Emili Saló

• Professor (Full) at University of Barcelona

The capability to simultaneously apply different molecular tools to visualize a wide variety of changes in genetic expression and tissue composition in Schmidtea mediterranea has always been of great interest. The most commonly used techniques are fluorescent in situ hybridization (FISH) and immunofluorescence (IF) detection. Here, we describe a no...

For successful regeneration, the identity of the missing tissue must be specified according to the pre-existing tissue. Planarians are ideal for the study of the mechanisms underlying this process; the same field of cells can regrow a head or a tail according to the missing body part. After amputation, the differential activation of the Wnt/β-caten...

Forkhead box (Fox) genes belong to the “winged helix” transcription factor superfamily. The function of some Fox genes is well known, such as the role of foxO in controlling metabolism and longevity and foxA in controlling differentiation of endodermal tissues. However, the role of some Fox factors is not yet well characterized. Such is the case of...

The forkhead box (Fox) genes encode transcription factors that control several key aspects of development. Present in the ancestor of all eukaryotes, Fox genes underwent several duplications followed by loss and diversification events that gave rise to the current 25 families. However, few Fox members have been identified from the Lophotrochozoa cl...

An organizer is defined as a group of cells that secrete extracellular proteins that specify the fate of surrounding cells according to their concentration. Their function during embryogenesis is key in patterning new growing tissues. Although organizers should also participate in adult development when new structures are regenerated, their presenc...

Embryonic specification of the first body axis requires the formation of an Organizer, a group of cells with the ability to instruct fates in the surrounding tissue. The existence of organizing regions in adults, i.e. during regeneration, which also requires patterning of new tissues, remains unstudied. To that aim, we study regeneration in planari...

The forkhead box (Fox) genes encode transcription factors that control several key aspects of development. Present in the ancestor of all eukaryotes, Fox genes underwent several duplications followed by loss and diversification events that gave rise to the current 25 families. However, few Fox members have been identified from the Lophotrochozoa cl...

Although many examples of simulated and real microgravity demonstrating their profound effect on biological systems are described in literature, few reports deal with hypergravity and vibration effects, the levels of which are severely increased during the launch preceding the desired microgravity period. Here, we used planarians, flatworms that ca...

Control of cell number is crucial to define body size during animal development and to restrict tumoral transformation. The cell number is determined by the balance between cell proliferation and cell death. Although many genes are known to regulate those processes, the molecular mechanisms underlying the relationship between cell number and body s...

In the last years, the Hippo signaling has emerged as an essential pathway to control tissue and organ homeostasis, and its deregulation leads to tumoral transformation in all animals studied. However, the underlying cellular and molecular mechanism of this transformation are still not clear. The core of the Hippo pathway includes a kinase cassette...

The possibility of humans to live outside of Earth on another planet has attracted the attention of numerous scientists around the world. One of the greatest difficulties is that humans cannot live in an extra-Earth environment without proper equipment. In addition, the consequences of chronic gravity alterations in human body are not known. Here,...

Tissue transplantation is an important tool for in vivo studies of pattern organization and axis establishment or maintenance in planarians. Further, transplants can inform on cell movements under different regenerative conditions or genetic backgrounds. Here, we detail classical grafting strategies that have been variously employed in planarian re...

Freshwater planarians are well known for their remarkable plasticity and regenerative capabilities. Most studies of planarian regeneration have specifically examined regeneration after transverse or longitudinal sectioning or during homeostasis in intact adults. However, tissue transplantation, first performed over a century ago, constitutes anothe...

The Hippo pathway plays a key role in regulating cell turnover in adult tissues, and abnormalities in this pathway are consistently associated with human cancers. Hippo was initially implicated in the control of cell proliferation and death, and its inhibition is linked to the expansion of stem cells and progenitors, leading to larger organ size an...

Cellular and molecular analysis of overgrowths and unpigmented regions in hippo (RNAi) animals. (A) Analysis of overgrowths. FISH combined with immunostaining showing the localization of smedwi-1 mRNA and SMEDWI-1 protein. Colocalization of both signals appears to be concentrated in the overgrowths, indicating that they consist of undifferentiated...

