The Tomato Fruit Cell Wall : II. Polyuronide Metabolism in a Nonsoftening Tomato Mutant

A nonsoftening tomato (Lycopersicon esculentum L.) variety, dg, was examined to assess the physiological basis for its inability to soften during ripening. Total uronic acid levels, 18 milligrams uronic acid/100 milligrams wall, and the extent of pectin esterification, 60 mole%, remained constant throughout fruit development in this mutant. The proportion of uronic acid susceptible to polygalacturonase in vitro also remained constant. Pretreatment of heat-inactivated dg fruit cell walls with tomato pectinmethylesterase enhances polygalacturonase susceptibility at all ripening stages. Pectinesterase activity of cell wall protein extracts from red ripe dg fruit was half that in extracts from analogous tissue of VF145B. Polygalacturonase activities of cell wall extracts, however, were similar in both varieties. Diffusion of uronic acid from tissue discs of both varieties increased beginning at the turning stage to a maximum of 2.0 milligrams uronic acid released/gram fresh weight at the ripe stage. The increased quantity of hydrolytic products released during ripening suggests the presence of in situ polygalacturonase activity. Low speed centrifugation was employed to induce efflux of uronide components from the cell wall tree space. In normal fruit, at the turning stage, 2.1 micrograms uronic acid/gram fresh weight was present in the eluant after 1 hour, and this value increased to a maximum of 8.2 micrograms uronic acid/gram fresh weight at the red ripe stage. However, centrifuge-aided extraction of hydrolytic products failed to provide evidence for in situ polygalacturonase activity in dg fruit. We conclude that pectinesterase and polygalacturonase enzymes are not active in situ during the ripening of dg fruit. This could account for the maintenance of firmness in ripe fruit tissue.     

Increase in acid phosphatase activity in the fat body during larval and pharate pupal development in Calliphora erythrocephala

Biochemical evidence was obtained for an increase in acid phosphatase activity in the larval fat body of Calliphora erythrocephala during larval and pharate pupal instars. This observation is in conflict with published data indicating a decreasing enzyme activity in late third stage larvae. Centrifugation and filtration studies showed that the pH of the homogenisation medium has a strong influence on the solubilisation of acid phosphatase and its distribution in homogenate components. Differences in biochemical techniques including the pH value may explain the discrepancy between the published results and the present findings.The observed increase in acid phosphatase activity is related to the activity of the lysosomal system in the period immediately preceding pupal-adult apolysis.     

Hydrolysis of juvenile hormone in diapausing and non-diapausing larvae of the southwestern corn borer,Diatraea grandiosella

Summary The role of juvenile hormone (JH) esterases in relation to the diapause state of the southwestern corn borer,Diatraea grandiosella, was examined. The facultative larval diapause of this insect is dependent upon the presence of JH. Plasma, fat body, midgut, and body wall extracts metabolized [³H]JH I and [³H]JH III to JH-acid in vitro. JH-diol, JH-acid-diol, or conjugated polar metabolites were not detected. A longer half life of [³H]JH I was found in vitro in the plasma of diapausing larvae than in that of non-diapausing larvae. Although JH hydrolytic activity was relatively low in the plasma of pre-diapausing and diapausing larvae, systematic changes were observed suggesting that JH esterases may be involved in regulating the JH titer during this period. The JH hydrolytic activity found in the plasma of diapausing larvae was 3 to 5 times lower than that found in the plasma of mid-last instar non-diapausing larvae. Gel filtration profiles obtained from the plasma of diapausing and non-diapausing larvae suggested that JH esterases and -naphthyl-acetate esterases are different enzymes. Multiple overlapping peaks of JH hydrolytic activity with an apparent molecular weight range of 43,000 to 75,000 were detected, whereas 2 separate peaks of -naphthyl-acetate hydrolytic activity (apparent mol. wt. ca. 54,000, and 120,000) were detected. Gel filtration of supernatants of fat body indicated that JH was hydrolyzed at a lower rate by the fat body of pre-diapausing larvae than by that of non-diapausing larvae.     

