The BMP pathway is essential for re-specification and maintenance of the dorsoventral axis in regenerating and intact planarians

The bone morphogenetic protein (BMP) pathway has been shown to play an important role in the establishment of the dorsoventral axis during development in both vertebrate and invertebrate species. In an attempt to unravel the role of BMPs in pattern formation during planarian regeneration, we studied this signaling pathway in Schmidtea mediterranea. Here, we functionally characterize planarian homologues of two key elements of the pathway: Smed-BMP and Smed-Smad1. Whole-mount in situ hybridization showed that Smed-BMP is expressed at the planarian dorsal midline, suggesting a role in dorsoventral patterning, while Smed-Smad1 is widely expressed throughout the mesenchyme and in the central nervous system. RNA interference (RNAi) knockdowns of Smed-BMP or Smed-Smad1 led to the disappearance of dorsal markers along with the ectopic expression of ventral markers on the dorsal side of the treated animals. In almost all cases, a duplicated central nervous system differentiated dorsally after Smed-BMP or Smed-Smad1 RNAi. These defects were observed not only during regeneration but also in intact non-regenerating animals. Our results suggest that the BMP signaling pathway is conserved in planarians and that it plays a key role in the regeneration and maintenance of the dorsoventral axis.     

Spoltud-1 is a chromatoid body component required for planarian long-term stem cell self-renewal

Freshwater planarians exhibit a striking power of regeneration, based on a population of undifferentiated totipotent stem cells, called neoblasts. These somatic stem cells have several characteristics resembling those of germ line stem cells in other animals, such as the presence of perinuclear RNA granules (chromatoid bodies). We have isolated a Tudor domain-containing gene in the planarian species Schmidtea polychroa, Spoltud-1, and show that it is expressed in neoblast cells, germ line cells and central nervous system, and during embryonic development. Within the neoblasts, Spoltud-1 protein is enriched in chromatoid bodies. Spoltud-1 RNAi eliminates protein expression after 3 weeks, and abolishes the power of regeneration of planarians after 7 weeks. Neoblast cells are eliminated by the RNAi treatment, disappearing at the end rather than gradually during the process. Neoblasts with no detectable Spoltud-1 protein are able to proliferate and differentiate. These results suggest that Spoltud-1 is required for long term stem cell self renewal.     

Expression and functional analysis of musashi-like genes in planarian CNS regeneration

The remarkable regenerative ability of planarians is made possible by a system of pluripotent stem cells. Recent molecular biological and ultrastructural studies have revealed that planarian stem cells consist of heterogeneous populations, which can be classified into several subsets according to their differential expression of RNA binding protein genes. In this study, we focused on planarian musashi family genes. Musashi encodes an evolutionarily conserved RNA binding protein known to be expressed in neural lineage cells, including neural stem cells, in many animals. Here, we investigated whether planarian musashi-like genes can be used as markers for detecting neural fate-restricted cells. Three musashi family genes, DjmlgA, DjmlgB and DjmlgC (Dugesia japonica musashi-like gene A, B, C), and Djdmlg (Dugesia japonica DAZAP-like/musashi-like gene) were obtained by searching a planarian EST database and 5′ RACE, and each was found to have two RNA recognition motifs. We analyzed the types of cells expressing DjmlgA, DjmlgB, DjmlgC and Djdmlg by in situ hybridization, RT-PCR and single-cell RT-PCR analysis. Although Djdmlg was expressed in X-ray-sensitive stem cells and various types of differentiated cells, expression of the other three musashi-like genes was restricted to neural cells, as we expected. Further detailed analyses yielded the unexpected finding that these three planarian musashi family genes were predominantly expressed in X-ray-resistant differentiated neurons, but not in X-ray-sensitive stem cells. RNAi experiments suggested that these planarian musashi family genes might be involved in neural cell differentiation after neural cell-fate commitment.     

Wnt signaling is required for antero-posterior patterning of the planarian brain

Wnt signaling functions in axis formation and morphogenesis in various animals and organs. Here we report that Wnt signaling is required for proper brain patterning during planarian brain regeneration. We showed here that one of the Wnt homologues in the planarian Dugesia japonica, DjwntA, was expressed in the posterior region of the brain. When DjwntA-knockdown planarians were produced by RNAi, they could regenerate their heads at the anterior ends of the fragments, but formed ectopic eyes with irregular posterior lateral branches and brain expansion. This suggests that the Wnt signal may be involved in antero-posterior (A-P) patterning of the planarian brain, as in vertebrates. We also investigated the relationship between the DjwntA and nou-darake/FGFR signal systems, as knockdown planarians of these genes showed similar phenotypes. Double-knockdown planarians of these genes did not show any synergistic effects, suggesting that the two signal systems function independently in the process of brain regeneration, which accords with the fact that nou-darake was expressed earlier than DjwntA during brain regeneration. These observations suggest that the nou-darake/FGFR signal may be involved in brain rudiment formation during the early stage of head regeneration, and subsequently the DjwntA signal may function in A-P patterning of the brain rudiment.     

