Polyploid tissues in the nematode Caenorhabditis elegans

During larval development, the number of somatic nuclei in C. elegans hermaphrodites increases from 558 to 959 (J. E. Sulston and H. R. Horvitz, Dev. Biol. 56, 110-156, 1977; J. E. Sulston et al., Dev. Biol. 100, 64-119, 1983). At the same time, the animals increase about 60-fold in volume. We have measured the DNA contents of several classes of nuclei by quantitating the fluorescence of Hoescht 33258 stained DNA (D. G. Albertson et al., Dev. Biol. 63, 165-178, 1978). Probably all embryonic nuclei, including those of neurons, muscles, hypodermis, and intestine, are diploid at hatching. Neurons, muscles, and nondividing hypodermal nuclei remain diploid throughout larval development. The DNA content of the intestinal nuclei doubles at the end of each larval stage, reaching 32C by the adult stage. New hypodermal cells, generated by division of seam cells in the larval stages, undergo an additional round of DNA replication before fusing with the major syncytium (hyp7, Sulston et al., 1983). Thus the larval hyp7 syncytium comprises a fixed number of diploid embryonic nuclei plus an increasing number of tetraploid postembryonic nuclei. Some of the endoreduplications that occur in the intestinal and hypodermal lineages of C. elegans may correspond to nuclear or cellular divisions in another nematode Panagrellus redivivus (P. W. Sternberg and H. R. Horvitz, Dev. Biol. 93, 181-205, 1982).     

An essential role in molting and morphogenesis of Caenorhabditis elegans for ACN-1, a novel member of the angiotensin-converting enzyme family that lacks a metallopeptidase active site

Genome sequence analyses predict many proteins that are structurally related to proteases but lack catalytic residues, thus making functional assignment difficult. We show that one of these proteins (ACN-1), a unique multi-domain angiotensin-converting enzyme (ACE)-like protein from Caenorhabditis elegans, is essential for larval development and adult morphogenesis. Green fluorescent protein-tagged ACN-1 is expressed in hypodermal cells, the developing vulva, and the ray papillae of the male tail. The hypodermal expression of acn-1 appears to be controlled by nhr-23 and nhr-25, two nuclear hormone receptors known to regulate molting in C. elegans. acn-1(RNAi) causes arrest of larval development because of a molting defect, a protruding vulva in adult hermaphrodites, severely disrupted alae, and an incomplete seam syncytium. Adult males also have multiple tail defects. The failure of the larval seam cells to undergo normal cell fusion is the likely reason for the severe disruption of the adult alae. We propose that alteration of the ancestral ACE during evolution, by loss of the metallopeptidase active site and the addition of new protein modules, has provided opportunities for novel molecular interactions important for post-embryonic development in nematodes.     

Ultrastructural analysis of pathologic lesions in sterol-deficient Nippostrongylus brasiliensis larvae

A variety of cellular lesions were manifested by the free-living larval stages of Nippostrongylus brasiliensis cultured axenically in medium lacking cholesterol. Pathologic changes developed rapidly and were most apparent in intestinal cells which displayed generalized degradation of membranous organelles. Mitochondria, endoplasmic reticulum, and Golgi complexes became disassociated and vacuolated. Autophagosomes appeared within intestinal cells and contained a wide variety of cellular components. By the 5th day gross vacuolization and degeneration of intestinal cells occurred and the hypodermis and lateral cords displayed lysed cytoplasmic regions. The latter structures are concerned with synthesis of cuticle and their degeneration correlates with the suppression of molting and the abnormal molts that occurred.     

LIN-42/PERIOD controls cyclical and developmental progression of C. elegans molts

Background

Biological timing mechanisms that integrate cyclical and successive processes are not well understood. C. elegans molting cycles involve rhythmic cellular and animal behaviors linked to the periodic reconstruction of cuticles. Molts are coordinated with successive transitions in the temporal fates of epidermal blast cells, which are programmed by genes in the heterochronic regulatory network. It was known that juveniles molt at regular 8–10 hr intervals, but the anticipated pacemaker had not been characterized.

Results

We find that inactivation of the heterochronic gene lin-42a, which is related to the core circadian clock gene PERIOD (PER), results in arrhythmic molts and continuously abnormal epidermal stem cell dynamics. The oscillatory expression of lin-42a in the epidermis peaks during the molts. Further, forced expression of lin-42a leads to anachronistic larval molts and lethargy in adults.

Conclusions

Our results suggest that rising and falling levels of LIN-42A allow the start and completion, respectively, of larval molts. We propose that LIN-42A and affiliated factors regulate molting cycles in much the same way that PER-based oscillators drive rhythmic behaviors and metabolic processes in mature mammals. Further, the combination of reiterative and stage-specific functions of LIN-42 may coordinate periodic molts with successive development of the epidermis.     

