Epidural extension failure in obese women is comparable to that of non-obese women.

BACKGROUND: Management of labor epidurals in obese women is difficult and extension to surgical anesthesia is not always successful. Our previous retrospective pilot study found epidural extension was more likely to fail in obese women. This study used a prospective cohort to compare the failure rate of epidural extension in obese and non-obese women and to identify risk factors for extension failure. METHODS: One hundred obese participants (Group O, body mass index ≥ 40 kg/m2 ) were prospectively identified and allocated two sequential controls (Group C, body mass index ≤ 30 kg/m2 ). All subjects utilized epidural labor analgesia and subsequently required anesthesia for cesarean section. The primary outcome measure was failure of the labor epidural to be used as the primary anesthetic technique. Risk factors for extension failure were identified using Chi-squared and logistic regression. RESULTS: The odds ratio (OR) of extension failure was 1.69 in Group O (20% vs. 13%; 95% CI: 0.88-3.21, P = 0.11). Risk factors for failure in obese women included ineffective labor analgesia requiring anesthesiologist intervention, (OR 3.94, 95% CI: 1.16-13.45, P = 0.028) and BMI > 50 kg/m2 (OR 3.42, 95% CI: 1.07-10.96, P = 0.038). CONCLUSION: The failure rate of epidural extension did not differ significantly between the groups. Further research is needed to determine the influence of body mass index > 50 kg/m2 on epidural extension for cesarean section.

Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion.

Cancer cell vascular invasion is a crucial step in the malignant progression toward metastasis. Here we used a genome-wide RNA interference screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these Krtaps have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/ß4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a Krtap can serve as a switch toward malignant progression.

Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling.

Metastasis of cancer cells involves multiple steps, including their dissociation from the primary tumor and invasion through the endothelial cell barrier to enter the circulation and finding their way to distant organ sites where they extravasate and establish metastatic lesions. Deficient contact inhibition is a hallmark of invasive cancer cells, yet surprisingly the vascular invasiveness of commonly studied cancer cell lines is regulated by the density at which cells are propagated in culture. Cells grown at high density were less effective at invading an endothelial monolayer than cells grown at low density. This phenotypic difference was also observed in a zebrafish model of vascular invasion of cancer cells after injection into the yolk sac and extravasation of cancer cells into tissues from the vasculature. The vascular invasive phenotypes were reversible. A kinome-wide RNA interference screen was used to identify drivers of vascular invasion by panning small hairpin RNA (shRNA) library-transduced noninvasive cancer cell populations on endothelial monolayers. The selection of invasive subpopulations showed enrichment of shRNAs targeting the large tumor suppressor 1 (LATS1) kinase that inhibits the activity of the transcriptional coactivator yes-associated protein (YAP) in the Hippo pathway. Depletion of LATS1 from noninvasive cancer cells restored the invasive phenotype. Complementary to this, inhibition or depletion of YAP inhibited invasion in vitro and in vivo. The vascular invasive phenotype was associated with a YAP-dependent upregulation of the cytokines IL6, IL8 and C-X-C motif ligand 1, 2 and 3. Antibody blockade of cytokine receptors inhibited invasion and confirmed that they are rate-limiting drivers that promote cancer cell vascular invasiveness and could provide therapeutic targets.

Small molecule inhibitors of ezrin inhibit the invasive phenotype of osteosarcoma cells.

Ezrin is a multifunctional protein that connects the actin cytoskeleton to the extracellular matrix through transmembrane proteins. High ezrin expression is associated with lung metastasis and poor survival in cancer. We screened small molecule libraries for compounds that directly interact with ezrin protein using surface plasmon resonance to identify lead compounds. The secondary functional assays used for lead compound selection included ezrin phosphorylation as measured by immunoprecipitation and in vitro kinase assays, actin binding, chemotaxis, invasion into an endothelial cell monolayer, zebrafish and Xenopus embryonic development, mouse lung organ culture and an in vivo lung metastasis model. Two molecules, NSC305787 and NSC668394, that directly bind to ezrin with low micromolar affinity were selected based on inhibition of ezrin function in multiple assays. They inhibited ezrin phosphorylation, ezrin-actin interaction and ezrin-mediated motility of osteosarcoma (OS) cells in culture. NSC305787 mimicked the ezrin morpholino phenotype, and NSC668394 caused a unique developmental defect consistent with reduced cell motility in zebrafish. Following tail vein injection of OS cells into mice, both molecules inhibited lung metastasis of ezrin-sensitive cells, but not ezrin-resistant cells. The small molecule inhibitors NSC305787 and NSC668394 demonstrate a novel targeted therapy that directly inhibits ezrin protein as an approach to prevent tumor metastasis.

