Forkhead containing transcription factor Albino controls tetrapyrrole-based body pigmentation in planarian

Abstract

Pigmentation processes occur from invertebrates to mammals. Owing to the complexity of the pigmentary system, in vivo animal models for pigmentation study are limited. Planarians are capable of regenerating any missing part including the dark-brown pigments, providing a promising model for pigmentation study. However, the molecular mechanism of planarian body pigmentation is poorly understood. We found in an RNA interference screen that a forkhead containing transcription factor, Albino, was required for pigmentation without affecting survival or other regeneration processes. In addition, the body color recovered after termination of Albino double stranded RNA feeding owing to the robust stem cell system. Further expression analysis revealed a spatial and temporal correlation between Albino and pigmentation process. Gene expression arrays revealed that the expression of three tetrapyrrole biosynthesis enzymes, ALAD, ALAS and PBGD, was impaired upon Albino RNA interference. RNA interference of PBGD led to a similar albinism phenotype caused by Albino RNA interference. Moreover, PBGD was specifically expressed in pigment cells and can serve as a pigment cell molecular marker. Our results revealed that Albino controls planarian body color pigmentation dominantly via regulating tetrapyrrole biogenesis. These results identified Albino as the key regulator of the tetrapyrrole-based planarian body pigmentation, suggesting a role of Albino during stem cell-pigment cell fate decision and provided new insights into porphyria pathogenesis.

Keywords: FoxP; PBGD; body color; pigmentation; planarian; tetrapyrrole.

Figure 1

Planarian pigmentation process and transcription factors involved. (a) Experimental design of RNAi screen; worms were fed dsRNA-mixed liver on days 1, 4, 7 and 10 for four times and pictures were collected from the twelfth day post first RNAi. (b) Genes identified from the RNAi screen that affect the pigmentary system during homeostasis. Scale bar: 200 μm. (c) Conserved domain analysis revealed a FOXP-CC domain and a Forkhead domain within protein TF_FK_025. (d) ClustX2 analysis of TF_FK_025 with human FOXP family proteins (Star indicates same amino acid).

Figure 2

Albino is required for pigmentation without affecting survival or regeneration. (a) Albino RNAi worms lost body color. Scale bar: 200 μm. (b) Regenerated Albino RNAi trunk fragment with albino phenotype. Scale bar: 200 μm. (c) Transmission electron micrographs showing the pigment loss in Albino RNAi worms. Scale bar: 20 μm. Yellow arrows indicate pigment granules. (d) Survival curve of RNAi worms. (e) ISH of stem cell progeny and tissue-specific markers of control and Albino RNAi intact planarian. Scale bar: 500 μm. (f) ISH of stem cell progeny and tissue-specific markers of control and Albino RNAi-amputated trunks. Scale bar: 500 μm.

Figure 3

Albino enriches at epidermal region and during regenerating blastema. (a) WISH in intact animals. Scale bar: 500 μm. (b) Vibration sections of FISH animals. Section thickness: 80 μm and scale bar: 500 μm. Images are z-stacks of 10 μm. (c) Double FISH with AGAT1 and Albino in wild-type animals showing the dorsal body wall. Scale bar: 20 μm. Cartoon indicates region of interest. Images are single confocal sections. (d) Double FISH with smedwi-1 and Albino in wild-type animals showing a transect section. Scale bar: 25 μm. Yellow arrows indicate smedwi-1 and Albino double-positive cells. Images are single confocal sections. (e) ISH in regenerating animals detecting Albino. Yellow dashes indicate amputation sites. Scale bar: 500 μm.

Figure 4

Albino regulates expression of tetrapyrrole biosynthetic enzymes in planarian. (a) Expression fold changes of tetrapyrrole biosynthetic enzymes upon Albino RNAi. Shown are averages of three independent experiments; error bars=s.d. (b) WISH for tetrapyrrole biosynthetic enzymes in worms upon control or Albino RNAi, indicating the reduction of tetrapyrrole biosynthetic enzyme expressions. WISH samples were collected 7 days post fourth RNAi. Scale bar: 500 μm. (c) Representative double FISH results of ALAD, ALAS and PBGD with Albino in wild-type animals. Scale bar: 20 μm. Images are single confocal sections. (d) Representative double FISH results of ALAD and ALAS with PBGD in wild-type animals. Scale bar: 20 μm. Images are single confocal sections. (e) ISH of either wild-type worms or worms received 9 days of continuous direct light for Albino, PBGD, ALAD and ALAS.

Figure 5

PBGD RNAi results in albinism. (a) RNAi of tetrapyrrole biosynthetic enzymes. Yellow arrows indicate regressions. Scale bar: 200 μm. (b) Survival curve of RNAi worms. (c) PBGD RNAi worms lost body color gradually and got totally albino ~60 days post first RNAi. (d) Transmission electron micrographs showing the pigment lost in PBGD RNAi worms. Scale bar: 20 μm. Yellow arrows indicate pigment granules.

Figure 6

PBGD labels pigment cells in planarian. (a) WISH and frozen section of PBGD in wild-type animals showing an epidermal-specific expression pattern. Scale bar: 200 μm. (b) Relative expression level to gapdh. Shown are averages of three independent experiments; error bars=s.d. (c) Double FISH for PBGD with mhc, troponin and prog2 in wild-type animals at dorsal body wall showing PBGD-positive cells lying in between muscle cells. Scale bar: 20 μm. Images are single confocal sections. (d) WISH showing expression patterns of PBGD in regenerating control or Albino RNAi worms. Red dashes indicate amputation sites. Scale bar: 200 μm.

Figure 7

Albino mediates neoblast-pigment cell differentiation. (a) Double FISH for smedwi-1 with Albino at different times of regeneration showing that smedwi-1 and Albino colocalize at the blastema during regeneration. Scale bar: 20 μm. Yellow circles indicate double-positive cells, whereas red and green circles indicate Albino and smedwi-1 single-positive cells, respectively. Images are single confocal sections. (b) Quantification of smedwi-1 and Albino double-positive cells. Cells in 0.1 mm² were counted in three independent experiments. Error bar=s.d.; *P<0.0001; significance determined with Student’s t-test. (c) Cartoon illustrates that Albino controls the expression of PBGD, and smedwi-1-positive neoblasts initially specialize into smedwi-1 and Albino double-positive cells and then fully differentiate into pigment cells expressing Albino and PBGD. During regeneration, Albino-expressing neoblasts accumulate in the blastema at 3 dpa and initiate the expression of tetrapyrrole biogenesis enzymes. The Albino and PBGD double-positive cells accumulate within blastema since 7 dpa, and finally these cells generate tetrapyrroles that get the planarians pigmented.