Yorkie may be one of the Hippo pathway effectors in planarians. (A) Western blot of protein extracts from hippo (RNAi), yorkie (RNAi), and control planarians immunoblotted with anti-Yorkie and anti-α-Tubulin antibodies. Extracts correspond to intact animals after 3 weeks (hippo (RNAi)) or 1 week (yorkie (RNAi)) of RNAi. The corresponding quantifica...

Hippo is essential for G2/M transition and M exit in planarians. (A) Cartoon illustrating the EdU pulse procedure. Animals were starved for 1 week, injected with hippo dsRNA for 3 weeks, and then injected with EdU and fixed 16 h later. (B) EdU labeling in transverse sections combined with immunostaining with anti-H3P antibody in the pharynx region...

A hippo-yki signal regulates cell differentiation during planarian regeneration. (A) Cartoon illustrating the RNAi procedure in regenerating conditions. Animals were starved for 1 week before the experiment and then injected on 3 consecutive days. The following week, animals were injected again on 3 consecutive days, cut the next day, and fixed at...

Bcat2 staining of a planarian following hippo RNAi. Movie showing anti-Bcat2 immunostaining of a region corresponding to an overgrowth formed following hippo inhibition. Nuclei are labeled with DAPI. Anti-Bcat2 antibody nonspecifically labels adherens junctions in epithelial cells and muscle fibers. Accumulation of nuclei can be observed from the i...

Bcat2 and H3P staining of a hippo (RNAi) planarian. Movie showing anti-Bcat2 and anti-H3P immunostaining of a region corresponding to an overgrowth in a hippo (RNAi) animal. Both antibodies are shown in red. The H3P signal but not the Bcat2 signal colocalizes with nuclear DAPI staining. Anti-Bcat2 nonspecifically labels adherens junctions in epithe...

Bcat2 and H3P staining in a control planarian. Movie showing anti-Bcat2 and H3P immunostaining in a control animal. Both antibodies are shown in red. The H3P signal but not the Bcat2 signal colocalizes with nuclear DAPI staining. Anti-Bcat2 nonspecifically labels adherens junctions in epithelial cells and muscle fibers. H3P+ nuclei are observed in...

All individual numerical values that underlie the summary data shown in the following figures: Fig 1B, 1C, 1D and 1E; Fig 2A, 2B and 2C; Fig 3E; Fig 4A and 4C; Fig 5A, 5B′, 5B″ and 5C; Fig 6A, 6B and 6C; S1D and S1E Fig; S2B and S2D Fig; S5 Fig; S6D Fig; S7A and S7B Fig; S8D Fig; S9C and S9D Fig; and S10A, S10C and S10D Fig. (XLSX)

Hippo is ubiquitously expressed in planarians and hippo RNAi produces overgrowths. (A) hippo expression levels in different cell types according to a single-cell RNAseq analysis (74). (B) In situ hybridization for hippo reveals a ubiquitous expression pattern. (C) Cartoon illustrating the experimental design used for hippo RNAi during planarian hom...

Hippo controls both cell death and mitotic levels in planarians. (A) Whole-mount TUNEL showing apoptotic cell death in planarians subjected to hippo RNAi for 3 weeks (n ≥ 5). Images correspond to confocal Z-projections. (B) Quantification of caspase-3 activity after 1, 2, and 3 weeks of hippo inhibition. Results are presented as units of caspase-3...

Inhibition of hippo increases in vivo PI incorporation. Staining of dead cells using PI in live control and hippo (RNAi) animals. Nuclei are stained with Hoechst. Magnifications of the indicated area are shown below. Arrowhead indicates some cells positive for PI. A stereomicroscopic view of live control and hippo (RNAi) animals used in the experim...

Analysis of mitotic cells and epidermal lineage markers. (A) FISH showing colocalization of hippo with smedwi-1, nb32, and agat. (B) Immunostaining using anti-H3P antibody in transverse sections. Nuclei are stained with DAPI. White arrows indicate H3P+ cells. (C) FISH with smedwi-1, nb32, and agat combined with immunostaining with anti-H3P. Nuclei...