The STAT3 inhibitor NSC 74859 is effective in hepatocellular cancers with disrupted TGF-beta signaling.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide, with few effective therapeutic options for advanced disease. At least 40% of HCCs are clonal, potentially arising from STAT3+, NANOG+ and OCT3/4+ liver progenitor/stem cell transformation, along with inactivation of transforming growth factor-beta (TGF-beta) signaling. Here we report significantly greater signal transducer and activator of transcription 3 (STAT3) and tyrosine phosphorylated STAT3 in human HCC tissues (P<0.0030 and P<0.0455, respectively) than in human normal liver. Further, in HCC cells with loss of response to TGF-beta, NSC 74859, a STAT3-specific inhibitor, markedly suppresses growth. In contrast, CD133(+) status did not affect the response to STAT3 inhibition: both CD133(+) Huh-7 cells and CD133(-) Huh-7 cells are equally sensitive to NSC 74859 treatment and STAT3 inhibition, with an IC(50) of 100 muM. Thus, the TGF-beta/beta2 spectrin (beta2SP) pathway may reflect a more functional 'stem/progenitor' state than CD133. Furthermore, NSC 74859 treatment of Huh-7 xenografts in nude mice significantly retarded tumor growth, with an effective dose of only 5 mg/kg. Moreover, NSC 74859 inhibited tyrosine phosphorylation of STAT3 in HCC cells in vivo. We conclude that inhibiting interleukin 6 (IL6)/STAT3 in HCCs with inactivation of the TGF-beta/beta2SP pathway is an effective approach in management of HCCs. Thus, IL6/STAT3, a major signaling pathway in HCC stem cell renewal and proliferation, can provide a novel approach to the treatment of specific HCCs.

The return of glomerular filtered albumin to the rat renal vein--the albumin retrieval pathway.

BACKGROUND: Recent studies have demonstrated that the normal glomerular capillary wall (GCW) is not charge selective to albumin. This means that albumin flux across the GCW is high. This has been confirmed in studies where albumin uptake by the tubules has been inhibited. Therefore, there must be a high capacity postglomerular retrieval pathway in normal kidneys that returns filtered albumin back to the blood supply. METHODS: This study identifies the presence of glomerular filtered albumin in the renal vein from the analysis of the decrease of radioactivity in the venous effluent after the injection of a pulse of tritium labeled albumin into the renal artery in vivo and in the isolated perfused kidney (IPK). RESULTS: The glomerular filtered albumin is returned to the blood supply by a high capacity pathway that transports this albumin at a rate of 1830+/-292 microg/min rat kidney (n= 14) (mean+/-SEM). This pathway has been identified under physiological conditions in vivo and in the IPK. The pathway is specific for albumin as it does not occur for horseradish peroxidase (HRP). The pathway is inhibited in a non-filtering kidney. The pathway is also inhibited by NH4Cl, an inhibitor of protein uptake. CONCLUSIONS: The high capacity retrieval pathway for albumin is most likely associated with transtubular cell transport. It is also apparent that most albuminuric states could be accounted for by the malfunctioning of this pathway without resorting to any change in glomerular permselectivity.

Minimally invasive subclavian/axillary artery to coronary artery bypass (SAXCAB): review and classification.