Multicellularity, stem cells, and the neoblasts of the planarian Schmidtea mediterranea

All multicellular organisms depend on stem cells for their survival and perpetuation. Their central role in reproductive, embryonic, and post-embryonic processes, combined with their wide phylogenetic distribution in both the plant and animal kingdoms intimates that the emergence of stem cells may have been a prerequisite in the evolution of multicellular organisms. We present an evolutionary perspective on stem cells and extend this view to ascertain the value of current comparative studies on various invertebrate and vertebrate somatic and germ line stem cells. We suggest that somatic stem cells may be ancestral, with germ line stem cells being derived later in the evolution of multicellular organisms. We also propose that current studies of stem cell biology are likely to benefit from studying the somatic stem cells of simple metazoans. Here, we present the merits of neoblasts, a largely unexplored, yet experimentally accessible population of stem cells found in the planarian Schmidtea mediterranea. We introduce what we know about the neoblasts, and posit some of the questions that will need to be addressed in order to better resolve the relationship between planarian somatic stem cells and those found in other organisms, including humans.     

The Drosophila RNA-binding protein ELAV is required for commissural axon midline crossing via control of commissureless mRNA expression in neurons

Drosophila ELAV is the founding member of an evolutionarily conserved family of RNA-binding proteins considered as key inducers of neuronal differentiation. Although several ELAV-specific targets have been identified, little is known about the role of elav during neural development. Here, we report a detailed characterization of the elav mutant commissural phenotype. The reduced number of commissures in elav mutant embryos is not due to loss or misspecification of neural cells but results from defects in commissural axon projections across the midline. We establish a causal relationship between the elav mutant commissural phenotype and a reduction in the expression of commissureless, a key component of the Robo/Slit growth cone repulsive signalling pathway. In the nerve cord of elav mutant embryos, comm mRNA expression is strongly reduced in neurons, but not in midline glial cells. Furthermore, specific expression of an elav transgene in posterior neurons of each segment of an elav mutant nerve cord restores comm mRNA expression in these cells, as well as the formation of posterior commissures. Finally, forced expression of comm in specific commissural neuron subsets rescues the midline crossing defects of these neurons in elav mutant embryos, further indicating that elav acts cell autonomously on comm expression.     

A mortalin-like gene is crucial for planarian stem cell viability

In adult organisms, stem cells are crucial to homeostasis and regeneration of damaged tissues. In planarians, adult stem cells (neoblasts) are endowed with an extraordinary replicative potential that guarantees unlimited replacement of all differentiated cell types and extraordinary regenerative ability. The molecular mechanisms by which neoblasts combine long-term stability and constant proliferative activity, overcoming the impact of time, remain by far unknown. Here we investigate the role of Djmot, a planarian orthologue that encodes a peculiar member of the HSP70 family, named Mortalin, on the dynamics of stem cells of Dugesia japonica. Planarian stem cells and progenitors constitutively express Djmot. Transient Djmot expression in differentiated tissues is only observed after X-ray irradiation. DjmotRNA interference causes inability to regenerate and death of the animals, as a result of permanent growth arrest of stem cells. These results provide the first evidence that an hsp-related gene is essential for neoblast viability and suggest the possibility that high levels of Djmot serve to keep a p53-like protein signaling under control, thus allowing neoblasts to escape cell death programs. Further studies are needed to unravel the molecular pathways involved in these processes.     

