LIM-HD transcription factors control axial patterning and specify distinct neuronal and intestinal cell identities in planarians.

Adult planarians can regenerate the gut, eyes and even a functional brain. Proper identity and patterning of the newly formed structures require signals that guide and commit their adult stem cells. During embryogenesis, LIM-homeodomain (LIM-HD) transcription factors act in a combinatorial 'LIM code' to control cell fate determination and differentiation. However, our understanding about the role these genes play during regeneration and homeostasis is limited. Here, we report the full repertoire of LIM-HD genes in Schmidtea mediterranea. We found that lim homeobox (lhx) genes appear expressed in complementary patterns along the cephalic ganglia and digestive system of the planarian, with some of them being co-expressed in the same cell types. We have identified that Smed-islet1, -lhx1/5-1, -lhx2/9-3, -lhx6/8, -lmx1a/b-2 and -lmx1a/b-3 are essential to pattern and size the planarian brain as well as for correct regeneration of specific subpopulations of dopaminergic, serotonergic, GABAergic and cholinergic neurons, while Smed-lhx1/5.2 and -lhx2/9.2 are required for the proper expression of intestinal cell type markers, specifically the goblet subtype. LIM-HD are also involved in controlling axonal pathfinding (lhx6/8), axial patterning (islet1, lhx1/5-1, lmx1a/b-3), head/body proportions (islet2) and stem cell proliferation (lhx3/4, lhx2/9-3, lmx1a/b-2, lmx1a/b-3). Altogether, our results suggest that planarians might present a combinatorial LIM code that controls axial patterning and axonal growing and specifies distinct neuronal and intestinal cell identities.

Colorimetric Whole-Mount In Situ Hybridization in Planarians.

Whole-mount in situ hybridization (WISH) is an extremely useful technique for visualizing specific mRNA targets and solving many biological questions. In planarians, this method is really valuable, for example, for determining gene expression profiles during whole-body regeneration and analyzing the effects of silencing any gene to determine their functions. In this chapter, we present in detail the WISH protocol routinely used in our lab, using a digoxigenin-labelled RNA probe and developing with NBT-BCIP. This protocol is basically that already described in Currie et al. (EvoDevo 7:7, 2016), which put together several modifications developed from several laboratories in recent years that improved the original protocol developed in the laboratory of Kiyokazu Agata in 1997. Although this protocol, or slight modifications of it, is the most common protocol in the planarian field for NBT-BCIP WISH, our results show that key steps such as the use and time of NAC treatment to remove the mucus need to be taken into account depending on the nature of the gene analyzed, especially for the epidermal markers.

FoxK1 is Required for Ectodermal Cell Differentiation During Planarian Regeneration.

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 FoxK genes, which are mainly studied in mammals and have been implicated in diverse processes including cell proliferation, tissue differentiation and carcinogenesis. Planarians are free-living flatworms, whose importance in biomedical research lies in their regeneration capacity. Planarians possess a wide population of pluripotent adult stem cells, called neoblasts, which allow them to regenerate any body part after injury. In a recent study, we identified three foxK paralogs in the genome of Schmidtea mediterranea. In this study, we demonstrate that foxK1 inhibition prevents regeneration of the ectodermal tissues, including the nervous system and the epidermis. These results correlate with foxK1 expression in neoblasts and in neural progenitors. Although the triggering of wound genes expression, polarity reestablishment and proliferation was not affected after foxK1 silencing, the apoptotic response was decreased. Altogether, these results suggest that foxK1 would be required for differentiation and maintenance of ectodermal tissues.

CREB-binding protein (CBP) gene family regulates planarian survival and stem cell differentiation.

In developmental biology, the regulation of stem cell plasticity and differentiation remains an open question. CBP(CREB-binding protein)/p300 is a conserved gene family that functions as a transcriptional co-activator and plays important roles in a wide range of cellular processes, including cell death, the DNA damage response, and tumorigenesis. The acetyl transferase activity of CBPs is particularly important, as histone and non-histone acetylation results in changes in chromatin architecture and protein activity that affect gene expression. Many studies have described the conserved functions of CBP/p300 in stem cell proliferation and differentiation. The planarian Schmidtea mediterranea is an excellent model for the in vivo study of the molecular mechanisms underlying stem cell differentiation during regeneration. However, how this process is regulated genetically and epigenetically is not well-understood yet. We identified 5 distinct Smed-cbp genes in S. mediterranea that show different expression patterns. Functional analyses revealed that Smed-cbp-2 appears to be essential for stem cell maintenance. On the other hand, the silencing of Smed-cbp-3 resulted in the growth of blastemas that were apparently normal, but remained largely unpigmented and undifferentiated. Smed-cbp-3 silencing also affected the differentiation of several cell lineages including neural, epidermal, digestive, and excretory cell types. Finally, we analysed the predicted interactomes of CBP-2 and CBP-3 as an initial step to better understand their functions in planarian stem cell biology. Our results indicate that planarian cbp genes play key roles in stem cell maintenance and differentiation.