The induction of lysosomal enzyme activity in the fat body of Calliphora erythrocephala: Changes in the internal environment

The activity of the lysosomal marker enzyme acid phosphatase in the larval fat body of Calliphora erythrocephala increases during development, but not at the same rate throughout the tissue. During the feeding stage, the posterior region has a higher acid phosphatase activity than the anterior region. When the larvae cease feeding on the 5th day of development, the acid phosphatase activity of the inactive anterior lobe increases rapidly in a mosaic-cell pattern. When 4-day-old feeding stage larvae are starved, this increase occurs one day earlier than normally. After the emptying of the gut, the acid phosphatase activity of all the anterior cells both in normal and in starved larvae exceeds that of those in the posterior region.Transplantation experiments indicate that the induction of acid phosphatase activity in the fat body during normal development, especially in the anterior region, is caused by a change in the internal environment when the larvae cease feeding. Both RNA and protein synthesis are involved in this induction process. Inductive factors are present in 5-day-old larvae as well as during formation of the puparium. The competence of the feeding-stage fat cells to develop high acid phosphatase activity is acquired before the actual induction takes place.     

Juvenile hormone acid synthesis and HMG-CoA reductase activity in corpora allata of Manduca sexta prepupae

Corpora allata (CA) of last instar larvae of Manduca sexta switch from juvenile hormone (JH) to JH acid secretion just before the onset of wandering behavior. JH acid secretion peaked during the prepupal period and ceased prior to pupal ecdysis. HMG-CoA reductase activity also peaked during the prepupal period and then declined. However, substantial enzyme activity was present in pupal and pharate adult glands. Removal of the brain at the wandering stage caused a reduction in JH acid secretion by prepupal CA. The profile of HMG-CoA activity in CA of debrained larvae resembled that of sham-operated larvae except that the prepupal peak was smaller than in control larvae. Addition of brain extracts to CA maintained in vitro neither stimulated not inhibited JH acid secretion and HMG-CoA reductase activity. It is suggested that the brain regulates CA activity in post-wandering stages via intact nerves.     

Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages

Moderate lysosomal membrane permeabilization (LMP) is an important inducer of apoptosis. Macrophages are professional scavengers and are rich in hydrolytic enzymes and iron. In the present study, we found that LMP by lysosomotropic detergent MSDH resulted in early up-regulation of lysosomal cathepsins, oxidative stress and ferritin up-regulation, and cell death. Lysosomotropic base NH₄Cl reduced the ferritin induction and oxidative stress in apoptotic cells induced by MSDH. Cysteine cathepsin inhibitors significantly protected cell death and oxidative stress, but had less effect on ferritin induction. We conclude that oxidative stress induced by lysosomal rupture causes ferritin induction with concomitant mitochondrial damage, which are the potential target for prevention of cellular oxidative stress and cell death induced by typical lysosomotropic substances in different disorders.     

Siramesine triggers cell death through destabilisation of mitochondria,but not lysosomes

A sigma-2 receptor agonist siramesine has been shown to trigger cell death of cancer cells and to exhibit a potent anticancer activity in vivo. However, its mechanism of action is still poorly understood. We show that siramesine can induce rapid cell death in a number of cell lines at concentrations above 20 μM. In HaCaT cells, cell death was accompanied by caspase activation, rapid loss of mitochondrial membrane potential (MMP), cytochrome c release, cardiolipin peroxidation and typical apoptotic morphology, whereas in U-87MG cells most apoptotic hallmarks were not notable, although MMP was rapidly lost. In contrast to the rapid loss of MMP above 20 μM siramesine, a rapid increase in lysosomal pH was observed at all concentrations tested (5–40 μM); however, it was not accompanied by lysosomal membrane permeabilisation (LMP) and the release of lysosomal enzymes into the cytosol. Increased lysosomal pH reduced the lysosomal degradation potential as indicated by the accumulation of immature forms of cysteine cathepsins. The lipophilic antioxidant α-tocopherol, but not the hydrophilic antioxidant N-acetyl-cysteine, considerably reduced cell death and destabilisation of mitochondrial membranes, but did not prevent the increase in lysosomal pH. At concentrations below 15 μM, siramesine triggered cell death after 2 days or later, which seems to be associated with a general metabolic and energy imbalance due to defects in the endocytic pathway, intracellular trafficking and energy production, and not by a specific molecular event. Overall, we show that cell death in siramesine-treated cells is induced by destabilisation of mitochondria and is independent of LMP and the release of cathepsins into the cytosol. Moreover, it is unlikely that siramesine acts exclusively through sigma-2 receptors, but rather through multiple molecular targets inside the cell. Our findings are therefore of significant importance in designing the next generation of siramesine analogues with high anticancer potential.     

