The Zn Finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis

Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian Schmidtea mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates.     

The maintenance and regeneration of the planarian excretory system are regulated by EGFR signaling

The maintenance of organs and their regeneration in case of injury are crucial to the survival of all animals. High rates of tissue turnover and nearly unlimited regenerative capabilities make planarian flatworms an ideal system with which to investigate these important processes, yet little is known about the cell biology and anatomy of their organs. Here we focus on the planarian excretory system, which consists of internal protonephridial tubules. We find that these assemble into complex branching patterns with a stereotyped succession of cell types along their length. Organ regeneration is likely to originate from a precursor structure arising in the blastema, which undergoes extensive branching morphogenesis. In an RNAi screen of signaling molecules, we identified an EGF receptor (Smed-EGFR-5) as a crucial regulator of branching morphogenesis and maintenance. Overall, our characterization of the planarian protonephridial system establishes a new paradigm for regenerative organogenesis and provides a platform for exploring its functional and evolutionary homologies with vertebrate excretory systems.     

Early planarian brain regeneration is independent of blastema polarity mediated by the Wnt/β-catenin pathway

Analysis of anteroposterior (AP) axis specification in regenerating planarian flatworms has shown that Wnt/β-catenin signaling is required for posterior specification and that the FGF-like receptor molecule nou-darake (ndk) may be involved in restricting brain regeneration to anterior regions. The relationship between re-establishment of AP identity and correct morphogenesis of the brain is, however, still poorly understood. Here we report the characterization of two axin paralogs in the planarian Schmidtea mediterranea. Although Axins are well known negative regulators of Wnt/β-catenin signaling, no role in AP specification has previously been reported for axin genes in planarians. We show that silencing of Smed-axin genes by RNA interference (RNAi) results in two-tailed planarians, a phenotype previously reported after silencing of Smed-APC-1, another β-catenin inhibitor. More strikingly, we show for the first time that while early brain formation at anterior wounds remains unaffected, subsequent development of the brain is blocked in the two-tailed planarians generated after silencing of Smed-axin genes and Smed-APC-1. These findings suggest that the mechanisms underlying early brain formation can be uncoupled from the specification of AP identity by the Wnt/β-catenin pathway. Finally, the posterior expansion of the brain observed following Smed-ndk RNAi is enhanced by silencing Smed-APC-1, revealing an indirect relationship between the FGFR/Ndk and Wnt/β-catenin signaling systems in establishing the posterior limits of brain differentiation.     

Epidermis and sensory receptors of Temnocephala minor (Plathelminthes, Rhabdocoela, Temnocephalida): an electron microscopic study

 The epidermis, rhabditic glands and receptors of the commensal flatworm Temnocephala minor are described using electron microscopic techniques. The epidermis is syncytial and non-ciliated at the anterior body end; it bears folds, microvilli and other structures which differ according to the body side. The nuclei are located intraepithelially and distally from the basal membrane. Long cilia occur at the posterior end anteriorly from the sucker. All receptor structures described belong to a single morphological type and stand in groups arising from epidermal pits. On the tentacles these groups are regularly distributed. Each receptor has a single cilium and a long rootlet. More than 15 000 receptors of this type have been estimated to occur on the surface of a single medium-sized specimen of T. minor. Although the total number of receptor structures appears very high, the number of different receptor types is extremely low in comparison to other taxa of flatworms. Accepted: 8 July 1997     

Epigenetic regulation of planarian stem cells by the SET1/MLL family of histone methyltransferases

Chromatin regulation is a fundamental mechanism underlying stem cell pluripotency, differentiation, and the establishment of cell type-specific gene expression profiles. To examine the role of chromatin regulation in stem cells in vivo, we study regeneration in the freshwater planarian Schmidtea mediterranea. These animals possess a high concentration of pluripotent stem cells, which are capable of restoring any damaged or lost tissues after injury or amputation. Here, we identify the S. mediterranea homologs of the SET1/MLL family of histone methyltransferases and COMPASS and COMPASS-like complex proteins and investigate their role in stem cell function during regeneration. We identified six S. mediterranea homologs of the SET1/MLL family (set1, mll1/2, trr-1, trr-2, mll5–1 and mll5–2), characterized their patterns of expression in the animal, and examined their function by RNAi. All members of this family are expressed in the stem cell population and differentiated tissues. We show that set1, mll1/2, trr-1, and mll5–2 are required for regeneration and that set1, trr-1 and mll5–2 play roles in the regulation of mitosis. Most notably, knockdown of the planarian set1 homolog leads to stem cell depletion. A subset of planarian homologs of COMPASS and COMPASS-like complex proteins are also expressed in stem cells and implicated in regeneration, but the knockdown phenotypes suggest that some complex members also function in other aspects of planarian biology. This work characterizes the function of the SET1/MLL family in the context of planarian regeneration and provides insight into the role of these enzymes in adult stem cell regulation in vivo.     