Analysis of X-ray-irradiated/hippo(RNAi) animals. (A) Cartoon illustrating the irradiation and dsRNA injection procedures. Animals were starved for 1 week, exposed to different doses of radiation (60 Gy and 17.5 Gy), and injected with hippo or gfp dsRNA for 4 consecutive days. Animals were fixed 7 days after the beginning of the experiment. (B) In...

Molecular analysis of h2b/hippo (RNAi) animals. (A) Relative expression of h2b and hippo, as determined by qRT-PCR, in planarians 4 days postinjection. Values represent the means of 3 biological replicates. Error bars represent standard deviation. Data were analyzed by Student t test. *p < 0.05; ***p < 0.001. (B) EdU labeling in transverse paraffin...

Up- and down-regulation in the hippo (RNAi) RNAseq of genes involved in the cell cycle. Selected up- or down-regulated genes in the RNAseq of hippo (RNAi) animals involved in cell cycle. Each column contains the following information: PlanMine code (74), gene name, gene symbol, E-val (indicating homology with the predicted gene), fold change, adjus...

Why some animals can regenerate and others not has fascinated biologists since the first examples of regeneration were reported. Although many animal phyla include species with some regenerative ability, mainly restricted to particular cell types or tissues, there are some other species capable of regenerating complex structures, such as the verteb...

How adult stem cells differentiate into different cell types remains one of the most intriguing questions in regenerative medicine. Pioneer factors are transcription factors that can bind to and open chromatin, and are among the first elements involved in cell differentiation. We used the freshwater planarian Schmidtea mediterranea as a model syste...

The freshwater planarian Schmidtea mediterranea is recognised as a valuable model for research into adult stem cells and regeneration. With the advent of the high-throughput sequencing technologies, it has become feasible to undertake detailed transcriptional analysis of its unique stem cell population, the neoblasts. Nonetheless, a reliable refere...

The phylum Platyhelminthes comprises dorso-ventrally flattened worms commonly known as flatworms (from the Greek platys, meaning flat, and helminthos, meaning worm) (for a general overview of this phylum, see Hyman 1–951; Rieger et al. 1–991). Platyhelminthes are one of the largest animal phyla after arthropods, mollusks, and chordates and includes...

Some species of Platyhelminthes have become model systems in which to study whole–013;body regeneration in adults. Before describing how this capacity is distributed and varies within the phylum, however, it is important to introduce the adult pluripotent stem cells that confer this remarkable ability in flatworms, the so–013;called neoblasts.

Planarians are flatworms, which belong to the phylum Platyhelminthes. They have been a classical subject of study due to their amazing regenerative ability, which relies on the existence of adult totipotent stem cells. Nowadays they are an emerging model system in the field of developmental, regenerative, and stem cell biology. In this study we ana...

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique mo...

Matrix metalloproteinases (MMPs) are major executors of extracellular matrix remodeling and, consequently, play key roles in the response of cells to their microenvironment. The experimentally accessible stem cell population and the robust regenerative capabilities of planarians offer an ideal model to study how modulation of the proteolytic system...

Smed-mt-mmpB(RNAi) does not produce any phenotype. (A) Bright-field images of head and tail fragments at 5 days of regeneration in S. mediterranea. Control: β-gal(RNAi), mt-mmpA/mt-mmpB(RNAi): simultaneous Smed-mt-mmpA(RNAi) and Smed-mt-mmpB(RNAi). No morphological difference between the β-gal(RNAi) controls and Smed-mt-mmpB(RNAi) animals is detect...

Characterization of mt-mmpA(RNAi) phenotype in D. japonica, at 5 days of regeneration. (A) Bright–field images of head and tail fragments, after two rounds of dsRNA injections and amputation. Control: β-gal(RNAi) fragments. Following Dj-mt-mmpA(RNAi) the fragments show a significantly reduced blastema. Relative blastema size between Dj-mt-mmpA(RNAi...

