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

Landmarks in Existing Tissue at Wounds Are Utilized to Generate Pattern in Regenerating Tissue

Regeneration in many organisms involves the formation of a blastema, which differentiates and organizes into the appropriate missing tissues. How blastema pattern is generated and integrated with pre-existing tissues is a central question in the field of regeneration. Planarians are free-living flatworms capable of rapidly regenerating from small body fragments [1]. A cell cluster at the anterior tip of planarian head blastemas (the anterior pole) is required for anterior-posterior (AP) and medial-lateral (ML) blastema patterning [2–4]. Transplantation of the head tip into tails induced host tissues to grow patterned head-like outgrowths containing a midline. Given the important patterning role of the anterior pole, understanding how it becomes localized during regeneration would help explain how wounds establish pattern in new tissue. Anterior pole progenitors were specified at the pre-existing midline of regenerating fragments, even when this location deviated from the ML median plane of the wound face. Anterior pole progenitors were specified broadly on the dorsal-ventral (DV) axis and subsequently formed a cluster at the DV boundary of the animal. We propose that three landmarks of pre-existing tissue at wounds set the location of anterior pole formation: a polarized AP axis, the pre-existing midline, and the dorsal-ventral median plane. Subsequently, blastema pattern is organized around the anterior pole. This process, utilizing positional information in existing tissue at unpredictably shaped wounds, can influence the patterning of new tissue in a manner that facilitates integration with pre-existing tissue in regeneration.National Institute of General Medical Sciences (U.S.) (Award T32GM007753)National Institutes of Health (U.S.) (Grant R01GM080639

CXCL12 Mediates CCR7-independent Homing of Central Memory Cells, But Not Naive T Cells, in Peripheral Lymph Nodes

Central memory CD8+ T cells (TCM) confer superior protective immunity against infections compared with other T cell subsets. TCM recirculate mainly through secondary lymphoid organs, including peripheral lymph nodes (PLNs). Here, we report that TCM, unlike naive T cells, can home to PLNs in both a CCR7-dependent and -independent manner. Homing experiments in paucity of lymph node T cells (plt/plt) mice, which do not express CCR7 ligands in secondary lymphoid organs, revealed that TCM migrate to PLNs at ∼20% of wild-type (WT) levels, whereas homing of naive T cells was reduced by 95%. Accordingly, a large fraction of endogenous CD8+ T cells in plt/plt PLNs displayed a TCM phenotype. Intravital microscopy of plt/plt subiliac lymph nodes showed that TCM rolled and firmly adhered (sticking) in high endothelial venules (HEVs), whereas naive T cells were incapable of sticking. Sticking of TCM in plt/plt HEVs was pertussis toxin sensitive and was blocked by anti-CXCL12 (SDF-1α). Anti-CXCL12 also reduced homing of TCM to PLNs in WT animals by 20%, indicating a nonredundant role for this chemokine in the presence of physiologic CCR7 agonists. Together, these data distinguish naive T cells from TCM, whereby only the latter display greater migratory flexibility by virtue of their increased responsiveness to both CCR7 ligands and CXCL12 during homing to PLN

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

The planarian wound epidermis gene equinox is required for blastema formation in regeneration

AbstractRegeneration often involves the formation of a blastema, an outgrowth or regenerative bud formed at the plane of injury where missing tissues are produced. The mechanisms that trigger blastema formation are therefore fundamental for regeneration. Here, we identify a gene, which we named equinox, that is expressed within hours of injury in the planarian wound epidermis. equinox encodes a predicted secreted protein that is conserved in many animal phyla. Following equinox inhibition, amputated planarians fail to maintain wound-induced gene expression and to subsequently undergo blastema outgrowth. Associated with these defects is an inability to reestablish lost positional information needed for missing tissue specification. Our findings link the planarian wound epidermis, through equinox, to regeneration of positional information and blastema formation, indicating a broad regulatory role of the wound epidermis in diverse regenerative contexts.</jats:p

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 wound-induced in the midline.

<p>(<b>A</b>) Wild-type animals were transversely amputated, fixed at six hours following wounding, and <i>FoxD</i> (purple) expression was analyzed by <i>in situ</i> hybridization. Red box in cartoon on the left shows the area imaged. All animals showed anterior and posterior <i>FoxD</i> expression at the midline domain following wounding (n>10). Animals are anterior up; ventral view. Area within dotted black box is enlarged in right panels. Scale bar, 500 µm. Right panels, scale bar, 100 µm. (<b>B</b>) Double FISH, <i>notum</i> (green) and <i>FoxD</i> (magenta) in wild-type (0 rads) and lethally irradiated animals (6,000 rads) at different time points following transverse amputation. Red box in cartoon on the left shows the area depicted. Images shown are maximal intensity projections. Images are representative of results seen in n>6 animals per panel. Dorsal view for the 72 hours time point, ventral view for all others. Dotted white line marks the approximate wound boundary. Scale bars, 100 µm. (<b>C</b>) Cartoons show injury types performed in wild-type animals. Dotted black line shows the wound site. Type of wound from left to right: parasagittal, sagittal, incision, wedge, dorsal midline puncture, lateral edge, and lateral puncture. Red box shows the region imaged below. Whole-mount <i>in situ</i> hybridization using <i>FoxD</i> (purple) RNA probe at six hours following wounding of wild-type animals. Red arrows indicate <i>FoxD</i> expression. Dotted black box is enlarged in the inset. Images are representative of results seen in >10 animals per wound type. Animals are anterior up, ventral view. Scale bars, 500 µm. Inset scale bars, 100 µm. (<b>D</b>) Double FISH, <i>FoxD</i> (magenta) and the immediate early gene <i>fos-1</i> (green) of wounded wild-type animals fixed at six hours following midline (upper panel) and lateral (lower panel) puncture injuries. Cartoon on the left shows type of wound, red box is area imaged. Dotted yellow line depicts the approximate midline, white line depicts the animal edge. Area within dotted white box is shown in the inset for <i>FoxD</i> expression. Yellow arrows point to <i>FoxD</i>-expressing cells at the midline. Images shown are maximal intensity projections. Images are representative of results seen in >10 animals per panel. Anterior is up, ventral view. Scale bars, 100 µm (<b>E</b>) Double FISH, <i>FoxD</i> (green for upper two panels, magenta all other panels) and midline, W2 genes or the muscle gene <i>collagen</i> of wild-type animals fixed six hours following transverse amputation. Percentage (mean ± SD) of <i>FoxD</i> co-expression with <i>slit</i> was 86.3±5.5% (n = 124 <i>FoxD⁺</i> cells examined), with collagen was 92.6±4.6% (n = 52 <i>FoxD⁺</i> cells examined). Red box shows the area imaged. Anterior wounds are up, ventral view. Insets show higher magnification of co-expressing cells, scale bars are 10 µm. For the double <i>FoxD</i>, <i>collagen</i> FISH, area within dotted white box is shown in the right panel. Images shown are maximal intensity projections. Images are representative of results seen in >5 animals per panel. Dotted white line marks the approximate wound boundary. Scale bars, 100 µm.</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

<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