Wnt and TGFβ coordinate growth and patterning to regulate size-dependent behaviour

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Abstract

Differential coordination of growth and patterning across metazoans gives rise to a diversity of sizes and shapes at tissue, organ and organismal levels. Although tissue size and tissue function can be interdependent1,2,3,4,5, mechanisms that coordinate size and function remain poorly understood. Planarians are regenerative flatworms that bidirectionally scale their adult body size6,7 and reproduce asexually, via transverse fission, in a size-dependent manner8,9,10. This model offers a robust context to address the gap in knowledge that underlies the link between size and function. Here, by generating an optimized planarian fission protocol in Schmidtea mediterranea, we show that progeny number and the frequency of fission initiation are correlated with parent size. Fission progeny size is fixed by previously unidentified mechanically vulnerable planes spaced at an absolute distance along the anterior–posterior axis. An RNA interference screen of genes for anterior–posterior patterning uncovered components of the TGFβ and Wnt signalling pathways as regulators of the frequency of fission initiation rather than the position of fission planes. Finally, inhibition of Wnt and TGFβ signalling during growth altered the patterning of mechanosensory neurons—a neural subpopulation that is distributed in accordance with worm size and modulates fission behaviour. Our study identifies a role for TGFβ and Wnt in regulating size-dependent behaviour, and uncovers an interdependence between patterning, growth and neurological function.

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Fig. 1: Planarian fission is a size-dependent behaviour.
Fig. 2: Wnt signalling and TGFβ signalling components modulate fission activity.
Fig. 3: Pre-established fission planes determine progeny size independently of Wnt and TGFβ signalling.
Fig. 4: Wnt and TGFβ signalling regulates fission frequency by size-dependent patterning of mechanosensory neurons in the CNS.

Data availability

Source data and construct sequences can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1356. All other data are available from the corresponding author upon reasonable request.

Code availability

Code for the Python 3.6 (https://www.python.org/) script used for a wrapper for FFMPEG (https://www.ffmpeg.org/) for the high-throughput recording of fission behaviour is available at the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1356.

Change history

  • 23 August 2019

    Owing to a technical error, this Letter was not published online on 14 August 2019, as originally stated, and was instead first published online on 15 August 2019. The Letter has been corrected online.

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Acknowledgements

We thank members of the A.S.A. laboratory for discussion and advice, F. Mann for providing unpublished reagents, and K. Si for comments. We are grateful to the Stowers Planarian and Microscopy core facilities for technical contributions and methods development. A.S.A. is an investigator of the Howard Hughes Medical Institute (HHMI) and the Stowers Institute for Medical Research. B.W.B.-P. is a Jane Coffin Childs Memorial Fund Postdoctoral Fellow. C.P.A. is a HHMI Postdoctoral Fellow. This work was supported in part by NIH R37GM057260 to A.S.A.

Author information

Correspondence to Alejandro Sánchez Alvarado.

Ethics declarations

Authors contributions

Conceptualization, data analysis and interpretation: C.P.A., B.W.B.-P. and A.S.A.; acquisition of data: C.P.A., B.W.B.-P. and J.J.L.; design and fabrication of planarian live-imaging systems: J.J.L.; software: C.J.W.; data curation: J.J.L.  and C.J.W.; writing of the original manuscript: C.P.A., B.W.B.-P.  and A.S.A.; supervision and funding acquisition: A.S.A.; and revision and editing of the manuscript: all authors.

Competing interests

The authors declare no competing interests.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Thomas Holstein and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Characterization of planarian fission biology.

a, Live imaging of large planarian worm during fission (representative of 12 experiments; see also Supplementary Video 1). b, Imaging of single individual large planarian and regenerating progeny 0, 4, 8 and 12 days after fission induction (experiment repeated 50 times). c, d, Anterior–posterior length of progeny (c) and time to fission event (d) since induction or the previous fission (n = 50 worms). Fission fragments binned by position along the anterior–posterior axis (the first fission is the most posterior). e, Schematic of fission induction and quantitative scoring system used to compare fission activity between different conditions. f, Cumulative fission fragments produced over 14 days by individual worms binned by parent size (n = 10 per bin). g, h, Time to first fission event (g) or time between sequential fission events (h) for worms 6–8 mm, 9–12 mm or 13–17 mm in length. i, Raw parent length measurement of planarian individuals 6–8 mm, 9–12 mm or 13–17 mm in length. j, Time between first and second fission events for worms 6–8 mm, 9–12 mm or 13–17 mm in length (n = 139 independent measurements from 30 worms). k, l, Time between induction and first fission (k) or between first and second fission (l) plotted relative to parent length (n = 26 and 21 independent measurements from 30 worms). PCC, linear regression and R2 values are provided. P values determined by determined by two-sided t-test. Data are mean ± s.e.m. (c, d, j).

