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Planarian mitochondria. I. Heterogeneity of cytochrome c oxidase subunit I gene sequences in the freshwater planarian, Dugesia japonica.

We have detected sequence heterogeneity in the cytochrome c oxidase subunit I (COI) gene of freshwater planarian, Dugesia japonica, collected in one locality. A part of the COI gene was amplified via the polymerase chain reaction (PCR) using template DNA prepared from a mixture of 500 individuals or from each of 18 individuals. Analyses of DNA sequences by standard strategies for cloning and sequencing or by direct sequencing clearly show that (1) considerable sequence heterogeneity exists in DNA prepared from the mixed individuals, (2) 11 individuals have almost identical sequences (type A), and (3) 7 individuals have sequences different from one another (Seq-D1 to Seq-D7; collectively called type D). Each of the Seq-D1-D7 sequences except for Seq-D5 shows some heterogeneity even in a single individual (heteroplasmy). A possible cause of the sequence heterogeneities is discussed.

Animals

Progress on the telomere-telomerase system in planarian neoblasts homeostasis and regeneration.

Planarians are flatworms with remarkable regenerative abilities, and their adult pluripotent stem cells, known as neoblasts, serve as the foundation for this regeneration. Neoblasts can rapidly migrate, proliferate, and undergo directed differentiation following tissue injury to complete regeneration. Telomeres are located at the ends of eukaryotic chromosomes and play a core role in maintaining chromosomal integrity. With each cell division, telomeres shorten. Telomerase is a reverse transcriptase that compensates for telomere loss during cell division by extending telomeric repeats. The telomere-telomerase system is one of the important mechanisms for maintaining stem cell homeostasis. As adult pluripotent stem cells of planarians, neoblasts are required to maintain a dynamic balance between high-frequency cell division and multilineage differentiation. The maintenance of their long-term proliferative capacity and genomic stability may depend on the precise regulation of the telomere-telomerase system. Thus, this system represents a critical entry point for understanding the remarkable regenerative ability of planarians. Integrating recent progress in regeneration, neoblast regulation, and the telomere-telomerase system, we systematically summarize the emerging evidence for telomere- telomerase involvement in neoblast homeostasis and regeneration. We aim to provide insights for research in regenerative medicine, stem cell regulation, and neural injury repair.

Animals

The mechanism of anterior-posterior polarity control in planarians.

The substance which inhibits brain formation in the regenerating planarian Dugesia etrusca was found to be a large molecule, at least in part protein, which electrophoreses as an electronegative moiety in pH 6.8 buffer. A model is presented, based on this finding and previous studies, which proposes an electrochemical mechanism for the control of polarity and possibly for the maintenance of tissue organization in planarians. It is proposed that a bioelectric field exists and moves the electronegative brain-inhibiting substance in a posterior direction, establishing polarity. This model explains the polarity reversal experiments using external fields and many of the previously unexplained classical planarian experiments. Data are presented demonstrating the existence, magnitude, and polarity of this bioelectric field, which is not greatly altered upon decapitation, all in accord with predictions of the model.

Animals

Conserved innate immunity components limit transgene expression in adult planarians.

The planarian flatworm Schmidtea mediterranea has become a powerful model for studying whole-body regeneration, tissue patterning, and stem cell regulation. Yet the absence of reliable tools for transgene expression still limits the elucidation of molecular mechanisms in in this system. Here, we establish a proof-of-principle system for plasmid-based expression of NanoLuciferase (NanoLuc) in S. mediterranea, employing commercially available transfection reagents and a panel of endogenous promoter sequences. Despite successful delivery, reporter expression remained low and transient. To identify biological barriers to robust transgene expression, we investigated the role of innate immune pathways. Candidate gene searches and biochemical pull-down of cytoplasmic DNA coupled to mass spectrometry identified several planarian homologs of conserved immune regulators and putative DNA sensors. Through RNA interference screening of conserved innate immune components, we uncover roles for S. mediterranea homologs of Tank-binding kinase 1 (TBK1) and macrophage mannose receptor 1 (MRC1) as potent repressors of transgene expression. Transcriptomic and functional analyses further implicate TBK1 in regulating broad innate immune and stress-response programs, akin to its vertebrate function. Together, our findings demonstrate that innate immune signaling limits transgene expression in S. mediterranea and suggest that modulating these pathways may be key to enabling stable and efficient genetic manipulation in planarians.

Animals

Investigating the Functions of Hox Genes Using Planarian Asexual Reproduction.