Mitochondrial and lysosomal pathways of lamprey (Lampetra fluviatilis L.) hepatocyte death

Mechanisms of mitochondrial and lysosomal pathways of natural death of lamprey hepatocytes are described at the spring period of the prespawn migration. The mitochondrial pathway (release of cytochrome c from mitochondria into cytosol and activation of caspases) is realized by the classic scheme of apoptosis. Comparatively recently, the lysosomal pathway of cell death associated with cathepsin B activation has been revealed in cells in pathologies, specifically in obstruction of gallbladder and bile ducts. A peculiarity of lamprey hepatocytes consists in that in the adult animal liver there takes place biliary atresia (the absence of gallbladder and bile ducts. Thereby, the lamprey hepatocytes are an excellent object for study of this new pathway of cell death. We have revealed development of the mitochondrial and the lysosomal pathways of cell death of lamprey hepatocytes.     

Lysosome biogenesis mediated by vps-18 affects apoptotic cell degradation in Caenorhabditis elegans

Appropriate clearance of apoptotic cells (cell corpses) is an important step of programmed cell death. Although genetic and biochemical studies have identified several genes that regulate the engulfment of cell corpses, how these are degraded after being internalized in engulfing cell remains elusive. Here, we show that VPS-18, the Caenorhabditis elegans homologue of yeast Vps18p, is critical to cell corpse degradation. VPS-18 is expressed and functions in engulfing cells. Deletion of vps-18 leads to significant accumulation of cell corpses that are not degraded properly. Furthermore, vps-18 mutation causes strong defects in the biogenesis of endosomes and lysosomes, thus affecting endosomal/lysosomal protein degradation. Importantly, we demonstrate that phagosomes containing internalized cell corpses are unable to fuse with lysosomes in vps-18 mutants. Our findings thus provide direct evidence for the important role of endosomal/lysosomal degradation in proper clearance of apoptotic cells during programmed cell death.     

Autophagy impairment as a key feature for acetaminophen-induced ototoxicity

Macroautophagy/autophagy is a highly conserved self-digestion pathway that plays an important role in cytoprotection under stress conditions. Autophagy is involved in hepatotoxicity induced by acetaminophen (APAP) in experimental animals and in humans. APAP also causes ototoxicity. However, the role of autophagy in APAP-induced auditory hair cell damage is unclear. In the present study, we investigated autophagy mechanisms during APAP-induced cell death in a mouse auditory cell line (HEI-OC1) and mouse cochlear explant culture. We found that the expression of LC3-II protein and autophagic structures was increased in APAP-treated HEI-OC1 cells; however, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence, and the activity of lysosomal enzymes decreased in APAP-treated HEI-OC1 cells. The degradation of p62 protein and the expression of lysosomal enzymes also decreased in APAP-treated mouse cochlear explants. These data indicate that APAP treatment compromises autophagic degradation and causes lysosomal dysfunction. We suggest that lysosomal dysfunction may be directly responsible for APAP-induced autophagy impairment. Treatment with antioxidant N-acetylcysteine (NAC) partially alleviated APAP-induced autophagy impairment and apoptotic cell death, suggesting the involvement of oxidative stress in APAP-induced autophagy impairment. Inhibition of autophagy by knocking down of Atg5 and Atg7 aggravated APAP-induced ER and oxidative stress and increased apoptotic cell death. This study provides a better understanding of the mechanism responsible for APAP ototoxicity, which is important for future exploration of treatment strategies for the prevention of hearing loss caused by ototoxic medications.Subject terms: Macroautophagy, Hair cell     