Extended Data Fig. 2 Quantification of fission behaviour across a range of worm sizes.

a, All individual timelines depicting fission activity over 9 days for worms ranging from 2 mm (bottom) to 12 mm (top) in length (n = 39 worms). b, Number of successful fission attempts per worm relative to parent length. c, d, Number of fission attempts (c) and time to first fission attempt (d) for worms binned into small (2–5 mm), medium (6–7 mm) and large (8–12 mm) groups (n = 16 small, 11 medium, 12 large worms). Data are mean ± s.e.m.

Extended Data Fig. 3 Strategy for a targeted RNAi screen to identify regulators of fission.

a, Detailed schematic of RNAi workflow. Worms are grown to an optimal size in the recirculation culture system and transferred to a flow system for RNAi feedings. After 3 RNAi feedings, worms were transferred to a 15-cm dish and worm length was recorded. The number of fissions were recorded daily for 14 days for each worm from each RNAi condition. For data analysis, the number of daily cumulative fissions were divided by initial body length and then normalized to the average of the control RNAi fissions. For data visualization, this normalized fission score for each day was converted to a heat colour code. Daily scores for each individual worm were aligned in ascending order along the y axis. The average score of each column is calculated and used to sort individual worms in ascending order along the x axis. The average fission score of each RNAi condition was then sorted in ascending order from left to right. The result is a heat-map visualization that ranks the effects of RNAi treatments on fission activity. b, Wnt, TGFβ and Hh signalling pathway diagrams focusing on components targeted for the RNAi screen. Green arrows indicate positive interactions; red arrows indicate inhibitory interactions.

Extended Data Fig. 4 Analysis of morphology and/or internal tissues in regenerating fragments and fissioning parents.

a, Representative images of regenerating tissue fragments from different positions along the anterior–posterior axis at 15 days post-amputation (dpa). Fraction of worms with pictured phenotype along with 1-mm scale bar depicted below each image. b, c, In situ staining of CNS (pc2), intestine (porc) and muscle (t-mus) tissues at day 15 of regeneration (b) or the fission assay (c). d, High-resolution image of body wall musculature (t-mus) in control RNAi and smad2/3 or β-catenin RNAi treated worms. Representative images (n = 7–13 worms) from a single experiment. All images are oriented ventral side up with anterior on the left side. Scale bars, 0.5 mm.

Extended Data Fig. 5 Effects of growth, starvation and regeneration on fission planes.

a, Image of planaria after incomplete fission, revealing ventral tear identical to compression planes (observed more than five independent times). b, Post-compression worms at 5, 18 and 30 days post-fertilization (dpf) (5 dpf image from same experiment as Fig. 3b). Data are from a single experiment. c, d, Bidirectional plot of compression planes versus worm length (n = 25 worms) (c), and relative distribution of planes (d) at 5, 18 or 30 dpf (n = 28, 18, 31, 15 and 19 worms (left to right in d)). e, Schematic of experiment tracking establishment of fission planes during tissue regeneration. f, g, Representative images (f) and bidirectional plot of compression planes versus worm length (g) after amputation (1 dpa, n = 15 worms), regeneration (8 dpa, n = 19 worms) and growth (fed 14 dpa and 25 dpa, n = 12 and 32 worms) or de-growth (starved 25 dpa, n = 15 worms). Data from a single experiment. hj, Worm length (h), number of compression planes (i) and relative distribution of planes (j) after amputation (1 dpa, n = 15 worms), regeneration (8 dpa, n = 19 worms) and growth (fed 14 dpa and 25 dpa, n = 12 and 32 worms) or de-growth (starved 25 dpa, n = 15 worms). Data are mean ± s.d. (c, g) or mean ± s.e.m (hj).

Extended Data Fig. 6 Effects of RNAi of Wnt and TGFβ signalling components on fission planes.

a, b, Relative plane distribution after RNAi treatment (n = 20 (a) and 10 (b) worms). c, Representative images of progeny within 24 h of fission and of remaining parent tissue at day 28 after fission induction for worms treated with control, β-catenin, actR-1 or smad2/3 RNAi (experiment independently performed twice). Scale bar, 1 mm. d, e, Length of the first fission progeny (d) or all subsequent progeny (e) in worms treated with control, β-catenin, actR-1 or smad2/3 RNAi (n = 85 fission fragments from 36 worms). P values determined by two-way ANOVA interaction factor (a, b) or two-sided t-test (d, e). Data are mean ± s.e.m.