A Xenopus tribbles orthologue is required for the progression of mitosis and for development of the nervous system

The product of the Drosophila gene tribbles inhibits cell division in the ventral furrow of the embryo and thereby allows the normal prosecution of gastrulation. Cell division is also absent in involuting dorsal mesoderm during gastrulation in Xenopus, and to ask whether the two species employ similar mechanisms to coordinate morphogenesis and the cell cycle, we isolated a putative Xenopus homologue of tribbles which we call Xtrb2. Extensive cDNA cloning identified long and short forms of Xtrb2, termed Xtrb2-L and Xtrb2-S, respectively. Xtrb2 is expressed maternally and in mesoderm and ectoderm at blastula and gastrula stages. Later, it is expressed in dorsal neural tube, eyes, and cephalic neural crest. Time-lapse imaging of GFP-tagged Xtrb2-L suggests that during cell division, it is associated with mitotic spindles. Knockdown of Xtrb2 by antisense morpholino oligonucleotides (MOs) disrupted synchronous cell divisions during blastula stages, apparently as a result of delayed progression through mitosis and cytokinesis. At later stages, tissues expressing the highest levels of Xtrb2 were most markedly affected by morpholino knockdown, with perturbation of neural crest and eye development.     

UNC-6 expression by the vulval precursor cells of Caenorhabditis elegans is required for the complex axon guidance of the HSN neurons

Netrin is an evolutionarily conserved axon guidance molecule that has both axonal attraction and repulsion activities. In Caenorhabditis elegans, Netrin/UNC-6 is secreted by ventral cells, attracting some axons ventrally and repelling some axons, which extend dorsally. One axon guided by UNC-6 is that of the HSN neuron. The axon guidance process for HSN neurons is complex, consisting of ventral growth, dorsal growth, branching, second ventral growth, fasciculation with ventral nerve cords, and then anterior growth. The vulval precursor cells (VPC) and the PVP and PVQ neurons are required for the HSN axon guidance; however, the molecular mechanisms involved are completely unknown. In this study, we found that the VPC strongly expressed UNC-6 during HSN axon growth. Silencing of UNC-6 expression in only the VPC, using a novel tissue-specific RNAi technique, resulted in abnormal HSN axon guidance. The expression of Netrin/UNC-6 by only the VPC in unc-6 null mutants partially rescued the HSN ventral axon guidance. Furthermore, the expression of Netrin/UNC-6 by the VPC and the ventral nerve cord (VNC) in unc-6 null mutants restored the complex HSN axon guidance. These results suggest that UNC-6 expressed by the VPC and the VNC cooperatively regulates the complex HSN axon guidance.     

Analysis of planarian Adh3 supports an intron-rich architecture and tissue-specific expression for the urbilaterian ancestral form

The alcohol dehydrogenase class 3 enzyme (ADH3) is the presumed ancestral form of the medium-chain dehydrogenase-reductase ADH family. This enzyme has been involved in formaldehyde and nitric oxide metabolism of a variety of deuterostomes and ecdysozoan protostomes. We have now characterized the structure and expression of the Adh3 gene in the lophotrochozoan Schmidtea mediterranea, a freshwater planarian. The planarian gene expands over 8.7 kb and is organized into 7 exons. The 1340 bp long Adh3cDNA contains a 1137 bp open reading frame corresponding to a deduced protein of 379 amino acids. The protein sequence is consistent with that expected for a typical class III enzyme. Twenty out of the twenty-two amino acid positions associated with enzymatic roles are strictly preserved, which suggests that the enzymatic capabilities have been conserved. In situ hybridization experiments show that Adh3 is expressed along the intestine of S. mediterranea specimens. This is consistent with the pattern observed in invertebrates and in contrast with the widespread expression of vertebrate Adh3. The comparative study across bilateria, which now includes a lophotrochozoan representative, further supports the idea that the urbilaterian Adh3 ancestor showed an intron-rich architecture and tissue-specific expression, and strengthens the view that widespread expression of Adh3 was a vertebrate innovation.     

Fgf19 regulated by Hh signaling is required for zebrafish forebrain development

Fibroblast growth factor (Fgf) signaling plays important roles in brain development. Fgf3 and Fgf8 are crucial for the formation of the forebrain and hindbrain. Fgf8 is also required for the midbrain to form. Here, we identified zebrafish Fgf19 and examined its roles in brain development by knocking down Fgf19 function. We found that Fgf19 expressed in the forebrain, midbrain and hindbrain was involved in cell proliferation and cell survival during embryonic brain development. Fgf19 was also essential for development of the ventral telencephalon and diencephalon. Regional specification is linked to cell type specification. Fgf19 was also essential for the specification of gamma-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes generated in the ventral telencephalon and diencephalon. The cross talk between Fgf and Hh signaling is critical for brain development. In the forebrain, Fgf19 expression was down-regulated on inhibition of Hh but not of Fgf3/Fgf8, and overexpression of Fgf19 rescued partially the phenotype on inhibition of Hh. The present findings indicate that Fgf19 signaling is crucial for forebrain development by interacting with Hh and provide new insights into the roles of Fgf signaling in brain development.     

