Neoblast Specialization in Regeneration of the Planarian Schmidtea mediterranea

Planarians can regenerate any missing body part in a process requiring dividing cells called neoblasts. Historically, neoblasts have largely been considered a homogeneous stem cell population. Most studies, however, analyzed neoblasts at the population rather than the single-cell level, leaving the degree of heterogeneity in this population unresolved. We combined RNA sequencing of neoblasts from wounded planarians with expression screening and identified 33 transcription factors transcribed in specific differentiated cells and in small fractions of neoblasts during regeneration. Many neoblast subsets expressing distinct tissue-associated transcription factors were present, suggesting candidate specification into many lineages. Consistent with this possibility, klf, pax3/7, and FoxA were required for the differentiation of cintillo-expressing sensory neurons, dopamine-β-hydroxylase-expressing neurons, and the pharynx, respectively. Together, these results suggest that specification of cell fate for most-to-all regenerative lineages occurs within neoblasts, with regenerative cells of blastemas being generated from a highly heterogeneous collection of lineage-specified neoblasts.National Institutes of Health (U.S.) (R01GM080639)National Science Foundation (U.S.). Graduate Research Fellowship Program (Grant 1122374

Eye Absence Does Not Regulate Planarian Stem Cells during Eye Regeneration

Dividing cells called neoblasts contain pluripotent stem cells and drive planarian flatworm regeneration from diverse injuries. A long-standing question is whether neoblasts directly sense and respond to the identity of missing tissues during regeneration. We used the eye to investigate this question. Surprisingly, eye removal was neither sufficient nor necessary for neoblasts to increase eye progenitor production. Neoblasts normally increase eye progenitor production following decapitation, facilitating regeneration. Eye removal alone, however, did not induce this response. Eye regeneration following eye-specific resection resulted from homeostatic rates of eye progenitor production and less cell death in the regenerating eye. Conversely, large head injuries that left eyes intact increased eye progenitor production. Large injuries also non-specifically increased progenitor production for multiple uninjured tissues. We propose a model for eye regeneration in which eye tissue production by planarian stem cells is not directly regulated by the absence of the eye itself. Keywords: planarian; regeneration; stem cell; eye; tissue turnover; target blind; progenitor; neoblastNational Institutes of Health (U.S.) (Grant R01GM080639

Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics

Patterns of gene expression can be used to characterize and classify neuronal types. It is challenging, however, to generate taxonomies that fulfill the essential criteria of being comprehensive, harmonizing with conventional classification schemes, and lacking superfluous subdivisions of genuine types. To address these challenges, we used massively parallel single-cell RNA profiling and optimized computational methods on a heterogeneous class of neurons, mouse retinal bipolar cells (BCs). From a population of ∼25,000 BCs, we derived a molecular classification that identified 15 types, including all types observed previously and two novel types, one of which has a non-canonical morphology and position. We validated the classification scheme and identified dozens of novel markers using methods that match molecular expression to cell morphology. This work provides a systematic methodology for achieving comprehensive molecular classification of neurons, identifies novel neuronal types, and uncovers transcriptional differences that distinguish types within a class

Neoblast Specialization in Regeneration of the Planarian Schmidtea mediterranea

Planarians can regenerate any missing body part in a process requiring dividing cells called neoblasts. Historically, neoblasts have largely been considered a homogeneous stem cell population. Most studies, however, analyzed neoblasts at the population rather than the single-cell level, leaving the degree of heterogeneity in this population unresolved. We combined RNA sequencing of neoblasts from wounded planarians with expression screening and identified 33 transcription factors transcribed in specific differentiated cells and in small fractions of neoblasts during regeneration. Many neoblast subsets expressing distinct tissue-associated transcription factors were present, suggesting candidate specification into many lineages. Consistent with this possibility, klf, pax3/7, and FoxA were required for the differentiation of cintillo-expressing sensory neurons, dopamine-β-hydroxylase-expressing neurons, and the pharynx, respectively. Together, these results suggest that specification of cell fate for most-to-all regenerative lineages occurs within neoblasts, with regenerative cells of blastemas being generated from a highly heterogeneous collection of lineage-specified neoblasts

A <i>forkhead</i> Transcription Factor Is Wound-Induced at the Planarian Midline and Required for Anterior Pole Regeneration