Extended Data Fig. 7 Wnt and TGFβ signalling components regulate the frequency of fission initiation.

ah, All individual timelines depicting fission activity over 9–10 days for worms treated with control (a, g), actR-1 (b), smad2/3 (c), β-catenin (d), dsh-B (e), apc (f) or wnt11-6 (h) RNAi. in, Graphs depicting the time between sequential fission attempts (i, j), the number of successful fission attempts (k, l) and the number of unsuccessful fission attempts (m, n) in worms fed double-stranded RNA (dsRNA) that targets regulators of fission (n = 421 fission events from 116 worms). Worms were given either 3 (af, i, k, m) or 18 (g, h, j, l, n) dsRNA feedings. Batched experiments are plotted separately. P values determined by two-sided t-test. Data are mean ± s.e.m.

Extended Data Fig. 8 The planarian anterior CNS regulates fission.

a, Whole-brain imaging of pc2 and fission regulator gene expression detected by double FISH (n = 2–4 worms; experiment independently repeated). Scale bars, 100 μm. b, Single-cell co-expression of pc2 and fission regulators in the posterior branches of the anterior CNS (n = 3–5 worms). Scale bar, 50 μm. c, Fission induction in intact, 100% head-amputated or 50% head-amputated worms over a 9-day observation period (n = 12 worms). df, Total number of fission progeny over 9 days (d), the time between fission induction and first fission (e), and the time between first and second fission (f) for intact, 100% head-amputated or 50% head-amputated worms (n = 94 fission events from 36 worms). g, Regeneration time course in 100% head-amputated worms showing recovery of anterior gene expression of pc2 co-localized with teashirt (n = 4–5 worms; experiment performed once). Scale bar, 500 μm. h, Heat maps depicting fission activity after treatment with coe RNAi. Normalized cumulative fissions over time are displayed for individual worms from each RNAi condition (n = 12 worms). i, Representative parent images on days 0 and 14 of the fission assay (n = 12, experiment independently performed twice). Scale bars, 1 mm. P value determined by two-sided t-test (df) or two-way ANOVA (h). Data are mean ± s.e.m. (df).

Extended Data Fig. 9 Comparison of neuronal subpopulations in worms of increasing size and after smad2/3 RNAi treatment.

a, Representative images of neuronal marker staining in small, medium and large worms (n = 3–5 worms; 1 experiment). b, Representative images of a subset of neuronal markers analysed in worms treated with smad2/3 RNAi (n = 3–5 worms; 1 experiment). Scale bars, 0.5 mm.

Extended Data Fig. 10 gabrg3L-2 negatively regulates the frequency of fission initiation.

a, b, All individual timelines depicting fission activity over 9 days for worms treated with control (a) or gabrg3L-2 (b) RNAi (n = recordings of 10 worms combined from 2 independent experiments).

Supplementary information

Supplementary Table 1

This file contains accession numbers for the genes used to clone RNAi/in situ probe constructs.

Reporting Summary

Video 1

Dual-camera imaging of planarian fission reveals complex behaviour. Video contains an image of dual camera imaging system, followed by two independently acquired videos of planarian fission imaged from two orthogonal views.

Video 2

Fission behaviour in animals of increasing size. Video contains representative videos of small (4mm), medium (6mm), and large (11mm) animals. Worms were placed in 6-well dishes with cameras mounted above the plates and images were acquired every 10 minutes for 9 days.

Video 3

Compression of adult animals reveals cryptic fission planes. Video contains live image capture of cryptic vulnerable planes revealed by mechanical compression, shown at ½ real time speed. Video was captured in real time using an iPhone 6.

Video 4

Control RNAi animal fission behaviour. Representative video of fission behavior in control (Unc-22) RNAi animals. Worms were placed in 6-well dishes with cameras mounted above the plates and images were acquired every 10 minutes for 9 days.

Video 5

Fission behaviour in TGFβ signaling RNAi treatments. Representative videos of fission behaviour in TGFβ signaling RNAi treatments: ActR-1 (n=16), and smad2/3 (n=16). Worms were placed in 6-well dishes with cameras mounted above the plates and images were acquired every 10 minutes for 9 days.

Video 6

Fission behaviour in Wnt signaling RNAi treatments. Representative videos of fission behaviour in Wnt signaling RNAi treatments: β-catenin (n=16), DshB (n=16), wnt11-6 (n=10), and APC (n=16). Worms were placed in 6-well dishes with cameras mounted above the plates and images were acquired every 10 minutes for 9 days.

Video 7

Fission behaviour in gabrg3L-2 RNAi animals. Representative video of fission behaviour in gabrg3L-2 RNAi animal (n=10). Worms were placed in 6-well dishes with cameras mounted above the plates and images were acquired every 10 minutes for 9 days.

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