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WIREs Dev Biol
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Nervous system development and regeneration in freshwater planarians

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Planarians have a long history in the fields of developmental and regenerative biology. These animals have also sparked interest in neuroscience due to their neuroanatomy, spectrum of simple behaviors, and especially, their almost unparalleled ability to generate new neurons after any type of injury. Research in adult planarians has revealed that neuronal subtypes homologous to those found in vertebrates are generated from stem cells throughout their lives. This feat is recapitulated after head amputation, wherein animals are capable of regenerating whole brains and regaining complete neural function. In this review, we summarize early studies on the anatomy and function of the planarian nervous system and discuss our present knowledge of the molecular mechanisms governing neurogenesis in planarians. Modern studies demonstrate that the transcriptional programs underlying neuronal specification are conserved in these remarkable organisms. Thus, planarians are outstanding models to investigate questions about how stem cells can replace neurons in vivo. WIREs Dev Biol 2017, 6:e266. doi: 10.1002/wdev.266

Overview of the anatomy of the planarian nervous system. All images are from Schmidtea mediterranea unless otherwise specified. (a) Illustration of the nervous system in Schmidtea polychroa. (Adapted from Ref ) Neural tissues are colored blue, reproductive organs in orange, and the digestive system in yellow. (b) Brain branches extend laterally from the brain of S. polychroa as depicted by Micoletzky. (Adapted from Ref ) Ovaries are colored orange. Regions of interest are labeled on the image. (c) Eye spots on a live planarian head (located on the dorsal surface) consist of pigment cups; photoreceptor neurons are located internally to an area devoid of pigment. Anti‐Arrestin antibody labels the photoreceptor neurons and their axons, which reveals the optic chiasm. The sensory neuron‐dense auricles are devoid of pigment and are located at the lateral edges of the head. (d) Anti‐Synapsin antibody labels the synapses of planarian neurons, highlighting the anterior commissure and neuropil of the brain, the ventral nerve cords and their transverse commissures and lateral branches, and the pharyngeal nerve net. 1H6 antibody labels the peripheral nervous system, including axons extending through the ventral nerve cords. Scale bar = 200 μm. (e) Cartoon depicting the anatomy of neural structures observed in a transverse cross section through the mid‐body of a planarian. Neural regions of interest are labeled on the image. (f) The subepidermal plexus (blue; stained with 1H6 antibody) sends projections in between epidermal cells (arrows, epidermal nuclei labeled with DAPI and pseudocolored yellow). The submuscular plexus is located beneath the body wall muscle (muscle labeled with 6G10 mAb and pseudocolored magenta). (g) Examples of neural projections observed in the subepidermal and submuscular plexuses (examples denoted with arrows, labeled with 1H6 mAb). Scale bar = 20 μm. ((f) and (g) were reprinted with permission from Ref . Copyright 2015 BMC)
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Development of the nervous system in planarians. Neurogenesis can be visualized by in situ hybridization to Spol‐elav‐4 in the developing Schmidtea polychroa embryo. Around Stage 4, early differentiating neurons can be detected. Early in Stage 5, these neurons form a neural primordium and, in late Stage 5, the developing brain rudiment and nerve net of the definitive pharynx primordium are readily detected. By Stage 7 (just prior to hatching), a defined brain, ventral nerve cords, and the nerve net of the definitive pharynx are visible. (Modified and reprinted with permission from Ref . Copyright 2015 Elsevier)
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Conserved polarity determinants regulate scaling of the planarian brain. (a) Smed‐notum (blue) and Smed‐wnt11‐6 (magenta) are expressed in neurons on the opposite anterior and posterior poles of the brain (indicated with arrows in i). Smed‐notum (blue) is expressed in muscle cells at the anterior pole (co‐expressed with Smed‐collagen, gray, white arrows in ii, yellow arrows in iii) and in the brain anterior commissure (yellow arrows in ii, white arrows in iii, co‐expressed with Smed‐chat, gray), whereas Smed‐wnt11‐6 (magenta) is expressed at the posterior end of each brain lobe (in Smed‐chat + cells, gray, arrows in iv). The green boxes in the cartoons indicate the region of the animal shown in the corresponding figures. (b) Smed‐notum inhibits Smed‐wnt11‐6 to control brain size during regeneration. At 21 days post‐amputation, trunks regenerate a smaller brain in Smed‐notum( RNAi ) animals, and larger brains in Smed‐wnt11‐6( RNAi ) and Smed‐notum;wnt11‐6(RNAi) animals compared to controls. Amputation schematic is illustrated in the cartoon. (c) Illustration depicting the regulation of brain size by notum and wnt11‐6. (All images are in Schmidtea mediterranea and are reprinted with permission from Ref . Copyright 2015 Company of Biologists)