Wnt signaling controls the stem cell-like asymmetric division of the epithelial seam cells during C. elegans larval development

Metazoan stem cells repopulate tissues during adult life by dividing asymmetrically to generate another stem cell and a cell that terminally differentiates. Wnt signaling regulates the division pattern of stem cells in flies and vertebrates. While the short-lived nematode C. elegans has no adult somatic stem cells, the lateral epithelial seam cells divide in a stem cell-like manner in each larval stage, usually generating a posterior daughter that retains the seam cell fate and an anterior daughter that terminally differentiates. We show that while wild-type adult animals have 16 seam cells per side, animals with reduced function of the TCF homolog POP-1 have as many as 67 seam cells, and animals with reduced function of the β-catenins SYS-1 and WRM-1 have as few as three. Analysis of seam cell division patterns showed alterations in their stem cell-like divisions in the L2-L4 stages: reduced Wnt signaling caused both daughters to adopt non-seam fates, while activated Wnt signaling caused both daughters to adopt the seam fate. Therefore, our results indicate that Wnt signaling globally regulates the asymmetric, stem cell-like division of most or all somatic seam cells during C. elegans larval development, and that Wnt pathway regulation of stem cell-like behavior is conserved in nematodes.     

The mir-84 and let-7 paralogous microRNA genes of Caenorhabditis elegans direct the cessation of molting via the conserved nuclear hormone receptors NHR-23 and NHR-25

The let-7 microRNA (miRNA) gene of Caenorhabditis elegans controls the timing of developmental events. let-7 is conserved throughout bilaterian phylogeny and has multiple paralogs. Here, we show that the paralog mir-84 acts synergistically with let-7 to promote terminal differentiation of the hypodermis and the cessation of molting in C. elegans. Loss of mir-84 exacerbates phenotypes caused by mutations in let-7, whereas increased expression of mir-84 suppresses a let-7 null allele. Adults with reduced levels of mir-84 and let-7 express genes characteristic of larval molting as they initiate a supernumerary molt. mir-84 and let-7 promote exit from the molting cycle by regulating targets in the heterochronic pathway and also nhr-23 and nhr-25, genes encoding conserved nuclear hormone receptors essential for larval molting. The synergistic action of miRNA paralogs in development may be a general feature of the diversified miRNA gene family.     

C. elegans peb-1 mutants exhibit pleiotropic defects in molting, feeding, and morphology

Caenorhabditis elegans PEB-1 is a novel DNA-binding protein expressed in most pharyngeal cell types and outside the pharynx in the hypodermis, hindgut, and vulva. Previous RNAi analyses indicated that PEB-1 is required for normal morphology of these tissues and growth; however, the peb-1 null phenotype was unknown. Here we describe the deletion mutant peb-1(cu9) that not only exhibits the morphological defects observed in peb-1(RNAi) animals, but also results in penetrant larval lethality characterized by defects in pharyngeal function and molting. Consistent with a function in molting, we found that PEB-1 was detectable in all hypodermal and hindgut cells underlying the cuticle. Comparison to molting-defective lrp-1(ku156) mutants revealed that the peb-1(cu9) mutants were particularly defective in shedding the pharyngeal cuticle, and this defect likely contributed to feeding defects and lethality. Most markers of pharyngeal cell differentiation examined were expressed normally in peb-1(cu9) mutants; however, g1 gland cell expression of a kel-1Colon, two colonsgfp reporter was reduced. As g1 gland cells have prominent functions during molting, we suggest defective gland cell differentiation contributes to peb-1(cu9) molting defects. In comparison, other peb-1 mutant phenotypes, including hindgut abnormalities, appeared independent of the molting defect. Similar phenotypes resulted from late loss of pha-4 function, suggesting that PEB-1 and PHA-4 have common functions in some tissues where they are co-expressed.     

The influence of intrinsic and extrinsic factors on developmental parameters and their relationships in the marine isopod Idotea linearis (Crustacea)

Developmental and reproductive parameters and their relationships were studied in the marine isopod Idotea linearis. We hypothesized that (1) the temporal patterns of molting and growth undergo complex and sex-specific changes with age as well as with the onset of sexual maturation, and that (2) sexual maturation (and dependent parameters) is controlled by the photoperiod. Both males and females were singly cultured in the laboratory at two alternative photoperiods (constant long and short days, respectively) from hatching until death. Males molted and grew throughout their life, showing a steady increase in stage duration and body size with each molt. Females, in contrast, showed much more complex modifications in molt chronology due to reproductive demands. There was some variability in the stage number, when females reached maturity. Reaching maturity early in the succession of molts was associated with smaller body size at maturity, smaller size of broods, but higher average number of broods per lifetime. Post-puberty molts in females occurred without further growth, and successive broods did not differ in size. The photoperiod strongly affected sexual maturation (and thus in turn molting and growth patterns) in females, while males remained completely unaffected by the photo regime.     

