Primitive ducts of renal dysplasia induced by culturing ureteral buds denuded of condensed renal mesenchyme.

Primitive ducts, the histological hallmark of human renal dysplasia, were induced in chick embryos by culturing ureteral buds denuded of condensed metanephrogenic mesenchyme.

Vertebrate regeneration system: culture in vitro.

With standard tissue-culture techniques and media, various components of the lizard tail regenerate have been maintained in culture for 8 months. Differentiation of two cell types, melanophores and striated muscle, has been obtained. Myoblast proliferation and fusion can be selectively controlled by altering the culture medium.

Frameshift mutations in the v-src gene of avian sarcoma virus act in cis to specifically reduce v-src mRNA levels.

A portion of the avian sarcoma virus (ASV) primary RNA transcripts is alternatively spliced in chicken embryo fibroblast cells to two different messages, the src and env mRNAs. Frameshift mutations of the viral genome causing premature translation termination within the src gene result in a decreased steady-state level of the src mRNA. In marked contrast, frameshift mutations at various positions of the env gene do not decrease the level of the env mRNA. We show that the src gene product is not required in trans for splicing and accumulation of src mRNA. Conversely, the truncated Src proteins do not act negatively in trans to decrease specifically the levels of src mRNA. Taken together, these results indicate that the frameshift mutations act in cis to reduce src mRNA levels. A double mutant with a lesion in the src initiator AUG and a frameshift within the src gene demonstrated wild-type RNA levels, indicating that the src mRNA must be recognized as a translatable mRNA for the effect on src mRNA levels to occur. Our results indicate that the reduced levels do not result from decreased cytoplasmic stability of the mature src mRNA. We also show that the src gene frameshift mutations affect src mRNA levels when expressed from intronless src cDNA clones. We conclude that the reduction of src mRNA levels triggered by the presence of frameshift mutations within the src gene occurs while it is associated with the nucleus. Our data also strongly suggest that this occurs at a step of RNA processing or transport independent of RNA splicing.

Proliferative and degenerative events in the early development of chick dorsal root ganglia. I. Normal development.

Development of the chick dorsal root ganglia was examined in 4.5- to 9.5-day embryos. Tritiated thymidine (3H-TdR) and autoradiography was used to analyze proliferative activity and the Feulgen procedure to analyze degenerative activity in ganglia 12-17. Proliferative activity was found to be elevated through 4.5 days of incubation when as many as 14% of the ganglionic cells become labelled following a one-hour exposure to 3H-TdR. By 6.5 to 7.5 days proliferative activity decreases to 2-4% in the lateroventral (LV) regions and to approximately 1% in the mediodorsal (MD) regions of the ganglia. However, there appears to be increased proliferative activity by the end of the experimental period at 9.5 days. Birthdate studies demonstrate that large-scale neuronal production occurs between 4.5 and 6.5 days in the LV regions and between 4.5 and 7.5 days in the MD regions. After those times ganglionic proliferative activity must be largely nonneuronal in nature. This nonneuronal proliferation is greater in LV than in MD regions and in brachial than in nonbrachial ganglia. Degenerative activiy was found to be absent from the ganglia until after 4.5 days of incubation. It then increases rapidly, and by 5.5 days 5% of the LV cells in nonbrachial ganglia are degenerating. Degenerative activity then declines but is still present at 9.5 days. In contrast to results of an earlier study (Hamburger and Levi-Montalcini, '49), degenerative activity was also found in the LV region of brachial ganglia and the MD regions of brachial and nonbrachial ganglia. The pattern of LV degenerative activity in brachial ganglia is similar to that in nonbrachial ganglia, but the level of activity is lower. In the MD regions degenerative activity increases throughout the experimental period, and by 9.5 days as many as 4% of the MD cells are degenerating.

Proliferative and degenerative events in the early development of chick dorsal root ganglia. II. Responses to altered peripheral fields.