Emergence of Motor Circuit Activity

In the developing nervous system, ordered neuronal activity patterns can occur even in the absence of sensory input and to investigate how these arise, we have used the model system of the embryonic chicken spinal motor circuit, focusing on motor neurons of the lateral motor column (LMC). At the earliest stages of their molecular differentiation, we can detect differences between medial and lateral LMC neurons in terms of expression of neurotransmitter receptor subunits, including CHRNA5, CHRNA7, GRIN2A, GRIK1, HTR1A and HTR1B, as well as the KCC2 transporter. Using patch-clamp recordings we also demonstrate that medial and lateral LMC motor neurons have subtly different activity patterns that reflect the differential expression of neurotransmitter receptor subunits. Using a combination of patch-clamp recordings in single neurons and calcium-imaging of motor neuron populations, we demonstrate that inhibition of nicotinic, muscarinic or GABA-ergic activity, has profound effects of motor circuit activity during the initial stages of neuromuscular junction formation. Finally, by analysing the activity of large populations of motor neurons at different developmental stages, we show that the asynchronous, disordered neuronal activity that occurs at early stages of circuit formation develops into organised, synchronous activity evident at the stage of LMC neuron muscle innervation. In light of the considerable diversity of neurotransmitter receptor expression, activity patterns in the LMC are surprisingly similar between neuronal types, however the emergence of patterned activity, in conjunction with the differential expression of transmitter systems likely leads to the development of near-mature patterns of locomotor activity by perinatal ages.     

Modulation of lysosomal enzyme levels in cultured cells: effects of alterations in cell density, balanced growth, and endocytosis

Factors which influence lysosomal enzyme accumulation in cultured cells have been studied. In cell types of both fibroblast (3T6) and epithelial (HeLa) origin, acid phosphatase and β-N-acetylglucosaminidase activities increase with increasing cell density. However, in other cell lines such as BHK or chick embryo fibroblasts, little or no accumulation of lysosomal enzymes occurred with increased cell density. Increased lysosomal enzyme activity need not necessarily be accompanied by alterations in rate of cell growth, rate of pinocytosis, or amount of internalized degradable macromolecules. The stimulus for lysosomal enzyme accumulation appears to require cell contact, since sparsely plated cells do not exhibit lysosomal enzyme accumulation. In 3T6 cells, lysosomal enzymes also accumulate during “step-down” conditions, such as amino acid or serum depletion, or during unbalanced growth resulting from inhibition of cytokinesis or DNA synthesis. Increases in the specific activity of lysosomal enzymes which occur during step-down conditions or unbalanced growth require cell contact, since they are not seen in sparse cells, but are observed in medium- and high-density cells incubated in serum-free medium. Studies employing actinomycin D suggest that lysosomal enzyme levels are regulated primarily via control of enzyme synthesis, rather than enzyme degradation.     

Acid phosphatase of HeLa cells: properties and regulation of lysosomal activity by serum.

Although the subcellular distribution profile of acid phosphatase in HeLa cells is typical of a lysosomal enzyme, different lysosomal (70–80%) and supernatant forms (20–30%) have been demonstrated by their differences in pH activity curves, substrate specificities, thermal stability, sensitivity to inhibitors, and kinetics. Enzymes of the lysosomal fraction displayed anomalous kinetics in the hydrolysis of p-nitrophenyl phosphate. The major lysosomal acid phosphatase activity appears to be associated with the membrane.The total acid phosphatase activity in the cell is controlled by the concentration of serum in the medium. The specific activity in the homogenates of cells grown in high serum concentration (30%) is about twice that of cells grown in low serum concentration (1%). This doubling of specific activity holds for the lysosomal enzyme (or enzymes), but little change occurs in the supernatant form (or forms). Two other lysosomal enzymes, β-glucuronidase and N-acetyl-β-d-hexosaminidase, do not increase in specific activity. The serum-dependent formation of acid phosphatase is sensitive to cycloheximide, actinomycin D, and cordycepin. Cycloheximide blocks the increase in enzymatic activity immediately, whereas cordycepin and actinomycin D have no effect for at least 8 h. These findings suggest that de novo protein synthesis is involved in the induction of lysosomal acid phosphatase by serum and that the mRNA for this enzyme is relatively stable.     

