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. 2022 Apr 1;149(7):dev200125.
doi: 10.1242/dev.200125. Epub 2022 Mar 30.

Src acts with WNT/FGFRL signaling to pattern the planarian anteroposterior axis

Nicolle A Bonar  1 , David I Gittin  1 , Christian P Petersen  1   2
Affiliations

Src acts with WNT/FGFRL signaling to pattern the planarian anteroposterior axis

Nicolle A Bonar et al. Development. .

Abstract

Tissue identity determination is crucial for regeneration, and the planarian anteroposterior (AP) axis uses positional control genes expressed from body wall muscle to determine body regionalization. Canonical Wnt signaling establishes anterior versus posterior pole identities through notum and wnt1 signaling, and two Wnt/FGFRL signaling pathways control head and trunk domains, but their downstream signaling mechanisms are not fully understood. Here, we identify a planarian Src homolog that restricts head and trunk identities to anterior positions. src-1(RNAi) animals formed enlarged brains and ectopic eyes and also duplicated trunk tissue, similar to a combination of Wnt/FGFRL RNAi phenotypes. src-1 was required for establishing territories of positional control gene expression in Schmidtea mediterranea, indicating that it acts at an upstream step in patterning the AP axis. Double RNAi experiments and eye regeneration assays suggest src-1 can act in parallel to at least some Wnt and FGFRL factors. Co-inhibition of src-1 with other posterior-promoting factors led to dramatic patterning changes and a reprogramming of Wnt/FGFRLs into controlling new positional outputs. These results identify src-1 as a factor that promotes robustness of the AP positional system that instructs appropriate regeneration.

Keywords: Axis formation; Patterning; Planarian; Positional information; Regeneration; Src; Wnt.

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Conflict of interest statement