Analysis of efficiency and specificity of each RNAi silencing in S. mediterranea. (A) Smed-mmp1 expression level as detected by WISH and Real Time RT-PCR in controls and Smed-mmp1(RNAi) demonstrates the efficiency of the treatment. Moreover, Smed-mmp1(RNAi) does not affect the expression level of Smed-mmp2, Smed-mt-mmpA and Smed-mt-mmpB. (B) Smed-m...

Immunostaining with anti-H3P antibody in Smed-mt-mmpA(RNAi) fragments, at 6 days of head regeneration. (A) β-gal(RNAi) control. (B) Smed-mt-mmpA(RNAi). After Smed-mt-mmpA(RNAi) silencing the regenerative blastema results reduced in size and a smaller number of anti-H3P-positive cells migrated into the blastema with respect to the control. Scale bar...

Amino acid sequence alignment of MMP1, MMP2, MT-MMPA and MT-MMPB from S. mediterranea and D. japonica. (A) S. mediterranea SMED-MMP1 and D. japonica DJ-MMP1. SMED-MMP1 is 495 amino acids in length. The pro-domain consists of 80 amino acids (1 to 80) and contains the pro-peptide cleavage site in position 18 and an atypical cysteine switch (GRCGGKD,...

Expression pattern of mmp1 and mmp2 in S. mediterranea. WISH depicting the distribution of Smed-mmp1 and Smed-mmp2 expression on intact planarians. (A) Smed-mmp1 antisense probe. (B) Smed-mmp2 antisense probe. Anterior to the left. To characterize further the cell types expressing Smed-mmp1 and Smed-mmp2, ISH experiments were carried out on wax lon...

Expression of mt-mmpA in S. mediterranea. (A) WISH with antisense Smed-mt-mmpA probe in intact planarians detects widespread expression, while lack of signal with sense Smed-mt-mmpA probe confirms the specificity of the expression. Anterior is up. Scale bar: 1 mm. (B) Real Time RT-PCR analysis of Smed-mt-mmpA expression level in lethally-irradiated...

Smed-mmp1(RNAi) does not inhibit regeneration. Bright-field images of head and tail fragments of water-injected controls and following Smed-mmp1(RNAi), as visualized at 3 days after amputation in S. mediterranea. Neither blastema formation nor blastema size are altered in Smed-mmp1(RNAi) regenerating head and tail fragments. Anterior is up. Scale b...

TUNEL assay after Dj-mmp1(RNAi) in D. japonica. Apoptotic cells are detected as red spots in longitudinal cryosections, visualized at the pre-pharyngeal level in an intact D. japonica. (A) A water-injected control. (B) A Dj-mmp1(RNAi) planarian. e: eye. Anterior is to the left. Consistent with the data obtained in S. mediterranea, a decrease in the...

Intron-exon organization and phylogeny of the planarian mmp genes. Exons shown in orange boxes are joined by introns denoted by grey lines. Green boxes represent 3′ UTRs. The number of the exon is indicated in the boxes. The diagram is to scale. Kbp: kilo base pairs. (A) Smed-mmp1 is organized in 10 exons and 9 introns. (B) Smed-mmp2 is organized i...

Characterization of Smed-mt-mmpA(RNAi) phenotypes in intact S. mediterranea. (A) Bright-field image of a β-gal(RNAi) control and a Dj-mt-mmpA(RNAi) planarian, at 8 days after the first injection. Dorsal view, anterior is up. Scale bar: 1 mm. (B) Real Time RT-PCR analysis reveals that the expression level of both Smedwi-1 and Smed-prog-1 is not affe...

Planarians of the species Schmidtea mediter- ranea are a well-established model for regeneration studies. In this paper, we first recall the morphological characters and the molecular mechanisms involved in the regeneration process, especially focussing on the Wnt pathway and the establishment of the antero- posterior axial polarity. Then, after an...

The term planarian is derived from the Latin planus (meaning flat) and refers to worms of the order Tricladida (phylum Platyhelminthes) whose body is flattened along the dorso-ventral axis. Planarians are best known for their ability to regenerate complete animals from tiny fragments of tissue, and for their capacity to continually remodel both the...