Cell death and tissue remodeling in planarian regeneration

Many long-lived organisms, including humans, can regenerate some adult tissues lost to physical injury or disease. Much of the previous research on mechanisms of regeneration has focused on adult stem cells, which give rise to new tissue necessary for the replacement of missing body parts. Here we report that apoptosis of differentiated cells complements stem cell division during regeneration in the planarian Schmidtea mediterranea. Specifically, we developed a whole-mount TUNEL assay that allowed us to document two dramatic increases in the rate of apoptosis following amputation—an initial localized response near the wound site and a subsequent systemic response that varies in magnitude depending on the type of fragment examined. The latter cell death response can be induced in uninjured organs, occurs in the absence of planarian stem cells, and can also be triggered by prolonged starvation. Taken together, our results implicate apoptosis in the restoration of proper anatomical scale and proportion through remodeling of existing tissues. We also report results from initial mechanistic studies of apoptosis in planarians, which revealed that a S. mediterranea homolog of the antiapoptotic gene BCL2 is required for cell survival in adult animals. We propose that apoptosis is a central mechanism working in concert with stem cell division to restore anatomical form and function during metazoan regeneration.     

Identification and expression analysis of a heat-shock protein 70 gene in Polycelis sp.

Heat-shock protein 70 (HSP70) is ubiquitously found in a variety of organisms and plays an important role in cytoprotection, environmental monitoring, and disease resistance. In this study, the full-length complementary DNA (cDNA) of hsp70 from planarian Polycelis sp. was first cloned using rapid amplification of cDNA ends (RACE). The expression levels of Pyhsp70 were analyzed in the presence of various stressors by real-time PCR, and its temporal-spatial expression patterns were also examined in both intact and regenerative animals by whole-mount in situ hybridization. The results show that (1) the deduced amino acid sequence of Pyhsp70 includes three typical HSP70 family signature motifs and is highly conserved during evolution; (2) Pyhsp70 expression is induced by prolonged starvation, tissue damage, and ionic liquid but inhibited by high or low temperatures; and (3) Pyhsp70 mRNA is mainly expressed in the head peripheral region and in the regenerating blastema during regeneration. These results suggest that the highly expressed Pyhsp70 gene may contribute to enhance cytoprotection and tolerance against stress-induced molecular damage, and the migration of neoblasts to the wound, which might also be involved in the proliferation and differentiation of neoblasts. Our work provides basic data for the study of stress responses and regenerative mechanism in freshwater planarians.     

Fgf19 is required for zebrafish lens and retina development

Fgf signaling plays crucial roles in morphogenesis. Fgf19 is required for zebrafish forebrain development. Here, we examined the roles of Fgf19 in the formation of the lens and retina in zebrafish. Knockdown of Fgf19 caused a size reduction of the lens and the retina, failure of closure of the choroids fissure, and a progressive expansion of the retinal tissue to the midline of the forebrain. Fgf19 expressed in the nasal retina and lens was involved in cell survival but not cell proliferation during embryonic lens and retina development. Fgf19 was essential for the differentiation of lens fiber cells in the lens but not for the neuronal differentiation and lamination in the retina. Loss of nasal fate in the retina caused by the knockdown of Fgf19, expansion of nasal fate in the retina caused by the overexpression of Fgf19 and eye transplantation indicated that Fgf19 in the retina was crucial for the nasal-temporal patterning of the retina that is critical for the guidance of retinal ganglion cell axons. Knockdown of Fgf19 also caused incorrect axon pathfinding. The present findings indicate that Fgf19 positively regulates the patterning and growth of the retina, and the differentiation and growth of the lens in zebrafish.     

Drosophila lacking the Cdk5 activator, p35, display defective axon guidance, age-dependent behavioral deficits and reduced lifespan

The cyclin-dependent kinase Cdk5 has attracted a great deal of attention both because of its roles in cell migration and axon patterning, and the extensive data implicating it in adult-onset neurodegeneration in mammals. Both the kinase activity and the biological effects of Cdk5 are absolutely dependent on association with an activating subunit, called p35. We show here that Drosophila lacking the Cdk5 activator, D-p35, display a wide range of defects in embryonic axon patterning. We further show that, while viable and fertile, p35 mutant adults display progressive, age-dependent loss of motor function and have a significantly shortened lifespan.