Ultrastructure of protonephridia in Xenotrichula carolinensis syltensis and Chaetonotus maximus (Gastrotricha: Chaetonotida): comparative evaluation of the gastrotrich excretory organs

In an attempt to obtain detailed information on the entire protonephridial system in Gastrotricha, we have studied the protonephridial ultrastructure of two paucitubulatan species, Xenotrichula carolinensis syltensis and Chaetonotus maximus by means of complete sets of ultrathin sections. In spite of some differences in detail, the morphology of protonephridia in both examined species shows a common pattern: Both species have one pair of protonephridia that consist of a bicellular terminal organ, a voluminous, aciliar canal cell and an adjacent, aciliar nephridiopore cell. The terminal organ consists of two monociliar terminal cells each with a distal cytoplasmic lobe. These lobes interdigitate and surround cilia and microvilli of the terminal cells. Where both lobes interdigitate, a meandering cleft is formed that is covered by the filtration barrier. We here term the entire structure composite filter. The elongated, in some regions convoluted protonephridial lumen opens distally to the outside via a permanent nephridiopore. A comparison with the protonephridia of other species of the Gastrotricha allows hypothesising the following autapomorphies of the Paucitubulata: The bicellular terminal organ with a composite filter, the convoluted distal canal cell lumen and the absence of cilia, ciliary basal structures and microvilli within the canal cell. Moreover, this comparative survey could confirm important characteristics of the protonephridial system assumed for the ground pattern of Gastrotricha like, for example, the single terminal cell with one cilium surrounded by eight microvilli.     

Ultrastructure and relationship between protonephridia and metanephridia in Phoronis muelleri (Phoronida)

Summary The actinotrocha of Phoronis muelleri has one pair of ectodermally derived, monociliated protonephridia. The duct runs mainly between the epidermis and the lining of the hyposphere coelom, pierces the septum and extends into the blastocoel. The proximal part is branched and closed up by terminal complexes consisting of two morphologically different cells which both serve filtration. During metamorphosis, the terminal complexes and the branches of the duct are cast off. The cells degenerate, pass into the remaining duct and are endocytosed by the duct cells. After metamorphosis the remaining part of the protonephridial duct is U-shaped, blindly closed and borders on the prospective lophophoral vessel. In a later stage the duct receives a ciliated funnel, which consists of monociliated epithelio-muscle cells and is a derivative of the lining of the metacoel. Thus, a part of the protonephridial duct of the larva and the whole metanephridial duct of the adult are identical. Aspects of a possible homology between phoronid nephridia and such organs in other bilaterians are discussed.     

SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development

Planarian flatworms have an impressive regenerative power. Although their embryonic development is still poorly studied and is highly derived it still displays some simple characteristics. We have identified SpolvlgA, a Schmidtea polychroa homolog of the DDX3/PL10 DEAD-box RNA helicase DjvlgA from the planarian species Dugesia japonica. This gene has been previously described as being expressed in planarian adult stem cells (neoblasts), as well as the germ line. Here we present the expression pattern of SpolvlgA in developing embryos of S. polychroa and show that it is expressed from the first cleavage rounds in blastomere cells and blastomere-derived embryonic cells. These cells are undifferentiated cells that engage in a massive wave of differentiation during stage 5 of development. SpolvlgA expression highlights this wave of differentiation, where nearly all previous structures are substituted by blastomere-derived embryonic cells. In late stages of development SpolvlgA is expressed in most proliferating and differentiating cells. Thus, SpolvlgA is a gene expressed in planarian embryos from the first stages of development and a good marker for the zygote-derived cell lineage in these embryos. Expression in adult worms is also monitored and is found in the planarian germ line, where it is showed to be expressed in spermatogonia, spermatocytes and differentiating spermatids.     