Responses of chick embryo dorsal root ganglia to early wing bud amputation were examined histologically using tritiated thymidine (3H-TdR) and autoradiography to analyze proliferation and the Feulgen procedure to visualize degenerating cells. Right wing buds were amputated at stage 15 or 16. At 4.5 to 9.5 days of incubation embryos were given a 1-hour exposure to 3H-TdR and fixed. Feulgen-stained autoradiographs were examined for percentage of cells labelled (labelling index) or degenerating (degeneration index) in lateroventral (LV) and mediodorsal (MD) regions of brachial (G14-16) and nonbrachial (G12, 13, 17) ganglia. The earliest response to amputation was a highly significant increase in degeneration indices of LV and MD regions of ipsilateral brachial ganglia at 5.5 days. Significant brachial LV responses were observed throughout the remainder of the experimental period. Two peaks occur in this response: at 5.5 days, corresponding to the peak seen in normal nonbrachial ganglia, and at 8.5 days, having no counterpart in normal development. In brachial MD regions significant degenerative responses occur at most times examined. Significant responses also occur at 7.5 and 8.5 days in MD regions of nonbrachial ganglia. The presence of MD responses in our material indicates that maturation of at least some MD neurons occurs earlier than previously thought. Significant labelling responses occur in brachial LV regions from 7.5 days on. Because other studies (Carr and Simpson, '78a) show that this time is after the end of large-scale neuronal production, this labelling response must be nonneuronal in nature. We conclude that this response is a secondary response to amputation, consequent to the greatly increased cellular degeneration. Results of experiments involving addition of limb buds at the brachial level are also presented.

Origin of spinal cord axons in the lizard regenerated tail: supernormal projections from local spinal neurons.

During tail regeneration most lizards also regenerate the tail spinal cord. The regenerated spinal cord primarily contains neuroepithelium (i.e., the ependymal tube which forms the central canal) and descending axons. The present experiments identify the source of the axons in the regenerated spinal cord. Application of HRP to normal tail spinal cord resulted in labeled cells in the nucleus paraventricularis, the interstitial nucleus of the fasciculus longitudinalis medialis, the nucleus ruber, the medullary reticular formation (including raphe nuclei), as well as in vestibular nuclei. HRP applied to the regenerated spinal cord labeled only 4% of the cells seen in normal animals, and these were confined to rhombencephalic nuclei. The lack of labeling of more rostral nuclei was not due to the death of descending neurons. Application of HRP immediately rostral to the regenerated spinal cord resulted in the labeling of a normal, and in some cases, greater than normal, number of neurons. To quantify the origin of axons in the regenerated spinal cord, electron microscopic montages of the regenerated spinal cord were made and the number of axons counted, before and after various spinal lesions. Only lesions within one spinal segment of the regenerated spinal cord had a significant effect on the number of axons in the regenerated tail spinal cord. This indicated that most of the regenerated axons were of local spinal origin. A significant increase in the number of labeled local spinal neurons was revealed following application of HRP to a regenerated tail spinal cord. These results suggest that while various portions of the lizard central nervous system can grow axons into the regenerating tail spinal cord, the great majority of axons in the regenerate are of local origin and that some of these arise from neurons that do not normally possess descending projections. Finally, to test whether new neurons were participating in the regeneration process, 3H-thymidine was injected during the regrowth of the tail. No labeled spinal cord cells were conclusively identified as neurons. Thus, the regenerating lizard tail spinal cord exhibits robust axonal sprouting from neurons near the site of a spinal transection in a manner reminiscent of sprouting in the mammalian CNS. This sprouting can develop into descending spinal projections that extend for significant distances into the regenerated tail spinal cord and provides a unique model for exploring the requirements for successful axon growth in an adult vertebrate CNS.

Axonal sprouting and frank regeneration in the lizard tail spinal cord: correlation between changes in synaptic circuitry and axonal growth.