Planarians represent an excellent model to study the processes of body axis and organ re-specification during regeneration. Previous studies have revealed a conserved role for the bone morphogenetic protein (BMP) pathway and its intracellular mediators Smad1/5/8 and Smad4 in planarian dorsoventral (DV) axis re-establishment. In an attempt to gain f...

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 identit...

During regeneration, lost structures are rebuilt and perfectly integrated within the remaining non-injured tissues. This fascinating process captured the attention of one of the founders of modern genetics, T.H. Morgan. He was particularly interested in understanding regeneration in freshwater planarians, which can regenerate a whole animal from a...

Details on Material and Methods. An extended description of the proteomics protocols applied to perform the analyses presented on this paper.

Image scans of all silver-stained 2D gel replicates. Image scans of different and independent silver-stained 2D gels used in the study. A to D and the respective zooms, for the regions delimited by red squares, I to L, come from 100 μg of loaded samples. E to H and the respective zooms M to P correspond to 500 μg loaded samples. A, C, E and G are c...

Comparing the results presented in this manuscript with previously published studies relating to stem cells. Comparison of candidate neoblast protein sequences presented in this paper with genes reported in other proteomic studied to be related to stem cells [25-30] and with specific neoblast-related genes identified in two different high-throughpu...

Table of peptide candidates. Listing of the sequence candidates obtained from the computational analysis of the raw proteomics data over the RefSeq and URF datasets (see the corresponding sheet on the spreadsheet file). Only those with a significant BLAST hit are shown (using BLASTP against NCBI-nr, min e-value = 0.001, min hsp length = 25). Genes...

The Wingless/Integrated (Wnt) signaling pathway controls multiple events during development and homeostasis. It comprises multiple branches, mainly classified according to their dependence on β-catenin activation. The Wnt/β-catenin branch is essential for the establishment of the embryonic anteroposterior (AP) body axis throughout the phylogenetic...

Planarians regenerate a whole animal from a small body piece within a few days. Recent studies have shown that the bone morphogenetic protein (BMP) pathway is required to reestablish the dorsoventral (DV) axis. In vertebrates, the specification of the DV axis depends on the coordinated action of a dual organizer defined by BMP and antidorsalizing m...

In recent years, planaria have emerged as an important model system for research into stem cells and regeneration. Attention is focused on their unique stem cells, the neoblasts, which can differentiate into any cell type present in the adult organism. Sequencing of the Schmidtea mediterranea genome and some expressed sequence tag projects have gen...

The emergence of regenerative medicine has raised the hope of treating an extraordinary range of diseases and serious injuries. However, to achieve such important goals, fundamental questions must first be answered, such as why regeneration occurs in some animals and not others. Thus, understanding the processes of cell proliferation, differentiati...

Summary report for the consensus set of 4,663 predicted transmembrane proteins including functional annotations. (Header: Sequence_ID = Protein sequence identifier, Sequence_AA = Amino acid sequence, Length[aa] = Length of amino acid sequence, Phobius_TM = Phobius prediction of number of transmembrane domains, Phobius_SP = Phobius prediction of sig...

List of 90e transcripts validated by RT-PCR. (Header: # = Number, CONTIG=90e contig ID, PRIMER_FORWARD = 5' to 3' sequence of the forward primer used, REVERSE_FORWARD = 5' to 3' sequence of the reverse primer used, AMPLICON SIZE = Size amplified in bp, SET = refers to the subset of origin of the 90e contig: no hit genome, hit genome, - blast (no BL...

Smed454 sequences matching known Schmidtea mediterranea genes. (Header: ACCESSION NUMBER = Known gene sequence identifier as target, NAME = Description for that sequence, LENGTH = Nucleotide length for that sequence, A&T CONTENT = Sequence composition, 454 90e CONTIG/SINGLETON = Smed454 sequence identifier as query, LENGTH = Nucleotide sequence len...

Splice sites for a subset of Smed454 sequences mapped onto the Schmidtea mediterranea genome. (Header: GID = Genomic contig IDentifier from WUSLv3.1 genome assembly--including the start and end nucleotide coordinates for the complete match--, CIG=90e contig IDentifier, INTNUM = Intron number within the 90e contig, EXO = splice signals found by exon...