Competing interests The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
src-1 restricts head and trunk identity to anterior positions. (A) src-1(RNAi) animals undergoing tail regeneration formed ectopic posterior eyes expressing opsin and tyrosinase. (B) src-1(RNAi) animals undergoing tail regeneration formed a larger brain, as evidenced by GluR expression, a marker of planarian brain branches. GluR also stains the pharynx (phx). Regenerating src-1(RNAi) head fragments formed a single pharynx, similar to control animals (10/10 animals). Right: quantification of brain branch length by GluR expression proportional to body length. *P<0.05 (unpaired two-tailed t-test). Box plot shows median values (middle bars) and first to third interquartile ranges (boxes); whiskers indicate 1.5× the interquartile ranges and dots are individual data points. (C) Regenerating src-1(RNAi) trunk fragments formed a posterior secondary pharynx (2 of 10 animals) as marked by laminin expression and a larger brain (10 of 10 animals) as marked by cintillo and chat expression. chat+ photoreceptor neuron staining showed that 114/204 animals had an extra set of eyes posteriorly. White arrows indicate ectopic structures. Scale bars: 150 µm (A); 300 µm (B,C).
Fig. 2.
Fig. 2.
Anterior and central PCG domains are modified by src-1 RNAi. Top: uninjured src-1(RNAi) or control RNAi animals stained for PCG domain expression by whole-mount in situ hybridization as indicated after 32 days of gene inhibition. Black bars indicate expression ranges modified from control conditions. Bottom: quantifications of expression domains measured as a fraction of total animal length. Below, the PCG expression domain features were measured from the anterior animal tip (ndk, ndl-5, ndl-3 anterior boundary, ptk7 anterior boundary, ndl-3 posterior boundary) or from the posterior tip (ptk7 posterior boundary, wntP-2, wnt11-1, fzd4). At least four animals were used in each measurement. *P<0.05, **P<0.01, ***P<0.001 (unpaired two-tailed t-test). n.s., P>0.05. axinB expression was continual across the axis and so could not be confidently scored in this way, and 4/4 animals appeared as shown. src-1 inhibition caused a posterior shift to the anterior and central PCG domains (ndk, ndl-5, ndl-3 anterior boundary, ptk7 anterior boundary) and no significant change to posterior PCG domains. Scale bars: 200 µm.
Fig. 3.
Fig. 3.
notum and src-1 can act independently to determine eye placement. (A) FISH to detect expression of opsin (green), a marker of photoreceptor neurons, and tyrosinase (red), a marker of pigment cup cells, in control, src-1, notum and src-1+notum(RNAi) regenerating head fragments. Hoechst (blue) was used as counterstain to detect nuclei. Ectopic eyes are marked by white arrows versus unmarked pre-existing eyes. notum(RNAi) caused formation of anterior ectopic eyes and src-1(RNAi) caused the formation of posterior ectopic eyes, whereas simultaneous inhibition of src-1 and notum(RNAi) resulted in a synthetic phenotype in 24/42 animals with both anterior and posterior ectopic eyes. (B) FISH to detect expression of cintillo (red), a marker of chemosensory neurons, in control, src-1, notum and src-1+notum(RNAi) regenerating head fragments. (C) Quantification of cintillo+ cell number normalized to animal size. *P<0.05 (unpaired two-tailed t-test). Box plot shows median values (middle bars) and first to third interquartile ranges (boxes); whiskers indicate 1.5× the interquartile ranges and dots are individual data points. notum(RNAi) caused the regeneration with reduced numbers of cintillo+ cells, and src-1(RNAi) caused formation of greater numbers of cintillo+ cells. Simultaneous inhibition of src-1 and notum resulted in an intermediate number of these cells. Scale bars: 150 µm.
Fig. 4.
Fig. 4.
Inhibition of src-1 or wnt11-6 but not ndk alters the location of eye regeneration. Uninjured animals were fed the indicated dsRNA 12 times over 6 weeks and eye resection was then performed to remove either a pre-existing original eye or a supernumerary posterior eye in either wnt11-6(RNAi), ndk(RNAi) or src-1(RNAi) conditions. Animals were then imaged to verify eye removal (yellow arrows) and tracked individually as they attempted eye regeneration over the course of 22 days. They were scored for the successful (green arrows, green numbers scoring animals shown on right) or unsuccessful (red arrows, red numbers scoring animals shown on right) eye regeneration. Removal of the pre-existing eye resulted in successful eye regeneration in 8/13 ndk(RNAi) animals but in 0/11 wnt11-6(RNAi) animals and 1/11 src-1(RNAi) animal. By contrast, removal of the supernumerary eyes resulted in regeneration in 6/9 wnt11-6(RNAi) animals and 5/11 src-1(RNAi) animals but only 2/13 ndk(RNAi) animals. The bottom panel summarizes the frequency of regeneration from each condition and eye type. Therefore, either wnt11-6 or src-1 RNAi treatments shift the target location of eye regeneration to a more posterior position, whereas ndk RNAi did not as strongly cause this shift. Scale bars: 150 µm.
Fig. 5.
Fig. 5.
src-1 inhibition sensitizes animals to AP pattern disruption and reprograms PCG activity. (A) FISH to detect expression of opsin (green), a marker of photoreceptor neurons, and tyrosinase (red), a marker of pigment cup cells, in head fragments at day 21 post amputation. Hoechst (blue) used as counterstain to detect nuclei. Simultaneous inhibition of src-1 with wnt11-6, ndk or fzd5/8-4 resulted in the formation of numerous ectopic eyes that extended posteriorly to a greater extent and number than in single-gene inhibitions. Right: quantification of eye cell number after each treatment. Ectopic eyes were increased in number after dual inhibition of src-1 and each tested gene compared with inhibition of each tested gene alone. ***P<0.001 (unpaired two-tailed t-test). Box plot shows median values (middle bars) and first to third interquartile ranges (boxes); whiskers show 1.5× interquartile ranges; dots are individual data points. (B) Day-21 regenerating trunk fragments stained with laminin riboprobe to mark the pharynx (red, central), along with FISH of cintillo (red, anterior) marking chemosensory neurons. Simultaneous inhibition of src-1 with wnt11-6, ndk, ndl-3, ptk7 or wntP-2 resulted in the formation of ectopic posterior pharynges at a greater penetrance than each RNAi condition alone. Numbers indicate fraction of animals with either a single pharynx or ectopic pharynges as shown. Scale bars: 300 μm.
Fig. 6.
Fig. 6.
src-1 acts with Wnt and FGFRLs to control AP axis identity. Model of src-1 participating in AP patterning along with Wnts and FGFRLs. Cartoons depict positional control gene domains from muscle that determine the normal animal pattern of eyes, brain and pharynx, as well as patterning phenotypes after inhibition of key factors. The synthetic phenotypes of src-1 and notum RNAi suggest that src-1 can act independently of some Wnt signaling for control of AP identity. Likewise, the distinct effects of src-1 RNAi and ndk RNAi on the location of eye regeneration after eye removal suggest that ndk likely does not act through src-1. However, it remains possible that src-1 could act downstream of Wnts in a notum-independent process and/or downstream of other factors acting in parallel. src-1 influences PCG domains and functions as a buffer to help define their territories and outputs, and thereby suppress anterior identity.
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