Effects of juvenile hormone on some phase characteristics in the common cutworm, Spodoptera litura

Phase characters of the common cutworm, Spodoptera litura, were influenced by different rearing densities from the 4th-larval instar. Primarily the final feeding period of isolated larvae was 1 day longer than that of crowded larvae causing an increase in pupal weight. Applications of juvenile hormone I, II, or methoprene to crowded larvae caused an increased feeding period similar to that of isolated larvae when the juvenile hormones were applied within 1 day after the last-larval ecdysis. Allatectomy of isolated Spodoptera during the moult to the final-larval instar decreased the duration of the final feeding period to that of intact crowded larvae. These results suggested that one of the characters of phase variation, pupal weight, is influenced by the differences in the regulation and activity of the corpora allata during the last-larval instar. Other characteristics of phase variation such as behaviour (feigned death) and colour were not affected by alteration in juvenile hormone levels after the last larva ecdysis.     

Some autophagic and apoptotic features of programmed cell death in the anterior silk glands of the silkworm,Bombyx mori

Programmed cell death has been subdivided into two major groups: apoptosis and autophagic cell death. The anterior silk gland of Bombyx mori degenerates during larval-pupal metamorphosis. Our findings indicate that two types of programmed cell death features are observed during this physiological process. During the prepupal period, pyknosis of the nucleus, cell detachment and membrane blebbing occur and they are the first signs of programmed cell death in the anterior silk glands. According to previous studies, all of these morphological appearences are common for both cell death types. Autophagy features are also exhibited during the prepupal period. One of the lysosomal marker enzymes, acid phosphatase, levels are high during this period then decrease gradually. Vacuole formation begins to appear first at the basal surface of the cell, then expands to the apical surface just before the larval pupal ecdysis. After larval-pupal ecdysis, DNA fragmentation, which is the obvious biochemical marker of apoptosis, is detected in agarose gel electrophoresis which also shows that caspase-like enzyme activities occur during the programmed cell death process of the anterior silk glands. Apoptosis and autophagic cell death interact with each other during the degeneration process of the anterior silk gland in Bombyx mori and this interaction occurs at a late phase of cell death. We suggest that only apoptotic cell death not enough for whole gland degeneration and that more effective degeneration occurs with this cooperation.

Addendum to: Goncu E, Parlak O. Morphological changes and patterns of ecdysone receptor B1 immunolocalization in the anterior silk gland undergoing programmed cell death in the silkworm, Bombyx mori. Acta Histochem 2008; In press.     

Monitoring of Vibrio harveyi quorum sensing activity in real time during infection of brine shrimp larvae

Quorum sensing, bacterial cell-to-cell communication, has been linked to the virulence of pathogenic bacteria. Indeed, in vitro experiments have shown that many bacterial pathogens regulate the expression of virulence genes by this cell-to-cell communication process. Moreover, signal molecules have been detected in samples retrieved from infected hosts and quorum sensing disruption has been reported to result in reduced virulence in different host–pathogen systems. However, data on in vivo quorum sensing activity of pathogens during infection of a host are currently lacking. We previously reported that quorum sensing regulates the virulence of Vibrio harveyi in a standardised model system with gnotobiotic brine shrimp (Artemia franciscana) larvae. Here, we monitored quorum sensing activity in Vibrio harveyi during infection of the shrimp, using bioluminescence as a read-out. We found that wild-type Vibrio harveyi shows a strong increase in quorum sensing activity early during infection. In this respect, the bacteria behave remarkably similar in different larvae, despite the fact that only half of them survive the infection. Interestingly, when expressed per bacterial cell, Vibrio harveyi showed around 200-fold higher maximal quorum sensing-regulated bioluminescence when associated with larvae than in the culture water. Finally, the in vivo quorum sensing activity of mutants defective in the production of one of the three signal molecules is consistent with their virulence, with no detectable in vivo quorum sensing activity in AI-2- and CAI-1-deficient mutants. These results indicate that AI-2 and CAI-1 are the dominant signals during infection of brine shrimp.     