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Abnormal fibroblast growth factor receptor (FGFR)‐like signaling leads to ectopic neurogenesis. (a) Following knockdown of Dj‐nou‐darake (ndk, a fibroblast growth factor receptor‐like gene) and head regeneration 16 days after amputation, ectopic eyespots (arrows in bleached image) are observed in locations posterior to the original photoreceptors of Dugesia japonica. The formation of these eyespots corresponds with ectopic photoreceptor neurons (labeled with anti‐Arrestin in green) that project to more posterior positions (arrowheads) along with the regeneration of brain tissue in posterior positions revealed by anti‐Synaptotagmin labeling (green, highlighted with arrows). Scale bar = 400 μm. (Reprinted with permission from Ref . Copyright 2002 Macmillan Publishers Ltd) (b) Knockdown of Smed‐ndk causes expansion of brain tissues in non‐regenerating animals. The brain branch marker Smed‐g‐protein alpha subunit (gpas, arrowheads) is ectopically expressed in regions posterior to the brain after 2 weeks of RNAi in Schmidtea mediterranea. Scale bars = 100 μm. White box indicates the location of images in (c). (Reprinted with permission from Ref . Copyright 2013 Company of Biologists) (c) Posterior expansion of neural tissues corresponds with an increase in neural progenitors. More Smed‐coe + neural progenitors (blue, arrowheads) are observed in the area anterior to the pharynx and between the brain lobes following 14 days of Smed‐ndk RNAi knockdown in S. mediterranea. (Reprinted with permission from Ref . Copyright 2013 Company of Biologists)
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Single cell analysis identifies conserved markers of neuronal progenitors in Schmidtea mediterranea. Homologs of conserved neural stem cell genes Smed‐embryonic lethal abnormal vision‐2 and Smed‐musashi‐1 (elav‐2 and msi‐1, respectively) were identified in the νNeoblast population. (a) Expression of elav‐2 and msi‐1 visualized in differentiated regions of the central nervous system. Boxed region in elav‐2 image indicates region of images in (b–d). (b–d) elav‐2 + and msi‐1 + νNeoblasts (gene expression shown in red) co‐express Smed‐piwi‐2 transcript (green), but very little Smed‐piwi‐1 (blue, a marker for neoblasts, in (b)). νNeoblasts, however, retain high expression of Smed‐PIWI‐1 protein (blue, a hallmark of progenitor cells, in (c)). νNeoblasts are BrdU + (green, following a 4 h chase, in (d)), indicating mitotic activity. (Reprinted with permission from Ref . Copyright 2016 BMC)
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Inhibition of genes expressed in neuronal progenitors or involved in nervous system patterning causes defects in nervous system regeneration. All images are from Schmidtea mediterranea unless otherwise specified. Illustrations of the experimental design are displayed above the corresponding panels. Where specified, images show uninjured worms following the specified period of RNAi exposure or of worms treated with RNAi, amputated (amputation site denoted by gray dashed lines) and allowed to regenerate for the specified time period. The blue dashed boxes in the cartoons indicate the region of the animals shown in the figures below. Smed‐coe knockdown results in loss of Smed‐cpp‐1 + neuropeptidergic cells in the head. Smed‐krüppel‐like factor (klf) RNAi causes a loss of Smed‐cintillo + putative sensory neuron gene expression (magenta) in the anterior of the head. Smed‐ap2 RNAi results in loss of Smed‐transient receptor potential A ( TrpA +, green) neurons in the brain. Smed‐pitx RNAi knock down results in loss of Smed‐serotonin transporter (sert +, serotonergic neurons) in the regenerated planarian. And, Dj‐DSCAM RNAi causes fasciculation impairments in the regenerated brain branches (labeled with anti‐G‐beta in Dugesia japonica; green). (Reprinted with permissions from: coe(RNAi) (Ref . Copyright 2013 Company of Biologists); klf( RNAi) (Ref . Copyright 2014 Elsevier);ap2 ( RNAi ) (Ref . Copyright 2012 CLHL); pitx(RNAi ) (Ref . Copyright 2013 Company of Biologists); DSCAM ( RNAi ) (Ref . Copyright 2006 John Wiley & Sons))