In our previous studies, we found that the number of supraspinal neurons projecting to the level of tail spinal cord increases by 74% during tail regeneration and that the number of local spinal neurons with descending projections increases 233%. However, only a small fraction of the supraspinal axons (less than 4%) and half of the local spinal axons actually enter the regenerated spinal cord. We suggested that this may be the result of "synaptic capture" in which regrowing axons make synapses on denervated targets rostral to the transection, aborting further regeneration. To examine this hypothesis, morphometric analysis of electron microscope (EM) photomontages was used to test for changes in synaptic distribution on ventral horn neurons rostral to regenerating tail spinal cord. In addition, 3H-thymidine and retrograde markers were used to determine whether the regenerate axons arose from cut axons, neurogenesis, or sprouting from uninjured neurons. 3H-thymidine injections during regeneration, combined with retrograde HRP pathway tracing, did not reveal the production of new neurons in the tail spinal cord. To test whether cut axons regenerate, fluorescein isothiocyanate conjugated latex beads were applied to the exposed end of the tail spinal cord. After tail regeneration, HRP was applied to the new spinal cord in the regenerated tail. Examination of local spinal neurons (the primary source of axons that enter the regenerated tail spinal cord) revealed that 28% of the neurons contained both labels. This indicated that cut axons successfully regrew into the new tail spinal cord. The regenerated axons that fail to enter the new tail spinal cord can be found in the normal spinal cord immediately rostral to the regenerated tail. To determine whether these axons were making synaptic contacts, lamina IX ventral horn neurons were examined. EM photomontages of the spinal cord rostral to the regenerate tail revealed the following properties: (1) neurons rostral to regenerated tails are larger in area compare to non-regenerates (mean increase = 112%); (2) axosomatic contacts cover a greater percentage of the neuronal soma following regeneration compared to normal (mean increase = 23%); and (3) this increased innervation is the result of an increase in the number of synaptic boutons rather than larger boutons. The number of synaptic contacts in regenerated lizards returned to normal following lumbar transection, indicating that supraspinal and/or long descending propriospinal afferents were the major source of the increased synaptic contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid appearance of labeled degenerating cells in the dorsal root ganglia after exposure of chick embryos to tritiated thymidine.

The interval between [3H]thymidine delivery and onset of cellular degeneration in 5.5 day embryonic chick brachial dorsal root ganglia was examined autoradiographically. Of the degenerating cells, 14% were labeled by 2 h after [3H]thymidine delivery. This percentage increased for 24 h. Wing bud amputation had no effect on this percentage through 9 h. Thus, some cause of cell death other than faulty peripheral connections may exist in at least some degenerating ganglionic cells.

Monoclonal antibody-based enzyme-linked immunoassays for the measurement of palytoxin in biological samples.

Mouse monoclonal and rabbit polyclonal antibodies were produced against conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Palytoxin haptens were produced by derivatization of the primary amino group with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or succinimidyl 3-(2-pyridyldithio)propionate. Selected antibodies were used to develop five palytoxin-specific enzyme-linked immunoassay formats for the quantitation of palytoxin in biological matrices, including crude extracts of Palythoa tuberculosa. The formats developed include an indirect competitive inhibition enzyme-linked immunoassay, two types of direct competitive inhibition enzyme-linked immunoassays, and both indirect and direct sandwich enzyme-linked immunosorbent assays. The sandwich enzyme-linked immunosorbent assays are capable of detecting as little as 10 pg palytoxin per test, but may be subject to matrix interference. The direct competitive inhibition enzyme-linked immunoassays detect as little as 30 pg palytoxin per test with a total assay time of only 4 hr. The enzyme-linked immunoassays do not cross-react with the other marine toxins tested, but do cross-react with certain non-toxic, treated preparations of palytoxin. The enzyme-linked immunoassays were used to quantitate palytoxin in P. tuberculosa extracts and to monitor toxin isolation. These enzyme-linked immunoassay systems can substitute for the mouse bioassay of palytoxin, providing a rapid, sensitive, and accurate means of toxin detection.

The impact of an intensive multisensory reading program on a population of learning-disabled delinquents.

The high prevalence of learning disabilities in the juvenile delinquent population has been well documented, but attempts to remediate and have an impact on recidivism of this population of delinquents has produced limited results. The present study is a replication of the remediation phase of the 1976 LD/JD Project with methodological refinements to control for treatment integrity and strength of treatment. Delinquents in two detention facilities were screened for a developmental reading disorder. Subjects were selected for the study based on normal intelligence, full English proficiency, and a discrepancy of 15 points between reading achievement and IQ. Subjects in the treatment group received 90 minutes of remedial reading instruction per day using a multisensory (Orton/Gillingham) approach. A comparison group received 45 minutes of daily reading instruction in the regular classroom. There was no significant difference between the two groups in mean age of first arrest, mean age, and mean hours of reading instruction. Based on pre- and posttesting in reading and arrest records one year following release, the treatment group made significantly greater growth in reading (.33 year growth vs -.05 year growth per 10 hours of instruction) and had a significantly lower rate of recidivism (41 percent vs 63 percent) than the comparison group. Results were discussed in terms of hours of instruction necessary to improve reading, intervening treatment variables, and cost effectiveness of remedial program.