BACKGROUND AND PURPOSE: Subclavian/axillary artery to coronary artery bypass (SAXCAB) surgery is defined as a minimally (or less) invasive coronary revascularization procedure where one or more grafts are anastomosed to the second or third parts of the subclavian artery or any of the three parts of the axillary artery (inflow source) and attached to one or more coronary arteries, and where there are two separate minimally invasive incisions to expose the target coronary artery and the inflow sources, respectively. The indications and contraindications for SAXCAB surgery are discussed, and the relevant chest wall anatomy and that of the subclavian and axillary arteries are reviewed. The effect of respiration and anatomic variability as they impact the SAXCAB graft are discussed. Three components of the anatomy that are important in SAXCAB surgery are discussed: The relation of the first rib to the clavicle insofar as it affects access to the third part of the subclavian artery, the anatomy of the subclavian and axillary arteries and their branches, and the anatomy of the chest wall and its movement. In addition, the different SAXCAB variations that have been applied clinically are reviewed and classified, and future aspects of SAXCAB research are discussed. SAXCAB surgery is unique among the different types of minimally invasive direct coronary artery bypass (MIDCAB) surgery because of the enormous diversity of the techniques that have been described. Based on these descriptions, a new classification of SAXCAB grafting is proposed depending on whether the graft is inside or outside the rib cage and whether or not the coronary artery is exposed by rib resection or through an intercostal space. The third part of the classification takes into consideration the mode of entry into the chest, whether it is by rib resection or through an intercostal space. METHODS: Inquiries were made by telephone and by mail in the year 2000 to a number of surgeons who had published details of their SAXCAB techniques, and informal information was obtained by a series of personal communications as to the estimated number of operations they had performed and the outcomes. Published data was also used to formulate a rough guide as to the international status of the procedure at this time. RESULTS: The total estimated international experience is about 100 cases and the patency is between 70 and 100 percent in the time frame of about one to two years. CONCLUSIONS: The MIDCAB technique in general has been successful in providing an alternative way to revascularize the coronary arteries, and the SAXCAB has proved to be one of the most interesting classes of MIDCAB surgery. SAXCAB grafts seem to be unique among coronary revascularization procedures and, indeed, probably almost all vascular procedures, in that there is enormous diversity in the route for the graft from the inflow source to the target coronary artery. Being knowledgeable about the different varieties of SAXCAB surgeries will help the surgeon during a rescue operation as the surgery can be tailored to suit a particular patient. The SAXCAB seems to be a very safe operation, and it is striking that so far no one has reported any major complications.

Chronic hypoosmolality induces a selective decrease in magnocellular neurone soma and nuclear size in the rat hypothalamic supraoptic nucleus.

The magnocellular neurones of the hypothalamo-neurohypophysial system (HNS) play a vital role in the maintenance of body homeostasis by regulating oxytocin (OT) and vasopressin (VP) secretion from the posterior pituitary. During hyperosmolality, OT and VP mRNA levels are known to increase by approximately two-fold, whereas during chronic hypoosmolality, OT and VP mRNA levels decrease to approximately 10-20% of basal levels. In these studies, we evaluated changes in cell size associated with these physiological conditions. Cell and nuclear sizes of neurones in the supraoptic nucleus (SON), the nucleus of the lateral olfactory tract (LOT) and the medial habenular nucleus (MHB) were measured from neurones identified by in situ hybridization histochemistry for beta(III)-tubulin mRNA, and measurements were made from OT and AVP magnocellular neurones in the SON after phenotypic identification by immunohistochemistry. Under hypoosmolar conditions, the cell and nuclear sizes of OT and VP magnocellular neurones decreased to approximately 60% of basal values, whereas cell and nuclear sizes of OT and VP neurones in hyperosmolar rats increased to approximately 170% of basal values. In contrast, neither hyperosmolality, nor hypoosmolality significantly affected cell and nuclear sizes in the LOT and MHB. These results confirm previous studies that showed that magnocellular neurones increase cell size in response to hyperosmolar conditions and, for the first time, demonstrate a marked decrease in cell size in the SON in response to chronic hypoosmolar conditions. These dramatic changes in cell and nuclear size directly parallel changes in OT and VP gene expression in the magnocellular neurones of the SON and, consequently, are consistent with the pronounced bidirectional changes in gene expression and cellular activity found during these osmotic perturbations. Our results therefore support the concept of global alterations in the synthetic activity of magnocellular OT and AVP neurones in response to extracellular osmolality.

Gene expression in the rat supraoptic nucleus induced by chronic hyperosmolality versus hyposmolality.