Analyzing pERK Activation During Planarian Regeneration.

Planarians are an ideal model in which to study stem cell-based regeneration. After amputation, planarian pluripotent stem cells surrounding the wound proliferate to produce the regenerative blastema, in which they differentiate into the missing tissues and structures. Recent independent studies in planarians have shown that Smed-egfr-3, a gene encoding a homologue of epidermal growth factor (EGF) receptors, and DjerkA, which encodes an extracellular signal-regulated kinase (ERK), may control cell differentiation and blastema growth. However, because these studies were carried in two different planarian species, the relationship between these two genes remains unclear. We have optimized anti-pERK immunostaining in Schmidtea mediterranea using the original protocol developed in Dugesia japonica. Both protocols are reported here as most laboratories worldwide work with one of these two species. Using this protocol we have determined that Smed-egfr-3 appears to be necessary for pERK activation during planarian regeneration.

The EGFR signaling pathway controls gut progenitor differentiation during planarian regeneration and homeostasis.

The planarian Schmidtea mediterranea maintains and regenerates all its adult tissues through the proliferation and differentiation of a single population of pluripotent adult stem cells (ASCs) called neoblasts. Despite recent advances, the mechanisms regulating ASC differentiation into mature cell types are poorly understood. Here, we show that silencing of the planarian EGF receptor egfr-1 by RNA interference (RNAi) impairs gut progenitor differentiation into mature cells, compromising gut regeneration and maintenance. We identify a new putative EGF ligand, nrg-1, the silencing of which phenocopies the defects observed in egfr-1(RNAi) animals. These findings indicate that egfr-1 and nrg-1 promote gut progenitor differentiation, and are thus essential for normal cell turnover and regeneration in the planarian gut. Our study demonstrates that the EGFR signaling pathway is an important regulator of ASC differentiation in planarians.

Reactive Oxygen Species in Planarian Regeneration: An Upstream Necessity for Correct Patterning and Brain Formation.

Recent research highlighted the impact of ROS as upstream regulators of tissue regeneration. We investigated their role and targeted processes during the regeneration of different body structures using the planarian Schmidtea mediterranea, an organism capable of regenerating its entire body, including its brain. The amputation of head and tail compartments induces a ROS burst at the wound site independently of the orientation. Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis. ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process. Stem cell proliferation was not affected, as indicated by histone H3-P immunostaining, fluorescence-activated cell sorting (FACS), in situ hybridization of smedwi-1, and transcript levels of proliferation-related genes. We showed for the first time that ROS modulate both anterior and posterior regeneration in a context where regeneration is not limited to certain body structures. Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes.

egr-4, a target of EGFR signaling, is required for the formation of the brain primordia and head regeneration in planarians.

During the regeneration of freshwater planarians, polarity and patterning programs play essential roles in determining whether a head or a tail regenerates at anterior or posterior-facing wounds. This decision is made very soon after amputation. The pivotal role of the Wnt/ß-catenin and Hh signaling pathways in re-establishing anterior-posterior (AP) polarity has been well documented. However, the mechanisms that control the growth and differentiation of the blastema in accordance with its AP identity are less well understood. Previous studies have described a role of Smed-egfr-3, a planarian epidermal growth factor receptor, in blastema growth and differentiation. Here, we identify Smed-egr-4, a zinc-finger transcription factor belonging to the early growth response gene family, as a putative downstream target of Smed-egfr-3. Smed-egr-4 is mainly expressed in the central nervous system and its silencing inhibits anterior regeneration without affecting the regeneration of posterior regions. Single and combinatorial RNA interference to target different elements of the Wnt/ß-catenin pathway, together with expression analysis of brain- and anterior-specific markers, revealed that Smed-egr-4: (1) is expressed in two phases - an early Smed-egfr-3-independent phase and a late Smed-egfr-3-dependent phase; (2) is necessary for the differentiation of the brain primordia in the early stages of regeneration; and (3) that it appears to antagonize the activity of the Wnt/ß-catenin pathway to allow head regeneration. These results suggest that a conserved EGFR/egr pathway plays an important role in cell differentiation during planarian regeneration and indicate an association between early brain differentiation and the proper progression of head regeneration.