Ultrastructural observations of the protonephridia of Polymerurus nodicaudus (Gastrotricha: Paucitubulatina)

Kieneke, A. and Hochberg, R. 2012. Ultrastructural observations of the protonephridia of Polymerurus nodicaudus (Gastrotricha: Paucitubulatina). —Acta Zoologica (Stockholm) 93 : 115–124. We studied different regions of the protonephridia of the limnic gastrotrich Polymerurus nodicaudus by means of light and electron microscopy to determine how freshwater species might differ from their marine relatives. Microscopic and ultrastructural characters are in accordance with another limnic species of Paucitubulatina, Chaetonotus maximus, whose protonephridial system has been previously reconstructed. Shared protonephridial characters of both species include the presence of highly elongate terminal organ cilia, microvilli, and the canal cell lumen as well as the presence of a conspicuous anterior loop of the protonephridial lumen. These features are not present in representatives of earlier, marine, paucitubulatan lineages (i.e., Xenotrichulidae) and so are assessed as evolutionary novelties that were likely important for the successful colonization of the freshwater environment.     

Immunocytochemical localization of histamine in flatworms

Summary Specific antibodies against histamine were used to demonstrate the occurrence and cellular distribution of histamine-like immunoreactivity in three species of flatworms (phylum Platyhelminthes). In the parasitic cestode Diphyllobothrium dendriticum, histamine-reactivity was found in neurons of the main nerve cords, and in cells lining the central and peripheral excretory ducts. In the free-living microturbellarian Microstomum lineare and in the planarian Polycelis nigra, histamine-immuno-reactivity was restricted to cells and fibres of the nervous system. The occurrence of histamine or a related substance in the nervous system of flatworms, which represent primary bilateria, indicates the importance of this neuroactive substance in the animal kingdom.     

Ultrastructure of the protonephridial system of Polystomoides malayi Rohde and P. renschi Rohde (Monogenea: Polystomatidae)

Rohde K. 1973. Ultrastructure of the protonephridial system of Polystomoides malayi Rohde and P. renschi Rohde (Monogenea : Polystomatidae). International Journal for Parasitology3: 329–333. Polystomoides malayi and P. renschi have three types of protonephridial flames. The first type is a typical flame cell with internal and external ribs connected by a weir membrane without nephrostomes, and with internal and external leptotriches. The second type is a flame cell complex consisting of at least two flames reaching into a common cavity. The third type is a non-terminal (= lateral) flame in the protonephridial ducts, consisting of loosely arranged cilia many of which have lateral tube-like extensions and whose tips have irregularly arranged filaments gradually decreasing in number. The number of cilia in all types of flames varies. The smallest capillaries are strongly convoluted and have a smooth or slightly reticulated surface, the larger ducts have strongly reticulated walls and single cilia may be found in the cavities of the reticulum.     

SMG-1 and mTORC1 act antagonistically to regulate response to injury and growth in planarians

Planarian flatworms are able to both regenerate their whole bodies and continuously adapt their size to nutrient status. Tight control of stem cell proliferation and differentiation during these processes is the key feature of planarian biology. Here we show that the planarian homolog of the phosphoinositide 3-kinase-related kinase (PIKK) family member SMG-1 and mTOR complex 1 components are required for this tight control. Loss of smg-1 results in a hyper-responsiveness to injury and growth and the formation of regenerative blastemas that remain undifferentiated and that lead to lethal ectopic outgrowths. Invasive stem cell hyper-proliferation, hyperplasia, hypertrophy, and differentiation defects are hallmarks of this uncontrolled growth. These data imply a previously unappreciated and novel physiological function for this PIKK family member. In contrast we found that planarian members of the mTOR complex 1, tor and raptor, are required for the initial response to injury and blastema formation. Double smg-1 RNAi experiments with tor or raptor show that abnormal growth requires mTOR signalling. We also found that the macrolide rapamycin, a natural compound inhibitor of mTORC1, is able to increase the survival rate of smg-1 RNAi animals by decreasing cell proliferation. Our findings support a model where Smg-1 acts as a novel regulator of both the response to injury and growth control mechanisms. Our data suggest the possibility that this may be by suppressing mTOR signalling. Characterisation of both the planarian mTORC1 signalling components and another PIKK family member as key regulators of regeneration and growth will influence future work on regeneration, growth control, and the development of anti-cancer therapies that target mTOR signalling.     