Identification of Cytoskeleton-Associated Proteins Essential for Lysosomal Stability and Survival of Human Cancer Cells

Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 siRNAs was preceded by lysosomal membrane permeabilization, and all identified siRNAs induced several changes in the endo-lysosomal compartment, i.e. increased lysosomal volume (KIF11, KIF20A, KIF25, MYO1G, MYH1), increased cysteine cathepsin activity (KIF20A, KIF25), altered lysosomal localization (KIF25, MYH1, TPM2), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide or cisplatin. Similarly to KIF11 siRNA, the KIF11 inhibitor monastrol induced lysosomal membrane permeabilization and sensitized several cancer cell lines to siramesine. While KIF11 inhibitors are under clinical development as mitotic blockers, our data reveal a new function for KIF11 in controlling lysosomal stability and introduce six other molecular motors as putative cancer drug targets.     

Caenorhabditis elegans PTR/PTCHD PTR-18 promotes the clearance of extracellular hedgehog-related protein via endocytosis

Spatiotemporal restriction of signaling plays a critical role in animal development and tissue homeostasis. All stem and progenitor cells in newly hatched C. elegans larvae are quiescent and capable of suspending their development until sufficient food is supplied. Here, we show that ptr-18, which encodes the evolutionarily conserved patched-related (PTR)/patched domain-containing (PTCHD) protein, temporally restricts the availability of extracellular hedgehog-related protein to establish the capacity of progenitor cells to maintain quiescence. We found that neural progenitor cells exit from quiescence in ptr-18 mutant larvae even when hatched under starved conditions. This unwanted reactivation depended on the activity of a specific set of hedgehog-related grl genes including grl-7. Unexpectedly, neither PTR-18 nor GRL-7 were expressed in newly hatched wild-type larvae. Instead, at the late embryonic stage, both PTR-18 and GRL-7 proteins were first localized around the apical membrane of hypodermal and neural progenitor cells and subsequently targeted for lysosomal degradation before hatching. Loss of ptr-18 caused a significant delay in GRL-7 clearance, causing this protein to be retained in the extracellular space in newly hatched ptr-18 mutant larvae. Furthermore, the putative transporter activity of PTR-18 was shown to be required for the appropriate function of the protein. These findings not only uncover a previously undescribed role of PTR/PTCHD in the clearance of extracellular hedgehog-related proteins via endocytosis-mediated degradation but also illustrate that failure to temporally restrict intercellular signaling during embryogenesis can subsequently compromise post-embryonic progenitor cell function.     

Fractionation of mouse T and B lymphocytes by preparative cell electrophoresis. Efficiency of the method

Mouse lymphocytes have been fractionated in preparative cell electrophoresis into two functionally viable populations, a high mobility cell (HMC) and a low mobility cell (LMC) population. The distribution of HMC in CBA spleen, blood, and lymph node corresponds to known proportions of θ-positive cells in these organs. The HMC carry the θ-isoantigen, respond to phytohemagglutinin in vitro, and induce a graft-versus-host reaction in newborn F₁ hybrid mice. Nearly all spleen LMC have complement receptors on their surface. About 70% of spleen LMC are sensitive to anti-MBLA serum and form “caps” when incubated with FITC-conjugated anti-Ig. Only LMC respond to E. coli lipopolysaccharide. Thus, T cells localize in the HMC population and B cells in the LMC population. There is no detectable contamination of T lymphocytes among the LMC, nor of B lymphocytes among the HMC.     

Evidence that some developing limb motoneurons die for reasons other than peripheral competition

In vertebrate embryos up to 75% of lateral motor column (LMC) cells die soon after innervating the limb bud. The hypothesis was tested that competition for unknown limb factors may decide which cells will survive. Removal of the future knee flexor motoneurons before the onset of cell death was attempted with varying success in Xenopus laevis tadpoles by removing a piece of spinal cord containing the rostral part of the left lumbar LMC. In normal tadpoles, hundreds of cells in the caudal part of the LMC temporarily project to the presumptive knee flexors and are among the first to die. The competition hypothesis predicts that they should remain alive after a successful operation. After maturation the most successful operations were found to have resulted in paralysis and hypoplasia of the knee flexors. Horseradish peroxidase tracing techniques confirmed that the knee flexors were not innervated. However, ankle and foot movements were normal indicating that the remaining caudal LMC cells had developed their normal projections to the distal limb. The failure to survive of the caudal LMC cells projecting to the knee flexors, despite the absence of rostral LMC cell innervation, shows that factors other than competition must control at least some LMC cell deaths.