Amphipod Life Histories: Community Structure, Impact of Temperature on Decoupled Growth and Maturation Rates, Productivity, and P:B Ratios

Data are presented on northern Bering Sea benthic amphipod growthrates and age at maturity, indicating that high latitude speciesgrow slowly, require 2 to 4 years to mature, reach a large size,and have long lifespans. Data are also presented demonstratingpredation by benthic amphipods on newly metamorphosed juvenilesof a potential space competitor, the northern sand dollar Echinarachniusparma. Such facultative predation by the predominantly herbivorousamphipods may, in part, explain the existence of alternativebenthic communities in the eastern Bering Sea. Incorporationof the high latitude results into a review of benthic amphipodlife histories revealed several important patterns. Amphipodgrowth rates and molting rates appear to be decoupled, resultingin small adults at warm temperatures and large adults at lowertemperatures. We posit that molting rate is temperature- sensitive,that at warm temperatures molting occurs rapidly regardlessof limited instar tissue growth, and that the amphipods reachsexual maturity after a fixed number of molts. Alternatively,gonad development is also temperature-dependent and may drivematuration, regardless of the number of molts experienced. Amphipodshave linear or exponential growth rates, as opposed to the familiarasymptotic curve. Consequently, secondary production is highlydependent upon the proportion of large individuals in the population,in contrast to organisms with asymptotic growth in which productionis largely determined by the proportion of young, growing individualspresent. Production in amphipods is significantly correlatedwith standing stock. P:B ratios are not correlated with productionin amphipods, and probably are not in other organisms with linearor exponential growth rates, and thus appear to have no comparativevalue other than as a possible index of generation times.     

A conserved metalloprotease mediates ecdysis in Caenorhabditis elegans

Molting is required for progression between larval stages in the life cycle of nematodes. We have identified four mutant alleles of a Caenorhabditis elegans metalloprotease gene, nas-37, that cause incomplete ecdysis. At each molt the cuticle fails to open sufficiently at the anterior end and the partially shed cuticle is dragged behind the animal. The gene is expressed in hypodermal cells 4 hours before ecdysis during all larval stages. The NAS-37 protein accumulates in the anterior cuticle and is shed in the cuticle after ecdysis. This pattern of protein accumulation places NAS-37 in the right place and at the right time to degrade the cuticle to facilitate ecdysis. The nas-37 gene has orthologs in other nematode species, including parasitic nematodes, and they undergo a similar shedding process. For example, Haemonchus contortus molts by digesting a ring of cuticle at the tip of the nose. Incubating Haemonchus larvae in extracted exsheathing fluids causes a refractile ring of digested cuticle to form at the tip of the nose. When Haemonchus cuticles are incubated with purified NAS-37, a similar refractile ring forms. NAS-37 degradation of the Haemonchus cuticle suggests that the metalloproteases and the cuticle substrates involved in exsheathment of parasitic nematodes are conserved in free-living nematodes.     

Relationship between an increase of juvenile hormone titer in early instars and the induction of diapause in fully grown larvae of Sesamia nonagrioides

The larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae) grown at 25 degrees C and long photoperiod (16:8h light:dark) pupate in the 5th or 6th (mostly) larval instar, while the larvae reared under a short photoperiod (12:12h) enter diapause during which they consume some food and undergo up to 12 (usually 3-4) stationary larval molts. Diapause programming includes an increase of juvenile hormone (JH) titer in the hemolymph from about 20 to 50 nM in the 4th and 5th instar larvae (titer in earlier instars was not measured). JH I, II, and III are present in approximate ratio 1-2:10:1. The JH titer drops to zero before pupation but remains around 20 nM during diapause. Perfect extra larval molts associated with a body weight increase can be induced in the non-diapausing larvae with a JH analogue (JHA). The weight rise is due to accumulation of reserves and not to a general body growth. The timing of extra molts is similar to the molting pattern of the diapausing larvae only when JHA is present since early larval instars. In the diapausing larvae, JHA application affects neither molting periodicity nor the body weight. It is concluded that (1) Increased JH titer in early larval instars is a part of diapause programming; (2) The extension of larval stage in the diapausing larvae, but not the timing pattern of extra molts, is due to continuously high JH titer; (3) The diapause program includes low food intake, maintenance of a certain body weight, and periodic larval molts.     

Cathepsin L is essential for embryogenesis and development of Caenorhabditis elegans.

Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterized Ce-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1 activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role for cpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematode Onchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes.