Tiling light sheet microscopy for imaging planarian central nervous system (CNS).

(A) Planarian expansion workflow. Planarians were fixed and stained with FISH or immunostaining, followed by tissue expansion and tiling light sheet microscopy imaging. Created with Biorender. (B) Segmentation of PI and neuron pool riboprobes labeled cells in eyes, brain, and pharynx. Scale bar, 600 μm. (C) Staining of anti-arrestin antibody for the planarian visual system. Scale bar, 600 μm. The lower image shows a magnification of the selected area in the upper image. Scale bar, 200 μm. (D) Neuron tracing of the upper image in panel C. (E) Tracing of single neurons in transverse view. Scale bar, 600 μm. (F) Traced axon projection trajectories from each eye. Scale bar, 600 μm. (G) Dual staining of glial cells (estrella+) and visual system (anti-arrestin+) in the head region of a wild-type planarian. Scale bar, 350 μm. (H) Xz view of the image in panel G. (I) Zoom in of panel G shows the glial cells close to the visual axons. Scale bar, 100 μm. (J) Reconstruction of the glial and visual system. Scale bar, 100 μm. (K1) Anti-SYT staining in a wild-type planarian. Scale bar, 600 μm. (K2) The 3D reconstructed image of ganglia in the anterior tip of the brain. Scale bar, 120 μm. (K3) The 3D reconstructed image of ganglia in the branch region of the brain. Scale bar, 30 μm. (L1) Anti-Phospho (Ser/Thr) staining in a wild-type planarian. Scale bar, 600 μm. (L2) Single plane image of the brain. Scale bar, 150 μm. (L3) 3D reconstruction of brain region. Green represents the visual neurons, and yellow represents the brain. Scale bar, 180 μm.

Changes in the number of planarian neurons during homeostasis.

(A) Workflow of sample collection of homeostatic planarians. Scale bar, 500 μm. (B) 3D reconstruction of a planarian with PI staining. Length, volume, and surface area were measured using reconstructed images. The planarian brain is segmented and shown in a black dotted box. Scale bar, 2700 μm. (C) Representative fluorescent images of planarians stained with neuron pool riboprobes and PI at sizes of 2 mm, 4 mm, 6 mm, and 8 mm. Scale bar, 6000 μm. (D) Dot plot shows the correlation of whole-body cell number with neuron number in different sizes of intact planarians during homeostasis. Two trendlines are shown to represent planarians with differing cell counts. (E) Zoomed grayscale (PI) and segmented neurons (green) of selected brain regions of planarians at sizes of 2mm, 4mm, and 8mm. Scale bar, 450 μm. (F) Correlation of total neuron number with neuron number in brains in different sizes of intact planarians. (G) Segmented images of octopaminergic neurons in the main brain region (blue) and brain branch region (red) of planarians with the indicated body length are shown with xy and yz views. Scale bar, 900 μm. (H) Dot plot shows the correlation of the total number of octopaminergic neurons with the octopaminergic neuron number in the brains (blue dots) and branches (red dots). (I) The plot illustrates the correlation between the angle of the brain lobe and the number of octopaminergic neurons in two groups of intact planarians: one with 80-160 octopaminergic neurons and the other with 160-220 octopaminergic neurons. n=8 in each condition. The data is shown as the mean ± SEM. Statistical significance was evaluated using the two-tailed unpaired Student’s t-test, with **p < 0.01, ***p < 0.001 indicating significance, while ns indicates lack of significance. (J) Segmented images of GABAergic neurons in the brains of planarians with the indicated body length are shown with xy and yz views. Scale bar, 900 μm. (K) Dot plot shows the correlation of the total number of GABAergic neurons with the GABAergic neuron number in the VM region (yellow dots) and DL region (green dots).

Changes in the number of planarian neurons during regeneration.

(A) Workflow of sample collection of regenerative planarians. (B) Reconstructed 3D brains segmented from PI-labeled regenerative tail fragments at various time points post-amputation. Scale bar, 900 μm. (C) Representative xy views of segmented cholinergic neurons on 0-, 3-, 9-, and 14-days post-amputation. Scale bar, 1500 μm. (D) Dot plot shows the increase of cholinergic neurons (chat) at different time points after amputation. N≥3 in each condition. The data is shown as the mean ± SEM. (E) Dot plot shows the increase of serotonergic neurons (tph) at different time points after amputation. N≥3 in each condition. The data is shown as the mean ± SEM. (F) Dot plot shows the increase of GABAergic (gad), octopaminergic (tbh), and dopaminergic (th) neurons at different time points after amputation. N≥3 in each condition. The data is shown as the mean ± SEM. (G) Representative xy and yz views of segmented GABAergic neurons on 4, 6, 8, 10, 12, and 14-day post-amputation. Scale bar, 900 μm. (H) Dot plot shows the correlation of the total number of brain GABAergic neurons with the neuron number in the VM region (yellow dots) and DL region (green dots) in regenerative planarians. (I) Representative xy and yz views of segmented octopaminergic neurons on 3, 5, 6, 8, 10, 13 days post-amputation. Scale bar, 900 μm. (J) Correlation of the total number of the brain octopaminergic neurons with the octopaminergic neuron number in the brains (blue dots) and branches (red dots) in regenerative planarians. (K) Percentage of each type of neuron in the total cell number between 14 dpa planarians and the same size homeostatic planarians. Statistical significance was assessed by the two-tailed unpaired Student’s t-test: **p < 0.01, ***p < 0.001; ns, not significant.

Distribution of planarian body-wall muscle fibers and their connections at the anterior and posterior poles.

(A) Illustration of the major five muscle fibers in wild-type planarians according to their orientation and distribution. A: anterior; P: posterior; D: dorsal; V: ventral. (B) Segmented fibers of planarian body-wall muscle. Scale bar, 200 μm. (C) Schematic depicting selected segmented areas of planarian body-wall muscle, with a chart depicting the number of different orientational fibers in those regions. d: dorsal region; v: ventral region. The data is shown as the mean ± SEM. Statistical significance was evaluated using the two-tailed unpaired Student’s t-test, with *p < 0.05, **p < 0.01, ***p < 0.001 indicating significance, while ns indicates lack of significance. (D) Planarian dorsal body-wall muscle fiber labeled with 6G10 antibody staining, with segmented circular (green), diagonal (blue), longitudinal (yellow), and D-V fibers (purple). Scale bar, 150 μm. (E) An xz view of the image in panel D. Scale bar, 150 μm. (F) Selected 100 μm depth region showing DV fiber (White arrows) located around diagonal fibers. Scale bar, 80 μm. (G) Body-wall muscle fiber of a region in the image of panel F. Segmented D-V fibers are shown in a tracked line. Scale bar, 50 μm. (H) An xz view of the segmented D-V fiber and its connecting fibers in panels F and G. Scale bar, 50 μm. (I) Xz projection of planarian anterior and posterior muscle fiber and their segmented muscle fibers in control planarian. Scale bar, 300 μm. (J) Xz projection of planarian anterior and posterior muscle fiber and their segmented muscle fibers in β-catenin-1 RNAi planarian. Scale bar, 300 μm.

Spatial connection of planarian neurons and glial cells with muscle fibers.

(A1) Dual staining for glial cells (estrella+) and muscles (6G10+) in a wild-type planarian. The representative brain region of xy is shown. (A2) Dual staining for glial cells (estrella+) and muscles (6G10+) in a wild-type planarian. The representative brain region of xz is shown. (A3) Dual staining for glial cells (estrella+) and muscles (6G10+) in a wild-type planarian. The representative brain region of yz is shown. Scale bar, 600 μm. (A4) A single slice of glial cells (estrella+) and muscles (6G10+) close to the epidermis of the anterior pole is shown. The arrowhead indicates the interaction of glial cells (estrella+) and muscles (6G10+). Scale bar, 250 μm. (A5) A single slice of glial cells (estrella+) and muscles (6G10+) close to the epidermis of the posterior pole is shown. The arrowhead indicates the interaction of glial cells (estrella+) and muscles (6G10+). Scale bar, 250 μm. (1-3) Zoomed in white dotted box region in A1 showing the estrella+ glial projection to the muscles. Scale bar, 150 μm. (B1) Dual staining for CNS (Anti-Phospho (Ser/Thr) staining) and muscles (6G10+) in a wild-type planarian. Scale bar, 600 μm. (B2) Single slice of anti-Phospho (Ser/Thr) and 6G10 expression around planarian’s eye. Scale bar, 300 μm. (B3) Magnification of selected region in panel B2. Scale bar, 200 μm. (B4) Single slice of Anti-Phospho (Ser/Thr) and 6G10 staining in a brain region. Scale bar, 200 μm. (B5) Volume rendering of selected region in panel B4. (B6) Reconstruction of B5. Scale bar, 60 μm. (C1) Dual staining for CNS (anti-SYT staining) and muscles (6G10+) in a wild-type planarian. Scale bar, 600 μm. (C2) Single slice of Anti-SYT and 6G10 expression in brain region. Scale bar, 600 μm. (C3) Single slice of Anti-SYT and 6G10 expression close to the epidermis in the middle part of the body. Scale bar, 450 μm. (C4) Single slice of Anti-SYT and 6G10 expression close to the epidermis in the anterior part of the body. Scale bar, 450 μm. (C5) Single slice of Anti-SYT and 6G10 expression close to the epidermis in the anterior part of the body. Scale bar, 450 μm.

Neuron and muscle defects in inr-1 RNAi and β-catenin RNAi planarians

(A) Representative images of dorsal and ventral muscle in egfp RNAi, inr-1 RNAi, and β-catenin-1 RNAi planarians. Scale bar, 120 μm. (B) Images depicting enhanced muscle fibers within dorsal and ventral regions of egfp RNAi, inr-1 RNAi, and β-catenin-1 RNAi planarian. Scale bar, 40 μm. (C) Schematic depicting selected segmented areas of planarian body-wall muscle in DV view.The plot shows the proportion of circular, longitudinal, and diagonal fibers in ventral and dorsal body muscle wall in egfp RNAi, inr-1 RNAi, and β-catenin-1 RNAi planarians. The data is shown as the mean ± SEM. Statistical significance was evaluated using the two-tailed unpaired Student’s t-test, with *p < 0.05, **p < 0.01, ***p < 0.001 indicating significance, while ns indicates lack of significance. (D) Illustration of egfp RNAi, inr-1 RNAi, and β-catenin-1 RNAi planarian glial cell structure and its connection with muscle fibers. (E) Anti-Phospho (Ser/Thr) and 6G10 expression and its reconstructed data in selected regions of egfp RNAi, inr-1 RNAi, and β-catenin-1 RNAi planarians (left). The estrella+ glia and 6G10+ muscles and their reconstructed images in selected regions of egfp RNAi, inr-1 RNAi, and β-catenin-1 RNAi planarians (right). Scale bar, 150 μm.

Key resources table

TLSM imaging and cell segmentation for planarian pharynx, brain, and epidermis.

(A) Chart displaying the resolution and speed for imaging planarians of different sizes and at different developmental stages. (B) Selected xy slices of a 3D planarian labeled with PI of different z positions. Scale bar, 1000 μm. (C) Grayscale image of a single image plane at the brain region of a wild-type planarian head. Scale bar, 300 μm. (D) Segmented image with individual cells from the image of panel D. Scale bar, 300 μm. (E) Isolated epidermal cells of the whole planarian labeled with PI. Scale bar, 1000 μm. (F) Isolated epidermal cells of a selected region in (E). (G) Segmentation of epidermal cells in the image of panel F. Scale bar, 150 μm. (H)-(J) 3D display, grayscale, and cell segmentation of a pharynx. Segmented cells are assigned with different colors. Scale bar, 150 μm. (K)-(M) Selected single image planes from regions 1-3 in panel I. Green arrows represent the cell boundaries in grayscale images. (N)-(P) Segmentation of body cells in the images of panels K-M from the regions 1-3. White arrows represent segmented cell boundaries corresponding to the green arrows in K-M. Scale bar, 150 μm. (Q) Comparison of cell counting results with original grayscale fluorescent images slice by slice. Cells are delineated with green lines. Scale bar, 150 μm. (R and S) Comparison of max intensity projections of body cells (R) with 3D segmentation reconstruction (S) in a 1050×450×450 µm3 region. Scale bar, 120 μm. (T) The plot shows the validation of the cell segmentation through automatic workflow and manual counting across six 1050×450 µm2 regions. Differences are indicated.

Five major neuron types in planarians.

(A) Distribution of cholinergic, serotonergic, GABAergic, dopaminergic, and octopaminergic neurons in wild-type planarians labeled with probes for chat, tph, gad, th, tbh, and their mixture (neuron pool). Scale bar, 600 μm. (B) FISH images (upper row) and segmentation (lower row) of cholinergic, serotonergic, GABAergic, dopaminergic, and octopaminergic neurons in the brain region. The unlabeled cells are in either the epidermis or non-brain regions. Scale bar, 900 μm. (C) Grayscale (upper row) and reconstruction (lower row) images of estrella+ glial cells. Scale bar, 120 μm.

Neuron changes during homeostasis.

(A) Representative live images of planarians ranging from 1 to 8 mm. Scale bar, 3500 μm. (B) Graph shows the correlation between the square root of surface area and the cube root of volume in homeostatic and regenerative planarians. (C) Graph shows the correlation between cell number and 3D volume in homeostatic planarians. (D) Graph shows the correlation between brain volume and body length in intact planarians. (E) Graph shows the correlation between brain volume and body volume in intact planarians. (F) Ratio of Neuron cells relative to planarians measuring 2-6 mm and 7-9 mm in the 7-9 mm group. The data is shown as the mean ± SEM. N=17 in the 2-6 mm group and N=7 in the 7-9 mm group. Statistical significance was assessed by the two-tailed unpaired Student’s t-test: ***p < 0.001. (G) Graph shows the correlation between GABAergic neuron number and total cell number. (H) Graph shows the correlation between octopaminergic neuron number and total cell number. (I) Graph shows the correlation between dopaminergic neuron number and total cell number. (J) Graph shows the correlation between serotonergic neuron number and total cell number.

Changes in the number of planarian neurons during tail regeneration.

(A) Live images of regenerative tail fragments from 0-14 days post-amputation (dpa). Scale bar, 3000 μm. (B) Graph shows the correlation between the square root of surface area and the cube root of volume in homeostatic and regenerative planarians. (C) Graph shows the correlation of brain volume with the regeneration time points. N≥3 in each condition. The data is shown as the mean ± SEM. (D) FISH images of regenerative tail fragments stained by probes for cholinergic, serotonergic, octopaminergic, GABAergic, and dopaminergic neurons at various time points post-amputation. Scale bar, 7500 μm. (E) Correlation of brain lobe angle with the octopaminergic neuron numbers in regenerative planarians (blue dots).

Distribution of planarian muscle fibers.

(A) Planarian body-wall muscles labeled with the antibody 6G10. Scale bar, 1200 μm. (B) Magnification image of selected region (left) in panel A. Scale bar, 210 μm. (C) Projection of planarian dorsal epidermis muscle. Scale bar, 150 μm. (D) Projection of planarian ventral epidermis muscle. Scale bar, 150 μm. (E) Magnification image of the ventral region. Scale bar, 40 μm. (F) Segmented DV muscle fibers. Scale bar, 40 μm. (G) Segmented DV muscle fibers and ventral muscle fibers. Scale bar, 40 μm. (H) A cartoon illustration depicting planarians’ intestines and eyes. The selected ROI is shown upright as the grayscale of the xy projection. (I) Max projection intensity of planarian eye and intestine region indicated in panel H. Scale bar, 1150 μm. (J) Selected segmented region of the indicated area in panel I. (K) The xy projection of pharynx muscle fibers. Scale bar, 150 μm. (L) Muscle fiber of 150 μm depth projection of pharynx at the dorsal plane. Scale bar, 150 μm. (M) Yz view of a 300 μm depth of planarian pharynx region. Scale bar, 90 μm. (N) Segmented muscle fiber of the connecting DV fibers and pharynx fibers in panel N. Scale bar, 90 μm. (O1) Illustration of muscle fiber regeneration process at 0, 2, and 4 days post-amputation (dpa). (O2) Xz view of blastema region with staining of neurons and muscles on 0, 2, and 4 dpa. Scale bar, 150 μm. (O3) Segmentation of single muscle fiber on 0,2,4 dpa. (P1) Staining of 6G10, neuron pool, and DAPI of planarian at 0 dpa. XZ view of the blastema is shown. Scale bar, 150 μm. 1 represents the xy view of the yellow dotted box in panel P1, and 2 represents the single slice of panel P1. Scale bar, 75 μm. (P2) Staining of 6G10, neuron pool, and DAPI of planarian at 2 dpa. XZ view of the blastema is shown. Scale bar, 150 μm. 1 represents the xy view of the yellow dotted box in panel P2, and 2 represents the single slice of panel P2. Scale bar, 75 μm. (P3) Staining of 6G10, neuron pool, and DAPI of planarian at 4 dpa. XZ view of the blastema is shown. Scale bar, 150 μm. 1 represents the xy view of the yellow dotted box in panel P3, and 2 represents the single slice of panel P3. Scale bar, 75 μm.

Muscle fiber distribution and estrella+ glia localization in inr-1 RNAi animals

(A) Length and width measurements of live egfp RNAi and inr-1 RNAi planarians. (B) Cholinergic, dopaminergic, octopaminergic, serotonergic, and GABAergic neuron labeling of egfp RNAi and inr-1 RNAi planarians. Scale bar, 450 μm (egfp RNAi) and 450 μm (inr-1 RNAi). (C) Representative images of estrella+ glial cells in egfp RNAi and inr-1 RNAi planarian. Scale bar, 400 μm. Magnifications in brain region and selected slices. Scale bars, 180 μm (up), 60 μm (down). (D) 6G10 staining in inr-1 RNAi planarian. Selected dorsal (left) and ventral (right) regions in white dotted line boxes are shown in magnification. Scale bars, 500 μm (left), 200 μm (middle and right). (E) Dual-staining of estrella and 6G10 in egfp RNAi (up) and inr-1 RNAi (down) planarians. Head regions are shown. Scale bar, 300 μm. (F) A representative region in egfp RNAi (up) and inr-1 RNAi (down) planarians with dual-staining of estrella and 6G10. (G) Single channel of estrella+ glia in egfp RNAi (up) and inr-1 RNAi (down) planarians shown in panel F. (H) Single channel of 6G10+ muscles in egfp RNAi (up) and inr-1 RNAi (down) planarians shown in panel F. Scale bar, 150 μm. (I) Xy view of isolated glial cell in egfp RNAi (up) and inr-1 RNAi (down) planarians. (J) Xz view of isolated glial cell in egfp RNAi (up) and inr-1 RNAi (down) planarians. Scale bar, 80 μm. (K) Segmented isolated glial cell in egfp RNAi (up) and inr-1 RNAi (down) planarians. Scale bar, 120 μm.