Changes in cell surface antigens during in vitro lizard myogenesis.

Indirect immunofluorescence has been used to examine surface antigens of lizard myogenic cells during in vitro differentiation. At least two developmental stage-specific surface alterations have been identified. One of these is a compositional change and involves the appearance of a cell-surface antigen(s) as the cells differentiate. This antigen(s) (Ag1422) is muscle specific and is characteristic of some rounded-up G0 myosin-positive myocytes, all stretched-back, G0 myosin-positive myocytes, and all identifiable myotubes. The antigen is not found on proliferating myoblasts, extended G1 (myosin-negative) cell-cycle-competent myoblasts or newly differentiated rounded-up, G0 myosin-positive myocytes. Pretreatment of cells with neuraminidase, trypsin, or proteinase K indicates the antigen is not present in "masked" form on normally nonreactive cells. Proteinase K is effective in the removal or destruction of the antigen, indicating it is at least partially protein in nature. The antigen is expressed in a similar developmental stage-specific fashion on early-passage myogenic cells taken from both adult lizard tail regenerates and embryonic muscle. The antibodies identifying Ag1422 can be removed by adsorption with homogenates of mature skeletal muscle. Therefore, Ag1422 is not an artifact due to in vitro conditions or the expression of a transformation antigen unique to the continuous culture line. The second alteration is an apparent restriction in the mobility of surface components (antigens and lectin receptors). Upon treatment with multivalent ligands, undifferentiated myosin-negative myoblasts exhibit rapid patching and capping of cell surface components while well-differentiated myocytes and myotubes do not. This mobility restriction is evident after the appearance of Ag1422. Treatment with cytochalasin B (15 micrograms/ml) and/or colchicine (100 microM) does not alter the restricted mobility of surface components seen on differentiated cells. Therefore, neither microfilaments nor microtubules seem to be involved in the mobility restriction. These observations are discussed in relation to current views of myogenesis.

In vivo test system for tumor production by cell lines derived from lower vertebrates.

The immune suppressed lizard, Anolis carolinensis, can be used to test for in vivo tumor production by cell lines derived from a variety of ectothermic vertebrates. Cell lines tested for tumor production were also assessed for loss of attachment-dependent proliferation and contact inhibition of cell overlap. The results demonstrate that the criteria standardly used to assess transformation and neoplastic change in cultured mammalian cells apply equally well to cultured cells from ectotherms.

3H-GABA administration during tail regeneration of lizards and autoradiographical localization.

Thirteen lizards (Anolis carolinensis and Scincella lateralis) were injected with 3H-GABA (tritiated gamma-aminobutyric acid) in order to study its localization inside the regenerating tail spinal cord (SC) by means of light and electron microscopic autoradiography. One and three hours after intraperitoneal injection, the radioactivity was essentially localized in regenerating nerves and pale cells, mostly contacting the central canal of the SC as typical Cerebrospinal-Fluid-Contacting-Neurons (CSFCNs). A smaller number of pale cells did not take up 3H-GABA and some of them appeared to be degenerating. Weaker radioactivity in regenerating SC was observed 6 hours post injection. Among non neural tissues, cells of the regenerating blastema, of the meninges and capillaries surrounding the regenerating SC, showed lower 3H-GABA uptake. This indicates that a certain amount of injected 3H-GABA has been converted to other metabolites. No 3H-GABA uptake was detected 1 or 6 hours post injection in normal SC and brain both in grey and white matter. This research indicates that CSFCNs of the regenerating tail in lizards uptake the aminoacid GABA or derived aminoacids, suggesting these cells subserve a high metabolic activity and may be GABA-ergic

Inhibition of RNA splicing at the Rous sarcoma virus src 3' splice site is mediated by an interaction between a negative cis element and a chicken embryo fibroblast nuclear factor.