<div><p>Planarian regeneration requires positional information to specify the identity of tissues to be replaced as well as pluripotent neoblasts capable of differentiating into new cell types. We found that wounding elicits rapid expression of a gene encoding a Forkhead-family transcription factor, <i>FoxD</i>. Wound-induced <i>FoxD</i> expression is specific to the ventral midline, is regulated by Hedgehog signaling, and is neoblast-independent. <i>FoxD</i> is subsequently expressed within a medial subpopulation of neoblasts at wounds involving head regeneration. Ultimately, <i>FoxD</i> is co-expressed with multiple anterior markers at the anterior pole. Inhibition of <i>FoxD</i> with RNA interference (RNAi) results in the failure to specify neoblasts expressing anterior markers (<i>notum</i> and <i>prep</i>) and in anterior pole formation defects. <i>FoxD(RNAi)</i> animals fail to regenerate a new midline and to properly pattern the anterior blastema, consistent with a role for the anterior pole in organizing pattern of the regenerating head. Our results suggest that wound signaling activates a <i>forkhead</i> transcription factor at the midline and, if the head is absent, <i>FoxD</i> promotes specification of neoblasts at the prior midline for anterior pole regeneration.</p></div

<i>FoxD</i> is expressed in anterior pole progenitors.

<p>Double FISH in wild-type head blastemas 72 hours after transverse amputation. Red box in cartoon shows the region imaged. Images shown are maximal intensity projections. Images are representative of n>5 animals per panel. Anterior is up, dorsal view. (<b>A</b>) <i>FoxD</i> (magenta) and PCGs (green). Percentages (mean ± SD) of <i>FoxD</i> cells co-expressing anterior patterning genes and anterior pole markers are as follows: 95±2% with <i>sFRP1</i>; 93±3% with <i>ndk</i>; 81±9% with <i>ndl-4</i>; 86±5% with <i>prep</i>; and 74±11% with <i>notum</i>; co-expression with the midline gene <i>admp</i> is 3±5% (n>120 <i>FoxD⁺</i> cells examined in each case). Scale bars, 100 µm. Inset shows higher magnification of co-expressing cells, scale bars are 10 µm. (<b>B</b>) <i>FoxD</i>, <i>notum</i>, and <i>prep</i> (magenta), <i>smedwi-1</i> (green). Percentage (mean ± SD) of <i>FoxD</i> cells co-expressing <i>smedwi-1</i> was 39.7±13.3% (n = 157 <i>FoxD⁺</i> cells examined); percentage of <i>notum</i> cells co-expressing <i>smedwi-1</i> was 22.9±6.1% (n = 111 <i>notum⁺</i> cells examined). Yellow arrows point to double-labeled cells. Scale bars in upper left panel, 100 µm; other panels, 10 µm.</p

<i>FoxD</i> is expressed with multiple other PCGs at the anterior pole.

<p>Wild-type intact animals were labeled in double whole-mount fluorescence <i>in situ</i> hybridization (FISH) with <i>FoxD</i> (magenta) and several anterior and midline patterning genes (green). Percentages (mean ± SD) of <i>FoxD</i> cells co-expressing anterior markers are: 56±5% with <i>sFRP1</i>; 66±9% with <i>ndk</i>, 43±9% with <i>ndl-4</i>, 72±19% with <i>prep</i>, and 68±11% with <i>notum</i>; co-expression with the midline genes <i>admp</i> is 2±4%, and <i>slit</i> is 5±8% (n>50 <i>FoxD⁺</i> cells examined in each case). Red box in cartoon on the left shows the area depicted. Animals are anterior up; dorsal view. Insets show higher magnification images of co-expressing cells. Images shown are maximal intensity projections. Images are representative of results seen in >6 animals per panel. Scale bars for all panels, 100 µm. Inset scale bars, 10 µm.</p

<i>FoxD(RNAi)</i> animals have normal expression of wound-induced genes and posterior genes.