Hox genes are highly conserved developmental regulators instrumental to the formation of a wide range of diverse body plans across metazoans. While significant progress in the field of Hox gene research has been made, persistent challenges in unraveling their mechanisms of action and full repertoire of functions remain. To date, investigations of Hox gene function have been primarily conducted in research models belonging to ecdysozoa and vertebrata. Herein we summarize recent findings on Hox genes' roles in the asexual reproduction of the regenerative flatworm planaria, a member of the understudied superphylum Spiralia. We detail our optimized methods for planarian culture, gene perturbation, and induction of asexual reproduction. We aim to provide an experimentally tractable means to dissect Hox gene adult tissue functions underlying planarian asexual reproduction with broader relevance to Hox genes' established and emerging roles in regulating cellular behaviors, developmental patterning, animal behavior, and tissue regeneration.

Animals

Planarian mitochondria. II. The unique genetic code as deduced from cytochrome c oxidase subunit I gene sequences.

The cytochrome c oxidase subunit I (COI) gene sequences from planarian (Dugesia japonica) DNA, most probably of mitochondrial origin, are heterogeneous. Taking advantage of the heterogeneity that occurs primarily in silent sites of the COI DNA sequences, amino acid assignments of several codons have been deduced as nonuniversal: UGA = Trp, AAA = Asp, and AGR (R: A or G) = Ser. In addition, UAA, a stop codon in the universal genetic code, is tentatively assumed to be a tyrosine codon, because three of the sequences examined have UAA at the well-conserved tyrosine site of UAY (Y: U or C) in other planarian sequences as well as in the mitochondria of human, Xenopus, sea urchin, Drosophila, Trypanosoma, and Saccharomyces cerevisiae. AUA would most probably be an isoleucine codon in these mitochondria, whereas it is a methionine codon in the majority of nonplant mitochondria.

Amino Acid Sequence

Structure and regeneration of the planarian basal lamina: an ultrastructural study.

The structure and regeneration of the planarian subepidermal basement membrane or basal lamina have been electron microscopically examined, particularly in relation to the changes of extracellular products at the wounded area. The intact basal lamina consists of three structural elements; namely, an electron-lucent zone, a limiting layer and a microfibrillar layer. Ultrastructural changes during wound healing have suggested that the amorphous material secreted in the interspace between the epidermal cells and blastema contains precursors of the basal lamina. Within the amorphous zone two distinct phases of the basal lamina regeneration are observed: one is a reconstitution of the limiting layer and the other is a polymerization of the microfibrils. The limiting layer arises from areas subjacent to newly developed hemidesmosomes of epidermal cells. The unit microfibrils are formed from an accumulation of the precursors through transitional smaller microfibrils. At the late stage, individual mature microfibrils are regularly lined with the limiting layer and cell membranes of the newly differentiated muscle fibres. On the basis of these observations we suggest that the planarian basal lamina is regenerated by the interaction between epidermal cells and myoblasts.

Animals

Cephalic mechanism for social control of fissioning in planarians: III. Central nervous system centers of facilitation and inhibition.

Previous studies have indicated that asexual reproduction (fissioning) in the planarian Dugesia dorotocephala is socially controlled through a cephalic mechanism: Isolation releases fissioning; grouping inhibits it; decapitation, at the level of the auricles, releases it even in grouped subjects. The brain is not necessary for programming the actual events of fissioning; these are orchestrated by the segmental plexus fissioning (SPF) system. Various surgical cuts were made to ablate selected portions of the central nervous system of isolated and grouped planarians in order to ascertain the inhibitory or facilitatory effects of these in the physiological mediation of such control on the SPF system. These results were synthesized into a model of this control system; the anterior lobes and optic regions of the brain inhibit the SPF system, and the anterior and caudal segmental plexuses facilitate it. These influences are partially tonic and partially contingent upon social stimulation.

Animals

Planarian homeobox genes: cloning, sequence analysis, and expression.

Freshwater planarians (Platyhelminthes, Turbellaria, and Tricladida) are acoelomate, triploblastic, unsegmented, and bilaterally symmetrical organisms that are mainly known for their ample power to regenerate a complete organism from a small piece of their body. To identify potential pattern-control genes in planarian regeneration, we have isolated two homeobox-containing genes, Dth-1 and Dth-2 [Dugesia (Girardia) tigrina homeobox], by using degenerate oligonucleotides corresponding to the most conserved amino acid sequence from helix-3 of the homeodomain. Dth-1 and Dth-2 homeodomains are closely related (68% at the nucleotide level and 78% at the protein level) and show the conserved residues characteristic of the homeodomains identified to data. Similarity with most homeobox sequences is low (30-50%), except with Drosophila NK homeodomains (80-82% with NK-2) and the rodent TTF-1 homeodomain (77-87%). Some unusual amino acid residues specific to NK-2, TTF-1, Dth-1, and Dth-2 can be observed in the recognition helix (helix-3) and may define a family of homeodomains. The deduced amino acid sequences from the cDNAs contain, in addition to the homeodomain, other domains also present in various homeobox-containing genes. The expression of both genes, detected by Northern blot analysis, appear slightly higher in cephalic regions than in the rest of the intact organism, while a slight increase is detected in the central period (5 days) or regeneration.