List of cell cycle, cell division, DNA repair or DNA damage candidates. Short list of candidates annotated as genes involved in cell cycle, cell division, DNA repair or DNA damage. (Header: ID = Smed454 sequence identifier, BLASTX HIT = Description of the best sequence hit, ACCESSION NUMBER = Sequence identifier of the best sequence hit, E-VALUE =...

List of neurotransmitter, peptide and hormone receptor sequence candidates. Complete complement of Smed454 dataset contigs and singletons showing homology to neurotransmitter and hormone receptors, totalling 287 sequences. (Header: ID = Smed454 sequence identifier, BLASTX HIT = Description of the best sequence hit, ACCESSION NUMBER = Sequence ident...

List of eye-related gene sequence candidates. Complete complement of Smed454 dataset contigs and singletons showing homology to eye-related genes, totalling 95 sequences. (Header: ID = Smed454 sequence identifier, BLASTX HIT = Description of the best sequence hit, ACCESSION NUMBER = Sequence identifier of the best sequence hit, E-VALUE = BLASTX e-v...

GO annotation for 90e contigs not mapping onto the WUSL 3.1 genome assembly. 8,831 90e contigs were not found in the genome. 3,480 had a BLASTX hit to a sequence of NCBI NRprot; yet only 2,401 had a hit to a protein functionally annotated in the GO database. This file contains the description of the best HSP for 71 of those annotated contigs, after...

Gene Ontology for all three Smed454 sets: 90, 98 and 90e. Level one and two GO codes are shown in order to simplify the listings. Although there are small changes in GO frequencies, annotation is consistent throughout all three sets. (Header: GO = Gene Ontology unique identifier, Count = Number of sequences with a given GO annotation, Freq% = Frequ...

Freshwater planarians are an attractive model for regeneration and stem cell research and have become a promising tool in the field of regenerative medicine. With the availability of a sequenced planarian genome, the recent application of modern genetic and high-throughput tools has resulted in revitalized interest in these animals, long known for...

beta-Catenin is a bifunctional protein participating in both cell adhesion and canonical Wnt signaling. In cell adhesion, it bridges the transmembrane cadherin and the actin-binding protein alpha-catenin and is essential for adherens junction formation, whereas in canonical Wnt signaling, it shuttles between the cytosol and nucleus and functions as...

Planarians are an ideal model system to study in vivo the dynamics of adult pluripotent stem cells. However, our knowledge of the factors necessary for regulating the 'stemness' of the neoblasts, the adult stem cells of planarians, is sparse. Here, we report on the characterization of the first planarian member of the LSm protein superfamily, Smed-...

Adult planarians are capable of undergoing regeneration and body remodelling in order to adapt to physical damage or extreme environmental conditions. Moreover, most planarians can tolerate long periods of starvation and during this time, they shrink from an adult size to, and sometimes beyond, the initial size at hatching. Indeed, these properties...

Planarian regeneration was one of the first models in which the gradient concept was developed. Morphological studies based on the analysis of the regeneration rates of planarian fragments from different body regions, the generation of heteromorphoses, and experiments of tissue transplantation led T.H. Morgan (1901) and C.M Child (1911) to postulat...

Planarians can regenerate a whole animal from only a small piece of their body, and have become an important model for stem cell biology. To identify regenerative processes dependent on Wnt growth factors in the planarian Schmidtea mediterranea (Smed), we analyzed RNAi phenotypes of Evi, a transmembrane protein specifically required for the secreti...

Noggin genes are mainly known as inhibitors of the Bone Morphogenetic Protein (BMP) signalling pathway. Noggin genes play an important role in various developmental processes such as axis formation and neural differentiation. In vertebrates, inhibition of the BMP pathway is usually carried out together with other inhibitory molecules: chordin and f...

The structural simplicity of the eye in planarians, combined with the regenerative abilities of these organisms, provides a unique system for dissecting the genetic mechanisms that allow a simple visual structure to be built and may provide insights into the morphogenesis of more complex eyes. Information emerging from the past few years of researc...