Regeneration of neuronal cell types in Schmidtea mediterranea: an immunohistochemical and expression study.

Freshwater planarians are unique in their ability to regenerate a complete Central Nervous System (CNS) from almost any small piece of their body in just a few days. The planarian CNS contains a pair of anterior cephalic ganglia lying on top of two ventral nerve cords that extend along the length of the animal. Studies of planarian CNS regeneration have generally used pan-neural markers, which provide only a general overview of the process. Nevertheless, some reports have started to characterize the genes that are required for this process. In this study, to obtain a more detailed description of planarian neural regeneration, we monitored the regeneration of neuronal populations specifically labelled with antibodies against serotonin, allatostatin, neuropeptide F, GYRFamide and FMRFamide. We also characterized the regeneration of dopaminergic and octopaminergic cell populations by in situ hybridization. Finally, we characterized the expression pattern of a set of receptors for neurotransmitters, neuropeptides and hormones that are suggested to play a role in the regeneration process itself. Together, these data provide a more detailed description of the cellular events occurring during anterior and posterior CNS regeneration in planarians and provide the foundations for future mechanistic studies into the regeneration process in this important model system.

EGFR signaling regulates cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis.

Similarly to development, the process of regeneration requires that cells accurately sense and respond to their external environment. Thus, intrinsic cues must be integrated with signals from the surrounding environment to ensure appropriate temporal and spatial regulation of tissue regeneration. Identifying the signaling pathways that control these events will not only provide insights into a fascinating biological phenomenon but may also yield new molecular targets for use in regenerative medicine. Among classical models to study regeneration, freshwater planarians represent an attractive system in which to investigate the signals that regulate cell proliferation and differentiation, as well as the proper patterning of the structures being regenerated. Recent studies in planarians have begun to define the role of conserved signaling pathways during regeneration. Here, we extend these analyses to the epidermal growth factor (EGF) receptor pathway. We report the characterization of three epidermal growth factor (EGF) receptors in the planarian Schmidtea mediterranea. Silencing of these genes by RNA interference (RNAi) yielded multiple defects in intact and regenerating planarians. Smed-egfr-1(RNAi) resulted in decreased differentiation of eye pigment cells, abnormal pharynx regeneration and maintenance, and the development of dorsal outgrowths. In contrast, Smed-egfr-3(RNAi) animals produced smaller blastemas associated with abnormal differentiation of certain cell types. Our results suggest important roles for the EGFR signaling in controlling cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis.

Smed454 dataset: unravelling the transcriptome of Schmidtea mediterranea.

BACKGROUND: 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 their amazing regenerative capabilities, which enable them to regrow even a new head after decapitation. However, a detailed description of the planarian transcriptome is essential for future investigation into regenerative processes using planarians as a model system. RESULTS: In order to complement and improve existing gene annotations, we used a 454 pyrosequencing approach to analyze the transcriptome of the planarian species Schmidtea mediterranea Altogether, 598,435 454-sequencing reads, with an average length of 327 bp, were assembled together with the ~10,000 sequences of the S. mediterranea UniGene set using different similarity cutoffs. The assembly was then mapped onto the current genome data. Remarkably, our Smed454 dataset contains more than 3 million novel transcribed nucleotides sequenced for the first time. A descriptive analysis of planarian splice sites was conducted on those Smed454 contigs that mapped univocally to the current genome assembly. Sequence analysis allowed us to identify genes encoding putative proteins with defined structural properties, such as transmembrane domains. Moreover, we annotated the Smed454 dataset using Gene Ontology, and identified putative homologues of several gene families that may play a key role during regeneration, such as neurotransmitter and hormone receptors, homeobox-containing genes, and genes related to eye function. CONCLUSIONS: We report the first planarian transcript dataset, Smed454, as an open resource tool that can be accessed via a web interface. Smed454 contains significant novel sequence information about most expressed genes of S. mediterranea. Analysis of the annotated data promises to contribute to identification of gene families poorly characterized at a functional level. The Smed454 transcriptome data will assist in the molecular characterization of S. mediterranea as a model organism, which will be useful to a broad scientific community.