Meis1 Controls the Differentiation of Eye Progenitor Cells and the Formation of Posterior Poles during Planarian Regeneration

Abstract

As a member of TALE family, Meis1 has been proven to regulate cell proliferation and differentiation during cell fate commitment; however, the mechanism is still not fully understood. The planarian, which has an abundance of stem cells (neoblasts) responsible for regenerating any organ after injury, is an ideal model for studying the mechanisms of tissue identity determination. Here, we characterized a planarian homolog of Meis1 from the planarian Dugesia japonica. Importantly, we found that knockdown of DjMeis1 inhibits the differentiation of neoblasts into eye progenitor cells and results in an eyeless phenotype with normal central nervous system. Furthermore, we observed that DjMeis1 is required for the activation of Wnt signaling pathway by promoting the Djwnt1 expression during posterior regeneration. The silencing of DjMeis1 suppresses the expression of Djwnt1 and results in the inability to reconstruct posterior poles. In general, our findings indicated that DjMeis1 acts as a trigger for the activation of eye and tail regeneration by regulating the differentiation of eye progenitor cells and the formation of posterior poles, respectively.

Keywords: DjMeis1; Djwnt1; Wnt; planarian; regeneration; stem cells.

Figure 1

The regeneration phenotypes of planarians after knockdown of Meis family genes. (A) Planarians were amputated into three fragments from the anterior and posterior sites of pharynx at 24 h after the last injection. (B) The head, trunk, and tail fragments of control planarians developed into normal and complete individuals at 7 dpa. (C) The newly regenerated eyes (red arrows) in trunk (30/30) and tail (30/30) fragments of DjMeis2 RNAi planarians were smaller than those in control groups at 7 dpa. (D) The trunk (24/30) and tail (23/30) fragments of DjMeis3 RNAi planarians regenerated a squared head with elongated eyes (white arrows) at 7 dpa. (E) The head (30/30) and trunk fragments (30/30) of DjMeis1 RNAi planarians failed to regenerate tails (red boxes). The trunk (30/30) and tail fragments (30/30) regenerated eyeless heads (white boxes) at 7 dpa. The images on the right were an enlarged view of the fragments in white and red boxes. Scale bars: 400 μm.

Figure 2

The effect of DjMeis1 RNAi on the anterior regeneration. (A) The expression pattern of ndl2 in trunk fragments by WISH. Only pre-pharyngeal regions were displayed here. DjMesi1 RNAi planarians (10/10) normally expressed ndl2 in the pre-pharyngeal regions. (B) Whole-mount ISH for sert in trunk fragments. DjMeis1 RNAi planarians (10/10) normally expressed sert in the newly regenerated head (yellow boxes). (C) The images of CNS (labeled with PC2) in trunk and tail fragments. DjMeis1 RNAi planarians (10/10) regenerated a proper bilobed brain (white boxes) in the newly regenerated head. (D) The regeneration of eye progenitor cells (labeled with Djovo) in trunk and tail fragments. DjMeis1 RNAi planarians (10/10) regenerated fewer numbers of eye progenitor cells (red arrows) than the control groups. (E) The regeneration of mature eye cells (labeled with opsin) in trunk and tail fragments. DjMeis1 RNAi planarians (10/10) did not express the mature eye cells marker, opsin, in anterior blastema (green arrows). (F) DjMeis1 RNAi planarians (6/6) failed to regenerate eyes (red boxes) at 7 days after eye resection. Scale bars: 400 μm.

Figure 3

DjMeis1 RNAi had no effect on wound-healing and cell proliferation. (A) The head (10/10), trunk (10/10), and tail (10/10) fragments of DjMeis1 RNAi planarians normally expressed the boundary marker LaminB, as indicated by WISH. (B,C) Phospho-H3 staining and quantitative statistical analysis of mitotic cells in planarian fragments. (B) DjMeis1 RNAi planarians (10/10) retained normal proliferative ability at 6 h after amputation. (C) DjMeis1 RNAi planarians (10/10) showed normal mitotic density compared to the control groups at 48 h after amputation. (D,E) Bromodeoxyuridine labeling experiments and quantitative statistical analysis of mitotic cells in the partial trunk fragments. (D) The anterior regions of pharynx (black box) of DjMeis1 RNAi planarians (10/10) displayed the same level of proliferative ability compared to the control groups at 6 h after amputation. (E) The number of mitotic cells in the posterior wound site (black box) of DjMeis1 RNAi planarians (10/10) had no significant difference with the control groups at 48 h after amputation. Statistical comparisons were conducted using the ANOVA test. Significant difference was defined as p < 0.05. ns p > 0.05. Scale bars: 400 μm.

Figure 4

DjMeis1 RNAi inhibited the reconstruction of posterior poles. (A) The images of CNS (stained with anti-synapsin) in trunk and tail fragments. DjMeis1 RNAi planarians (10/10) did not regenerate VNC posteriorly (white arrows). (B) DjMeis1 RNAi planarians did not regenerate (3/10) pharynx or regenerated incomplete pharynx (7/10), as indicated by mhc-1 WISH (red arrows) in head fragments. (C) Whole-mount ISH for fz4 in trunk fragments. DjMeis1 RNAi planarians (10/10) did not express fz4 (red boxes) in posterior blastema. (D) Whole-mount ISH for Djwnt1 in newly regenerated posterior blastema of trunk fragments. DjMeis1 RNAi suppressed the Djwnt1 expression at both 24 and 96 h after amputation (white boxes and yellow arrows). (E) The expression pattern of Djwnt11-2 in newly regenerated posterior blastema of trunk fragments by WISH. DjMeis1 RNAi planarians failed to express Djwnt11-2 at 96 h after amputation (yellow boxes). Scale bars: 400 μm.

Figure 5

β-catenin/DjMeis1 RNAi caused anteriorization of posterior poles in planarians. (A) Head fragments of β-catenin RNAi planarians (10/10) regenerated another head with a pair of eyes (red arrows) from posterior blastema. β-catenin/DjMeis1 RNAi planarians (10/10) regenerated eyeless heads (yellow arrows) from posterior blastema. (B) The expression pattern of sFRP1 (white boxes) in head fragments by WISH. sFRP1 was observed in both anterior and posterior blastema of β-catenin RNAi (10/10) and β-catenin/DjMeis1 RNAi planarians (10/10). (C) The images of CNS (labeled with PC2) in head fragments. Another pair of brain nerves (red boxes) were observed in posterior blastema of DjMeis1/β-catenin RNAi and β-catenin RNAi (10/10) planarians. (D) The regeneration of mature eye cells (labeled with opsin) in head fragments. Only β-catenin RNAi planarians (10/10) expressed opsin (green arrows) in posterior blastema. Scale bars: 400 μm.

Figure 6

Summary of DjMeis1 functions on regeneration. (A) DjMeis1 is required for the proper reconstruction of VNC and eye regeneration. (B) The model of Meis1 regulates the eye regeneration. (C) DjMeis1 is required for the Djwnt1 and Djwnt11-2 expression. (D) The model of Meis1 functions on posterior regeneration.