An intriguing,new planarian species from Tasmania,with a discussion on protandry in triclad flatworms (Platyhelminthes,Tricladida)

An account is given of an unusual new species of freshwater planarian from the Hartz Mountains in Tasmania, Australia, Romankenkius flaccidus Sluys, sp. nov. The species is characterized, among other features, by an asymmetrical penis papilla, an extremely large, elongated copulatory bursa, and by the absence of testes in animals with fully developed male and female copulatory apparatus. Instigated by the sexual cycle of this new species, the study opportunely reviews whether planarian flatworms generally are simultaneous or sequential—in particular protandrous—hermaphrodites. It is concluded that real protandry does not occur in triclads and that even less extreme cases of sexual segregation, such as complete reduction of the testes or the more or less complete separation of male and female functionality as present in some species, have only sparsely and convergently evolved within the group of the triclad flatworms.     

A small scale expression screen identifies tissue specific markers in the Dugesia japonica strain Pek-1

Freshwater planaria has tremendous capacity to reform the missing part of the body and therefore is considered as one of the most important model organism for regeneration study.At present,Schmidtea mediterranea and Dugesia japonica are the two major species utilized for laboratory manipulations.Dugesia japonica flatworms are widely distributed in the Far East including Cherry Valley region in the north-west area of Beijing,China.We reported here the establishment of an asexual Dugesia japonica strain Pek-1,as a suitable system for regeneration study.Using morphological,karyotypical as well as phyiogenetic analyses,we confirmed that these flatworms indeed belonged to Dugesia japonica.We went on to show that the commonly used in situ probes and immunohistochemistry reagents and protocols were applicable to the Pek-1 strain.Using this strain,we carried out small scale analysis on EST,RNAi and gene expression.We identified 193 unique EST sequences and 65 of them had not been reported in planarian.By RNAi analysis,we showed that 48 genes,when down-regulated individually,had no effect on regeneration.Furthermore,we identified 3 groups of tissue specific expressing genes that were useful for cell lineage analysis.We concluded that the Dugesia japonica Pek-1 swain could be another suitable animal model to regeneration research.     

The evolution of the protonephridial terminal organ across Rotifera with particular emphasis on Dicranophorus forcipatus,Encentrum mucronatum and Erignatha clastopis (Rotifera: Dicranophoridae)

Riemann, O. and Ahlrichs, W.H. 2009. The evolution of the protonephridial terminal organ across Rotifera with particular emphasis on Dicranophorus forcipatus, Encentrum mucronatum and Erignatha clastopis (Rotifera: Dicranophoridae). —Acta Zoologica (Stockholm) 91 : 199–211 We report on the ultrastructure of the protonephridial terminal organ in three species of dicranophorid rotifers (Dicranophorus forcipatus, Encentrum mucronatum and Erignatha clastopis). Differences between the three species relate to shape and size, the morphology of the filter region and the number of microvilli and cilia inside the terminal organ. A comparison across Rotifera indicates that the terminal organs in D. forcipatus display a number of plesiomorphic characters, but are modified in E. mucronatum and Er. clastopis. This is in accordance with the results of phylogenetic analyses suggesting a basal position of D. forcipatus compared with the more derived species E. mucronatum and Er. clastopis. Moreover, we survey available data on the terminal organ in Rotifera and discuss its evolutionary transformations. The protonephridial terminal organ in the common ancestor of Rotifera consisted of a cytoplasmic cylinder with cilia united into a vibratile flame and a single circle of circumciliary microvilli. Depending on the topology on which characters are optimized, the site of ultrafiltration was formed by longitudinal cytoplasmic columns spanned by a fine filter diaphragm or by pores in the wall of the terminal organ. In several taxa of Rotifera, the terminal organ – probably independently – lost its circumciliary microvilli.