In permissive Rous sarcoma virus-infected chicken embryo fibroblasts (CEF), approximately equimolar amounts of env and src mRNAs are present. In nonpermissive mammalian cells, the src mRNA level is elevated and env mRNA level is reduced. A cis element in the region between the env gene and the src 3' splice site, which we have termed the suppressor of src splicing (SSS), acts specifically in CEF but not in human cells to reduce src mRNA levels. The splicing inhibition in CEF is not caused by a base-paired structure which is predicted to form between the SSS and the src 3' splice site. To further investigate the mechanism of the inhibition, we have used human HeLa cell nuclear extracts to compare in vitro the rates of splicing of RNA substrates containing the Rous sarcoma virus major 5' splice site and either the env or src 3' splice sites. We show that the src 3' splice site is used approximately fivefold more efficiently than the env 3' splice site. The efficiency of in vitro splicing at the src 3' splice site is specifically reduced by addition of CEF nuclear extract. The inhibition is dependent on the presence of the SSS element and can be abrogated by addition of competitor RNA. We propose that the SSS region represents a binding site for a negative-acting CEF splicing factor(s).

Selection and characterization of replication-competent revertants of a Rous sarcoma virus src gene oversplicing mutant.

All retroviruses require both unspliced and spliced RNA for a productive infection. One mechanism by which Rous sarcoma virus achieves incomplete splicing involves suboptimal env and src 3' splice sites. We have previously shown that mutagenesis of the nonconsensus src polypyrimidine tract to a 14-nucleotide uninterrupted polypyrimidine tract results in an oversplicing phenotype and a concomitant defective replication in permissive chicken embryo fibroblasts. In this report, we show that splicing at the src 3' splice site (3'ss) is further negatively regulated by the suppressor of src splicing cis element which is located approximately 100 nucleotides upstream of the src 3'ss. The increase in splicing at the src 3'ss results in a corresponding increase in splicing at a cryptic 5'ss within the env gene. Two classes of replication-competent revertants of the src oversplicing mutant (pSAP1) were produced after infection, and these mutants were characterized by molecular cloning and sequence analysis. Class I revertants are transformation-defective revertants in which the src 3'ss and the src gene are deleted by homologous recombination at several different sites within the imperfect direct repeat sequences that flank the src gene. Cells infected with these transformation-defective revertants produce lower levels of virus particles than cells infected with the wild-type virus. Class II revertants bear small deletions in the region containing the branchpoint sequence or polypyrimidine tract of the src 3'ss. Insertion of these mutated sequences into pSAP1 restored inefficient splicing at the src 3'ss and efficient replication in chicken embryo fibroblasts. All of these mutations caused reduced splicing at the src 3'ss when they were tested in an in vitro splicing system. These results indicate that maintenance of a weak src 3'ss is necessary for efficient Rous sarcoma virus replication.

Bulbospinal and intraspinal connections in normal and regenerated salamander spinal cord.

The salamander is the only limbed adult vertebrate which can regenerate portions of cervical, thoracic, or lumbar spinal cord. While the salamander has been a popular model for regeneration of the spinal cord, it is still not known what portions of the nervous system participate in the regeneration process. In the experiments reported here we examine the bulbospinal and intraspinal projections to the lumbar spinal cord in normal and regenerated salamanders (Notophthalmus viridescens). HRP application to the lumbar enlargement of normal salamanders labeled cells in the ventral thalamus, the rostral tegmentum in the proposed homolog of the red nucleus, the reticular neurons of the rhombencephalon, and the midline regions of the rhombencephalon which are possibly equivalent to raphe nuclei of other vertebrates. In the brachial spinal cord HRP-labeled cells were located in dorsal, intermediate, and ventral regions of the spinal gray matter and tended to be located at the periphery of the gray matter. To examine the spinal circuitry of regenerated salamanders, animals received complete spinal transections at the junction of the thoracic and lumbar spinal cord, abolishing all spontaneous coordinated hindlimb and tail movements. Animals exhibited walking and swimming within 60 days at which time a pledget of HRP was inserted into a gap in the spinal cord made by a transection 10.0 mm (six animals) or 5.0 mm (one animal) caudal to the first lesion. On average, the number of HRP labeled brain stem neurons in regenerated animals was 40% of that found in normal animals. The number of labeled cells in the brachial spinal cord was within the range of normal animals.(ABSTRACT TRUNCATED AT 250 WORDS)