Amino Acid Sequence

Analysis of the progress of hormone-receptor amplification during differentiation in regenerating planarians.

Planarians developing from isolated regenerates, when pretreated with epinephrine at different periods of their second regeneration, show a higher rate of sugar uptake than the controls, which were not treated with epinephrine. The hormone receptor development can be influenced most effectively by pretreatments performed during the 1--3 and 6--9 day periods, though some effect can be evoked at any time in the course of the second regeneration. The head regenerates developing from the anterior end of the original midpiece (and regenerating tails the second time, B1) were found to be most sensitive, while the similar regenerates developing from the hindpiece of the original planarian (C) showed the weakest reactivity. The experiments provide further evidence that the hormone receptors' activity can be amplified in the course of regenerative development, leading thus--at a later stage--to a more intense sugar uptake response of the cells.

Animals

[DNA reproduction in the organism of planarians in post-injury recovery].

No mitotic figures were found during histological observations of intact as well as transsected planarians Dendrocoelum lacteum within 15 days after the operation. DNA content in intact and regenerating worms was measured. It has been shown, that the posttraumatic repair in planarians does not involve DNA synthesis or cell reproduction.

Animals

Reappearance of embryonal antigens in planarian regenerates.

A transient and apparently orderly re-expression of embryonal antigens in planarian regenerates has been demonstrated. This finding seems to corroborate the hypothesis that regeneration in planarians is based on a recapitulation of mechanisms that operate the embryogenesis of these animals, andgives some support to the concept of cancer as a misprogrammed regeneration when contemplated against the background of facts pointed out in the literature.

Animals

Content of mitochondrial protein in the planarian Polycelis nigra during starvation and feeding.

To discover the possible mechanisms determining the level of energy metabolism during changes in the body size of animals, the content of mitochondrial protein was studied in planarians during starvation and feeding. In the course of starvation, the relative content of mitochondrial protein decreases, whereas during feeding it increases. Comparison of the experimental results with previous observations on respiration of planarians under similar conditions shows that changes in the level of respiration during starvation and feeding correlate with the content of mitochondrial protein. However, the degree of the functional load on the mitochondria must be taken into account under these circumstances.

Animals

[The effect of repeated amputations on planarian regeneration in the presence of the heat-stable toxin from Bacillus thuringiensis].

The exotoxin of Bacillus thuringiensis, which is an inhibitor of RNA synthesis, inhibits Planarian regeneration. Planarians which have been cut twice at the same level are able to regenerate after the second section in the presence of the toxin. This indicates that the first amputation stimulates a synthesis of stable RNAs which thus are available for regeneration at the moment of the second section.

Animals

Identification of a 5-HT1A receptor positively coupled to planarian adenylate cyclase.

The adenylate cyclase system present in particulate fractions prepared from two planarian species was tested for sensitivity to various neurotransmitters. While dopamine and other catecholamines were ineffective, serotonin was capable of stimulating the enzyme. Among the various serotonin agonists tested, only 8-OH-DPAT resulted effective on the adenylate cyclase activity, thus suggesting the presence in planarians of a serotonin receptor of the type 5-HT1A.

8-Hydroxy-2-(di-n-propylamino)tetralin

Symptoms and causation of the planarian disease related to cancer.

The disease provoked by injections of water and other harmless liquids or wounds without tissue loss, is characterized by the regression of certain organs, opening in the epidermis, pigmentary anomalies and malignant tumors. The very few worms which recover spontaneously, regenerate the eliminated parts or develop supernumerary structures. Wound are producing the disease less frequently when planarians are undernourished. The disease can also manifest itself spontaneously, especially in the summer. Diseased worms become unable to regenerate. During a transitory period epimorphosis is still possible but is not followed by morphallaxis. The symptoms of the disease manifest themselves in the regenerated parts. A fragment cut out from a worm that just became diseased regenerates and recovers completely. The results give additional evidence to the earlier conclusions about the "type I cell" system of planarians. The disease is provoked by an excessive activity of this system with respect to the animal's actual need and is analogous both to cancer and the consequences of X-ray treatment.

Cell Division

Use of the p-nitrophenyl phosphate method for the demonstration of acid phosphatase during starvation and cell autolysis in the planarian Polycelis tenuis Iijima.

Acid phosphatase activity is demonstrated employing p-nitrophenyl phosphate as substrate and lead acetate as coupler. The fine structural localization of the enzyme in starved planarian tissues is described. The method is used to pin-point starvation - induced acid phosphatase activity in relation to autophagy and crinophagy in the gland cells; autophagy, autolysis and cell death in parenchymal and gastrodermal cells and basement membrane lysis. Attention is also payed to the demonstration of muscle lysis. The histochemical implications of the method are discussed.

4-Nitrophenylphosphatase