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Planarian brain regeneration visualized by staining animals with specific gene or protein markers. Species used for each experiment are indicated to the left of the images. (a) Live regenerating Schmidtea mediterranea. In the live images, the blastema is easily distinguishable at 2 days post head amputation (2d, arrowhead). As the blastema grows, eyespots are visible by 3 days post‐amputation (3d) and become well developed as regeneration continues (arrows in 3d and 5d). (b) Brain regeneration visualized by the appearance of neuronal cell bodies marked by expression of Smed‐th, which labels serotonergic neurons. The appearance of the brain primordia is observed as small clusters of cells beginning at day 2 of regeneration. These clusters are more defined as more neurons differentiate at day 3 of regeneration (arrows in 2d and 3d images). (c) Regeneration of the brain branches visualized by in situ hybridization to Dj‐517HH (a gene encoding a receptor protein tyrosine phosphatase). Branches extend laterally from the regenerating brain by 5 days post‐amputation (arrows in 5 days image), increase in length, and appear denser as regeneration proceeds. (d) Regeneration of the brain neuropil labeled with anti‐Dj‐Synaptotagmin antibody. In this regeneration time series, reconnection of the brain lobes is observed by day 3 of regeneration (arrowhead in day 3 image); the transverse commissures of the nerve cords appear ventrally in relationship to the cephalic ganglia (denoted with arrow in day 5 image) and the fully re‐established brain morphology is apparent by day 7 of regeneration. (e) Re‐establishment of neural network during brain regeneration visualized by labeling a subset of axonal projections with anti‐Neuropeptide F. At 2 days post‐amputation, the brain lobes are not yet connected at the anterior commissure (arrowhead in 2d image). By 3 days post‐amputation, the brain lobes begin to reconnect at the anterior commissure (arrowheads in 3d and 4d images) and the transverse commissures of the ventral nerve cords are also re‐established (arrow in 3d image). Anterior is to the top in all images. (Reprinted with permission from: Smed‐th and NPF (Ref . Copyright 2012 UBC Press); Dj‐517HH (Ref . Copyright 2011 Company of Biologists, which was reprinted from Ref . Copyright 2002 John Wiley & Sons); DjSYT (Ref . Copyright 2002 John Wiley & Sons)). (f) Schematic depicting some of the major events involved in whole‐brain regeneration.
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The planarian nervous system is comprised of distinct neuronal subtypes. The species used for each image are specified in the corresponding panels. (a) Whole‐mount in situ hybridization labeling of genes that encode enzymes required for the synthesis of neurotransmitter populations: Dj‐glutamic acid decarboxylase, GABAergic neurons; Dj‐tyrosine hydroxylase, dopaminergic neurons; Dj‐tryptophan hydroxylase, serotonergic neurons; Dj‐choline acetyl transferase, cholinergic neurons; Dj‐tyramine beta hydroxylase, octopaminergic neurons. (b) Antibody labeling of the enzymatic proteins found in the same neurotransmitter populations as in (a). ((a) and (b) are reprinted with permission from: GABAergic neurons (Ref . Copyright 2008 Elsevier); dopaminergic neurons (Ref . Copyright 2007 John Wiley & Sons; Ref . Copyright 2011 John Wiley & Sons); serotonergic neurons (Ref . Copyright 2007 Elsevier); cholinergic neurons (Ref . Copyright 2010 Elsevier); octopaminergic neurons (Ref . Copyright 2008 Elsevier)) (c) Double‐labeling of octopaminergic and dopaminergic neurons with antibody markers (same markers as specified in (b)) shows that these neuronal populations are unique. Arrowheads and arrows point to octopaminergic and dopaminergic neural cell bodies, respectively. Scale bar = 50 μm. (Reprinted with permission from Ref . Copyright 2008 Elsevier) (d) Glutamic Acid Decarboxylase+ (GABAergic neurons) and Tyrosine Hydroxylase+ (dopaminergic neurons) represent distinct and separate populations of neurons in the dorsal region of the brain. (Reprinted with permission from Ref . Copyright 2008 Elsevier) (e) The brain contains distinct spatial domains identified by expression of specific genes. Expression of the transcription factors Dj‐otxA, ‐otxB, and ‐otp is restricted to the medial region of the brain, the region of the brain below the photoreceptors, and the lateral branches of the brain, respectively. The cartoon depicts the mediolateral expression pattern of all three genes in the planarian brain. (Reprinted with permission from Ref . Copyright 1999 Springer) (f) Planarians have many distinct neuropeptidergic populations, including Smed‐ppp‐1 + , ‐spp‐1 +, and ‐npp‐2 + neurons, which are detected throughout the body and are abundant in the central nervous system (CNS) of Schmidtea mediterranea. (Reprinted with permission from Ref . Copyright 2010 PLOS) (g) Neurons expressing Smed‐npy‐8 target a distinct population of Smed‐npyr‐1 + cells in the CNS of S. mediterranea hermaphrodites. Distinct cell populations can be visualized by labeling animals with probes for specific neuropeptide genes (Smed‐npyr‐1, red; Smed‐npy‐8, green; DAPI, gray). Function of both of these genes is required for sexual maturation and differentiation of the germ cells. (Reprinted with permission from Ref . Copyright 2016 PLOS) Anterior is up in all images except for (g) where anterior is to the left.
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