Magnocellular neurons of the hypothalamo-neurohypophysial system play a fundamental role in the maintenance of body homeostasis by secreting vasopressin and oxytocin in response to systemic osmotic perturbations. During chronic hyperosmolality, vasopressin and oxytocin mRNA levels increase twofold, whereas, during chronic hyposmolality, these mRNA levels decrease to 10-20% of that of normoosmolar control animals. To determine what other genes respond to these osmotic perturbations, we have analyzed gene expression during chronic hyper- versus hyponatremia. Thirty-seven cDNA clones were isolated by differentially screening cDNA libraries that were generated from supraoptic nucleus tissue punches from hyper- or hyponatremic rats. Further analysis of 12 of these cDNAs by in situ hybridization histochemistry confirmed that they are osmotically regulated. These cDNAs represent a variety of functional classes and include cytochrome oxidase, tubulin, Na(+)-K(+)-ATPase, spectrin, PEP-19, calmodulin, GTPase, DnaJ-like, clathrin-associated, synaptic glycoprotein, regulator of GTPase stimulation, and gene for oligodendrocyte lineage-myelin basic proteins. This analysis therefore suggests that adaptation to chronic osmotic stress results in global changes in gene expression in the magnocellular neurons of the supraoptic nucleus.

Single cell reverse transcription-polymerase chain reaction analysis of rat supraoptic magnocellular neurons: neuropeptide phenotypes and high voltage-gated calcium channel subtypes.

Magnocellular neurosecretory cells (MNCs) in the hypothalamo-neurohypophysial system that express and secrete the nonapeptides oxytocin (OT) and vasopressin (VP) were evaluated for the expression of multiple genes in single magnocellular neurons from the rat supraoptic nucleus using a single cell RT-PCR protocol. We found that all cells representing the two major phenotypes, the OT and VP MNCs, express a small, but significant, amount of the other nonapeptide's messenger RNA (mRNA). In situ hybridization histochemical analyses confirmed this observation. A third phenotype, containing equivalent amounts of OT and VP mRNA, was detected in about 19% of the MNCs from lactating female supraoptic nuclei. Analyses of these phenotypes for other coexisting peptide mRNAs (e.g. CRH, cholecystokinin, galanin, dynorphin, and the calcium-binding protein, calbindin) generally confirmed expectations from the literature, but revealed cell to cell variation in their coexpression. Our results also show that the high voltage-activated calcium channel subunit genes, alpha1A-D, alpha2, and beta1-4 are expressed in virtually all MNCs. However, the alpha1E subunit gene is not expressed at detectable levels in these cells. The expression of all of the beta-subunit genes in each MNC may account for the variations in physiological and pharmacological properties of the high voltage-activated channels found in these neurons. (Endocrinology 140: 5391-5401, 1999)

The return of glomerular-filtered albumin to the rat renal vein.

BACKGROUND: Recent studies have demonstrated that the normal glomerular capillary wall (GCW) is not charge selective to albumin. This means that albumin flux across the GCW is high, and this has been confirmed in studies in which albumin uptake by the tubules has been inhibited. Therefore, there must be a high-capacity postglomerular retrieval pathway in normal kidneys that returns filtered albumin back to the blood supply. METHODS: This study identifies the presence of glomerular-filtered albumin in the renal vein from the analysis of the decrease of radioactivity in the venous effluent after the injection of a pulse of tritium-labeled albumin into the renal artery in vivo and in the isolated perfused kidney. RESULTS: The postglomerular filtered albumin is returned to the blood supply by a high-capacity pathway that transports this albumin at a rate of 1830 +/- 292 micrograms/min.rat kidney (N = 14, mean +/- SEM). This pathway has been identified under physiological conditions in vivo and in the isolated perfused kidney. The pathway is specific for albumin, as it does not occur for horseradish peroxidase. The pathway is inhibited in a nonfiltering kidney. The pathway is also inhibited by ammonium chloride (an agent that inhibits tubular protein uptake but does not alter glomerular size selectivity) and by albumin peptides (which compete for the tubular albumin receptor). CONCLUSIONS: The high-capacity retrieval pathway for albumin is most likely associated with transtubular cell transport. It is also apparent that most albuminuric states could be accounted for by the malfunctioning of this pathway without resorting to any change in glomerular permselectivity.

Restricted expression of the homeobox gene prox 1 in developing zebrafish.

Prox 1 is a vertebrate homeobox gene which is homologous to the Drosophila transcription factor, prospero. We have isolated a prox 1 cDNA from zebrafish, which encodes a protein that has 82%, 84% and 83% amino acid identity with chicken, mouse and human Prox 1, respectively. Antibodies raised against human Prox 1 cross-react with zebrafish Prox 1 and are used here to determine the expression patterns of Prox 1 during zebrafish embryogenesis by whole-mount immunohistochemistry. In the 10-somite embryo, Prox 1 is expressed over the prospective lens placode and over a broad region of epithelium extending from the eye to the otic vesicle. As embryogenesis proceeds, Prox 1 expression in the eye lens becomes intense, and is detected in maturing muscle pioneer cells and superficial muscle cells. In the CNS, Prox 1 is expressed in a stripe along the forebrain-midbrain boundary, in a segmented pattern in the ventral hindbrain, and in selected cells of the ventral spinal cord. Additional sites of Prox 1 expression include the lateral line primordium, the trigeminal ganglia, the otic vesicle and occasional endodermal cells.