<p>(<b>A</b>) Double FISH using <i>notum</i> (green) and <i>wnt1</i> (magenta) in trunk fragments of control and <i>FoxD</i> dsRNAi injected animals six hours following amputation. Red box in cartoon on the left shows the area imaged. Graph shows total number of cells expressing <i>notum</i>, or <i>wnt1</i> in the different RNAi conditions. Data are shown as mean± SD, n>5 animals per group. Anterior is up, ventral view. (<b>B</b>) Posterior blastemas at day seven of regeneration of <i>FoxD</i> dsRNA injected animals are shown; expression of posterior patterning gene <i>wnt11-2</i> (17/17) and the posterior pole gene <i>wnt1</i> (14/14) was normal in <i>FoxD(RNAi)</i> animals. Anterior is up, dorsal view. For all panels, images shown are maximal intensity projections. Scale bars, 50 µm.</p

Hh signaling is required for wound-induced <i>FoxD</i> expression.

<p>(<b>A</b>) Double FISH; <i>notum</i> (green) and <i>FoxD</i> (magenta) of RNAi fed animals fixed six hours following transverse amputation. Red box in cartoon shows region imaged. Images shown are maximal intensity projections. Dotted white line marks the approximate wound boundary. Anterior wounds are up, ventral view. Scale bars for all panels, 100 µm. (<b>B</b>) Graphs show number of cells expressing <i>FoxD, notum</i>, or <i>wnt1</i> in the different RNAi conditions. Data are shown as mean ± SD, and analyzed using a one-way ANOVA test; **p<0.01, ***p<0.001, n>10 animals per RNAi condition.</p

<i>FoxD(RNAi)</i> animals display abnormal anterior pole, head patterning and midline gene expression.

<p>(<b>A</b>) Upper panels: animals injected with <i>FoxD</i> dsRNA regenerated normally sized anterior blastemas with one eye (73/130) or smaller blastemas with no eyes (24/130) seven days following amputation. Scale bars, 200 µm. Lower panels: double FISH and immunostainings using anti-ARRESTIN (VC1) antibody in regenerating <i>FoxD</i> dsRNA injected animals. Defects in head regeneration of <i>FoxD(RNAi)</i> animals were observed; probes used: brain, <i>chat</i> (7/7 animals showed a medially collapsed or a very small brain); intestine, <i>mat</i> (3/7 animals showed some regeneration defect in the anterior blastema). Images shown are maximal intensity projections. Scale bars, 100 µm. Anterior is up, dorsal view. (<b>B</b>) Upper panels: double FISH (<i>smedwi-1</i> in green, <i>NB.21.11e</i> in magenta) and immunostaining with anti-phospho histone 3 (H3P in blue) of tail fragments fixed at 18 hours following amputation of eight weeks RNAi fed animals. Red box in cartoon on the left shows the area imaged. Lower panels: FISH (<i>notum</i>, green) and immunostaining with anti-H3P (blue) were performed at 48 hours following amputation in regenerating tail fragments of eight weeks RNAi fed animals. Anterior is up, dorsal view. Images shown are maximal intensity projections. Scale bars, 100 µm. Numbers of mitotic cells were counted and normalized by the tail area (mm²) and analyzed using a Student-<i>t</i>-test analysis; *p<0.05, n>10. (<b>C</b>) <i>FoxD</i> dsRNA injected animals displayed defects in anterior pole gene and PCG expression at 72 hours following amputation (<i>sFRP-1</i>, 5/9; <i>ndl-4</i>, 3/4; <i>notum</i>, 10/11; <i>prep</i>, 5/6). Dotted white line marks the approximate animal edge. Images shown are maximal intensity projections. Anterior is up, dorsal view. Scale bars, 50 µm. (<b>D</b>) Single or double FISH in day seven regenerating dsRNA injected animals. Defective expression of the anterior pole gene <i>notum</i> (8/20 no expression, 10/20 decreased expression) and <i>sFRP-1</i> (9/18 no expression, 7/18 reduced expression) is observed in <i>FoxD(RNAi)</i> animals. Reduced expression of the midline genes <i>slit</i> (6/11 severe defect, 2/11 mild defect), <i>admp</i> (8/10 reduced expression), and <i>ephrin receptor 1, ephR1</i> (7/14 severe reduction, 3/14 mild reduction) was observed in <i>FoxD(RNAi)</i> animals. Yellow arrows point to missing or aberrant expression. Images shown are maximal intensity projections. Anterior is up, dorsal view for all panels except for <i>admp</i> and <i>slit</i> FISH. Scale bars, 100 µm.</p