l5long, could rescue retinal and RPE defects in moe- mutants, but not brain ventricle formation. Injection of the Epb4.1l5long chimera (Epb4.1l5long+short_PBD), containing the PBD from Epb4.1l5short, rescued retinal defects, and to a large extent rescued RPE integrity. The only construct that caused a dominant phenotype in wild-type embryos, was Epb4.1l5long+short_PBD, which caused brain ventricle defects and edema that were similar to those observed in moe- mutants. Lastly, the morphology of rod photoreceptors in moe- mutants where embryonic defects were rescued by moe or epb4.1l5long mRNA injection is abnormal and their outer segments are larger than normal. CONCLUSION: Taken together, the data reveal tissue specificity for the function of the PBD in Epb4.1l5long, and suggest that additional C' terminal sequences are important for zebrafish retinal development. Additionally, our data provide further evidence that Moe is a negative regulator of rod outer segment size.

Mosaic Eyes is a novel component of the Crumbs complex and negatively regulates photoreceptor apical size.

Establishment of apical-basal cell polarity has emerged as an important process during development, and the Crumbs complex is a major component of this process in Drosophila. By comparison, little is known about the role of Crumbs (Crb) proteins in vertebrate development. We show that the FERM protein Mosaic Eyes (Moe) is a novel regulatory component of the Crumbs complex. Moe coimmunoprecipitates with Ome/Crb2a and Nok (Pals1) from adult eye and in vitro interaction experiments suggest these interactions are direct. Morpholino knockdown of ome/crb2a phenocopies the moe mutations. Moe and Crumbs proteins colocalize apically and this apical localization requires reciprocal protein function. By performing genetic mosaic analyses, we show that moe- rod photoreceptors have greatly expanded apical structures, suggesting that Moe is a negative regulator of Crumbs protein function in photoreceptors. We propose that Moe is a crucial regulator of Crumbs protein cell-surface abundance and localization in embryos.

The FERM protein Yurt is a negative regulatory component of the Crumbs complex that controls epithelial polarity and apical membrane size.

The Crumbs (Crb) complex is a key regulator of epithelial cell architecture where it promotes apical membrane formation. Here, we show that binding of the FERM protein Yurt to the cytoplasmic domain of Crb is part of a negative-feedback loop that regulates Crb activity. Yurt is predominantly a basolateral protein but is recruited by Crb to apical membranes late during epithelial development. Loss of Yurt causes an expansion of the apical membrane in embryonic epithelia and photoreceptor cells similar to Crb overexpression and in contrast to loss of Crb. Analysis of yurt crb double mutants suggests that these genes function in one pathway and that yurt negatively regulates crb. We also show that the mammalian Yurt orthologs YMO1 and EHM2 bind to mammalian Crb proteins. We propose that Yurt is part of an evolutionary conserved negative-feedback mechanism that restricts Crb complex activity in promoting apical membrane formation.

Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes.

Podocytes are specialized cells of the kidney that form the blood filtration barrier in the kidney glomerulus. The barrier function of podocytes depends upon the development of specialized cell-cell adhesion complexes called slit-diaphragms that form between podocyte foot processes surrounding glomerular blood vessels. Failure of the slit-diaphragm to form results in leakage of high molecular weight proteins into the blood filtrate and urine, a condition called proteinuria. In this work, we test whether the zebrafish pronephros can be used as an assay system for the development of glomerular function with the goal of identifying novel components of the slit-diaphragm. We first characterized the function of the zebrafish homolog of Nephrin, the disease gene associated with the congenital nephritic syndrome of the Finnish type, and Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome. Zebrafish nephrin and podocin were specifically expressed in pronephric podocytes and required for the development of pronephric podocyte cell structure. Ultrastructurally, disruption of nephrin or podocin expression resulted in a loss of slit-diaphragms at 72 and 96 h post-fertilization and failure to form normal podocyte foot processes. We also find that expression of the band 4.1/FERM domain gene mosaic eyes in podocytes is required for proper formation of slit-diaphragm cell-cell junctions. A functional assay of glomerular filtration barrier revealed that absence of normal nephrin, podocin or mosaic eyes expression results in loss of glomerular filtration discrimination and aberrant passage of high molecular weight substances into the glomerular filtrate.

Potential roles for BMP and Pax genes in the development of iris smooth muscle.

The embryonic optic cup generates four types of tissue: neural retina, pigmented epithelium, ciliary epithelium, and iris smooth muscle. Remarkably little attention has focused on the development of the iris smooth muscle since Lewis ([1903] J. Am. Anat. 2:405-416) described its origins from the anterior rim of the optic cup neuroepithelium. As an initial step toward understanding iris smooth muscle development, I first determined the spatial and temporal pattern of the development of the iris smooth muscle in the chick by using the HNK1 antibody, which labels developing iris smooth muscle. HNK1 labeling shows that iris smooth muscle development is correlated in time and space with the development of the ciliary epithelial folds. Second, because neural crest is the only other neural tissue that has been shown to generate smooth muscle (Le Lievre and Le Douarin [1975] J. Embryo. Exp. Morphol. 34:125-154), I sought to determine whether iris smooth muscle development shares similarities with neural crest development. Two members of the BMP superfamily, BMP4 and BMP7, which may regulate neural crest development, are highly expressed by cells at the site of iris smooth muscle generation. Third, because humans and mice that are heterozygous for Pax6 mutations have no irides (Hill et al. [1991] Nature 354:522-525; Hanson et al. [1994] Nat. Genet. 6:168-173), I determined the expression of Pax6. I also examined the expression of Pax3 in the developing anterior optic cup. The developing iris smooth muscle coexpresses Pax6 and Pax3. I suggest that some of the eye defects caused by mutations in Pax6, BMP4, and BMP7 may be due to abnormal iris smooth muscle.

Zebrafish mosaic eyes is a novel FERM protein required for retinal lamination and retinal pigmented epithelial tight junction formation.

Polarization is a common feature of many types of cells, and we are beginning to understand how cells become polarized. The role of cell polarity in development and tissue morphogenesis, however, is much less well understood. Our previous analysis of the mosaic eyes (moe) mutations revealed that moe is required for retinal lamination and also suggested that zebrafish moe function is required in the retinal pigmented epithelium (RPE) for the proper localization of adjacent retinal cell divisions at the apical neuroepithelial surface. To understand the function of moe in the RPE, we cloned the moe locus and show that it encodes a novel FERM (for 4.1 protein, ezrin, radixin, moesin) domain-containing protein. Expression of moe in the eye, kidney, and brain reflects phenotypes found in moe(-) mutants, including RPE and retinal lamination defects, edema, and small or absent brain ventricles. We show that moe function is required for tight junction formation in the RPE. We suggest that moe may be a necessary component of the crumbs pathway that regulates apical cell polarity and also may play a role in photoreceptor morphogenesis.