Restricted expression of the neuronal intermediate filament protein plasticin during zebrafish development.

In the adult goldfish visual pathway, expression of the neuronal intermediate filament (nIF) protein plasticin is restricted to differentiating retinal ganglion cells (RGCs) at the margin of the retina. Following optic nerve injury, plasticin expression is elevated transiently in all RGCs coincident with the early stages of axon regeneration. These results suggest that plasticin may be expressed throughout the nervous system during the early stages of axonogenesis. To test this hypothesis, we analyzed plasticin expression during zebrafish (Danio rerio) neuronal development. By using immunocytochemistry and in situ hybridization, we found that plasticin is expressed in restricted subsets of early zebrafish neurons. Expression coincides with axon outgrowth in projection neurons that pioneer distinct axon tracts in the embryo. Plasticin is expressed first in trigeminal, Rohon-Beard, and posterior lateral line ganglia neurons, which are among the earliest neurons to initiate axonogenesis in zebrafish. Plasticin is expressed also in reticulospinal neurons and in caudal primary motoneurons. Together, these neurons establish the first behavioral responses in the embryo. Plasticin expression also coincides with initial RGC axonogenesis and progressively decreases after RGC axons reach the tectum. At later developmental stages, plasticin is expressed in a subset of the cranial nerves. The majority of plasticin-positive neurons are within or project axons to the peripheral nervous system. Our results suggest that plasticin subserves the changing requirements for plasticity and stability during axonal outgrowth in neurons that project long axons.

Vsx-1 and Vsx-2: two Chx10-like homeobox genes expressed in overlapping domains in the adult goldfish retina.

The genetic linkages of the murine ocular retardation mutation with the Chx10 gene and the murine small eye mutation with the Pax-6 gene has demonstrated the importance of Paired class homeobox genes in the development of the mammalian retina. Previously, we identified a Paired-class homeobox gene, Vsx-1, whose expression in the adult goldfish retina is restricted to the inner nuclear layer (INL) and to postmitotic, differentiating progenitor cells in the growth zone at the retinal peripheral margin, where neurogenesis continues throughout life. Here, we report the molecular cloning and expression pattern of a new Paired class homeobox gene, Vsx-2, in the adult goldfish retina. Like Vsx-1, Vsx-2 expression is highly restricted to the retina in the adult goldfish and overlaps with Vsx-1 expression in the mature INL. At the peripheral margin, Vsx-2 is expressed in mitotically active neuronal progenitors and is downregulated as these cells become postmitotic and begin to differentiate. Comparison of the amino acid sequences of Vsx-2, Vsx-1, Chx10, and C. elegans ceh-10 reveal a conserved homeodomain and a unique domain termed the CVC domain. The similarities of the Vsx-2, Vsx-1, and Chx10 expression patterns suggest that genes containing the CVC domain have conserved functions during retinal development in vertebrates.

Neuronal and neuroendocrine expression of lim3, a LIM class homeobox gene, is altered in mutant zebrafish with axial signaling defects.

LIM class homeobox genes code for a family of transcriptional regulators that encode important determinants of cell lineage and cell type specificity. The lim3 gene from the zebrafish, Danio rerio, is highly conserved in sequence and expression pattern compared to its homologs in other vertebrates. In this paper we report immunocytochemical analysis of Lim3 protein expression in the pituitary, pineal, hindbrain, and spinal cord of the embryo, revealing an asymmetrical, lateral and late program of pituitary development in zebrafish, distinct from the pattern in other vertebrates. We studied Lim3 expression in no tail, floating head, and cyclops mutant embryos, all of which have midline defects, with special reference to spinal cord differentiation where Lim3 marks mostly motoneurons. cyclops embryos showed essentially normal Lim3 expression in the hindbrain and spinal cord despite the absence of the floor plate, while no tail mutant embryos, which lack a differentiated notochord, displayed an excess of Lim3-expressing cells in a generally normal pattern. In contrast, Lim3-positive cells largely disappeared from the posterior spinal cord in floating head mutants, except in patches that correlated with remnants of apparent floor plate cells. These results support the view that either notochord or floor plate signaling can specify Lim3-positive motoneurons in the spinal cord.

Motoneuron fate specification revealed by patterned LIM homeobox gene expression in embryonic zebrafish.

In zebrafish, individual primary motoneurons can be uniquely identified by their characteristic cell body positions and axonal projection patterns. The fate of individual primary motoneurons remains plastic until just prior to axogenesis when they become committed to particular identities. We find that distinct primary motoneurons express particular combinations of LIM homeobox genes. Expression precedes axogenesis as well as commitment, suggesting that LIM homeobox genes may contribute to the specification of motoneuronal fates. By transplanting them to new spinal cord positions, we demonstrate that primary motoneurons can initiate a new program of LIM homeobox gene expression, as well as the morphological features appropriate for the new position. We conclude that the patterned distribution of different primary motoneuronal types within the zebrafish spinal cord follows the patterned expression of LIM homeobox genes, and that this reflects a highly resolved system of positional information controlling gene transcription.

Plasticin, a newly identified neurofilament protein, is preferentially expressed in young retinal ganglion cells of adult goldfish.

The adult goldfish retina and optic nerve display continuous growth, plasticity, and the capacity to regenerate throughout the animal's life. The intermediate filament proteins in this pathway are different from those in adult mammalian nerves, which do not continuously grow or normally regenerate. One novel intermediate filament protein of the goldfish visual pathway is plasticin, which is synthesized in ganglion cells and transported into the optic nerve. Using specific polyclonal antibodies raised against a plasticin fusion protein, we investigated the distribution of this protein in the normal retina and nerve and in the retina and nerve following optic nerve crush. In the normal pathway, plasticin was localized predominantly to the axons of very young ganglion cells; however, there was considerable immunoreactivity in older axons as they approach the chiasm. In addition, following optic nerve crush, all ganglion cell somata and their axons proximal to the crush site became equally immunoreactive. The results suggest that plasticin may contribute to axonal growth, plasticity, and regeneration.

Restricted expression of a new paired-class homeobox gene in normal and regenerating adult goldfish retina.

We describe the cloning and expression pattern of a new paired-class homeobox gene, Vsx-1, in the continuously growing retina of the goldfish. Vsx-1 belongs to a subset of paired-class homeobox genes that lack a second DNA binding domain, the paired-domain, and is closely related to the C. elegans ceh-10 gene. In the adult goldfish, Vsx-1 expression is restricted to the neural retina. In the central, mature retina, Vsx-1 mRNA is synthesized in a subset of differentiated cells in the inner nuclear layer in a pattern suggestive of bipolar cells. In immature retina, adjacent to the retinal margin, Vsx-1 is expressed in a relatively broader subset of newly postmitotic cells but is downregulated in some of these cells to form the mature expression pattern. Following retinal injury, during the early phase of regeneration, Vsx-1 mRNA synthesis appears to be upregulated in cells in the inner nuclear layer and is expressed de novo in cells outside this layer. By virtue of its identity as a transcriptional regulatory gene and its patterns of expression, we speculate that Vsx-1 may stabilize the differentiated state of a subset of cells in the inner nuclear layer and may be involved in cellular differentiation during retinal development and regeneration.

Cloning of multiple forms of goldfish vimentin: differential expression in CNS.

In efforts to determine the primary structure of intermediate filament proteins in the goldfish visual pathway, we isolated clones from a retinal lambda gt11 cDNA expression library that represent goldfish vimentin. We show that there are at least two forms of goldfish vimentin, designated as vimentin alpha and vimentin beta. RNase protection assays indicate that vimentin alpha mRNA is expressed in low amounts in retina, optic nerve, and brain and in higher amounts in spinal cord. In contrast, vimentin beta mRNA is expressed in low amounts in retina, optic nerve, brain, and spinal cord and in very high amounts in eye lens. Immunohistochemical studies show that in the optic nerve, vimentin alpha is mainly restricted to blood vessels, meninges, and septa. Light staining is observed with this antibody in an astrocytic glial pattern throughout the optic nerve. Two-dimensional gel analysis shows that all of these goldfish vimentins are low abundant components of optic nerve cytoskeletal preparations.