Combined inactivation of pRB and hippo pathways induces dedifferentiation in the Drosophila retina

Abstract

Functional inactivation of the Retinoblastoma (pRB) pathway is an early and obligatory event in tumorigenesis. The importance of pRB is usually explained by its ability to promote cell cycle exit. Here, we demonstrate that, independently of cell cycle exit control, in cooperation with the Hippo tumor suppressor pathway, pRB functions to maintain the terminally differentiated state. We show that mutations in the Hippo signaling pathway, wts or hpo, trigger widespread dedifferentiation of rbf mutant cells in the Drosophila eye. Initially, rbf wts or rbf hpo double mutant cells are morphologically indistinguishable from their wild-type counterparts as they properly differentiate into photoreceptors, form axonal projections, and express late neuronal markers. However, the double mutant cells cannot maintain their neuronal identity, dedifferentiate, and thus become uncommitted eye specific cells. Surprisingly, this dedifferentiation is fully independent of cell cycle exit defects and occurs even when inappropriate proliferation is fully blocked by a de2f1 mutation. Thus, our results reveal the novel involvement of the pRB pathway during the maintenance of a differentiated state and suggest that terminally differentiated Rb mutant cells are intrinsically prone to dedifferentiation, can be converted to progenitor cells, and thus contribute to cancer advancement.

Figure 1. rbf wts double mutants have defects in differentiation.

(A) Schematic of the spatiotemporal model of differentiation during larval eye development. The morphogenetic furrow [(MF), dotted line] moves from the posterior (P) edge of the disc towards the anterior (A) compartment. As it moves into contact with cells they begin to undergo a progressive differentiation that occurs in stages. The first being a progressive recruitment period to develop cell identity (marked by bar); the second stage being a cell autonomous program to complete terminal neuronal differentiation and maintain mature neuronal identity. Therefore, the cells in the most posterior regions of the disc have been fully differentiated the longest time and those cells nearest to the MF are at the earliest stages of differentiation. (B–L) The position of the MF is shown by a white arrowhead and the posterior compartment is to the right in all images. All images of larval discs are projection images. Clones of mutant cells were induced with the FLP-FRT system and distinguished by the lack of GFP (green). (B–G) Eye discs were labeled for the Senseless (Sens) protein in red and the Elav protein in blue. (I,J) Eye discs were labeled for the Senseless (Sens) protein in blue and the Atonal (Ato) protein in red. (K,L) Eye discs were labeled for the Senseless (Sens) protein in blue and the Scabrous (Sca) protein in red. (B) A wild-type disc. (C,D) Photoreceptors differentiate normally in the eye disc hemizygous for the rbf^120a mutant allele (C) and in clones of wts^x1 mutant cells (D). (E) The number of Sens positive cells is reduced in the posterior of the rbf^120a wts^x1 mutant clones. White arrows point to Elav positive clusters of cells that lack any Sens positive cell. Elav expression reveals an incomplete complement of photoreceptors in the posterior of the double mutant tissue. (Ei) Quantification of Sens positive cells present in rbf^120a wts^x1 mutant clones (see Materials and Methods for details). A Student's t-test revealed that the differences between the rbf^120a wts^x1 double mutant cells and the wild-type population of Elav positive cells with zero or two Senseless positive cells were statistically significant with a p-value <0.05. (F,G) The loss of differentiation is specific to a genetic interaction between the pRB and Hippo pathways as photoreceptors differentiate normally in (F) tsc^f01910 and (G) rbf^120a tsc^f01910 mutant clones. Loss of tsc results in larger than normal cells, thus leading to increased spacing between adjacent ommatidia marked by Sens and Elav. (H) wts^x1 single mutant and rbf^120a wts^x1 double clones were generated at a low frequency with hs-FLP and examined in the adult eye. Arrows highlight the differences between the two tissue samples shown. The wts^x1 single mutant tissue is well differentiated and contains a high number of bristles (black dots). In contrast, the surface of the rbf^120a wts^x1 double mutant tissue is glossy and lacks bristles. (I) A wild-type eye disc. Senseless expression in pre-R8 cells requires that Atonal expression be initiated first. After the expression of Senseless has been able to define the R8 cell and to begin recruitment of the ensuing R cells, Atonal expression is lost. (J) Atonal expression is normal in the rbf^120a wts^x1 double mutant tissue suggesting that mature R8 cells expressing Senseless can develop normally. (K) A wild-type eye disc. Ommatidial cluster formation relies upon proper spacing to be present between clusters. This spacing is defined early in the recruitment and refinement stage of differentiation of the R8 cell when a Senseless positive cell expresses the glycoprotein Scabrous. (L) Scabrous expression appears normal in rbf^120a wts^x1 double mutant tissue. Therefore, rbf^120a wts^x1 mutant R8 cells can successfully be refined and can establish proper spacing for further recruitment of the ensuing R cells to take place.

Figure 2. Loss of differentiation markers is not due to apoptosis.

Apoptosis does not contribute to the reduction in the number of Sens positive cells. Clones of mutant cells were induced with the FLP-FRT technique and distinguished by the lack of GFP (green). (A) Apoptosis, as detected by the cleaved caspase-3 (C3) antibody (magenta), in the MF of a disc hemizygous for rbf^120a but not in the posterior compartment. (B) Lack of cleaved caspase-3 positive cells, as detected by the C3 antibody (magenta), in clones of rbf^120a wts^x1 double mutant cells within and posterior to the MF. (C) Overexpression of p35 within and posterior to the MF does not rescue the reduced number of Sens positive cells in the posterior of rbf^120a wts^x1 mutant clones.

Figure 3. Dedifferentiation of rbf wts double mutant cells.

Clones of mutant cells were induced with the FLP-FRT technique and distinguished by the lack of GFP (green). In all merged images Elav is blue, and when shown on a single channel it is on the gray scale. All images are projection images. (A–Ai) Expression of the protein Rough (Ro) (red) is detected at high levels in R2/5 and at lower levels in R3/4 cells in a wild-type disc. Elav expression is found in all mature R cells. Rough positive cells are developed and recruited by the proper refinement and resolution of R8 cells. Once developed, Rough positive cells take on a cell autonomous program to maintain cell identity. (B–Bi) The correct numbers (shown by white arrows in Bi) of Ro (red) positive cells are resolved in rbf^120a wts^x1 double mutant ommatidial clusters marked by Elav nearest the MF. However, a stochastic pattern of ommatidial cluster appearance begins further posterior from the MF, seen by the loss of both Ro and Elav expression in the rbf^120a wts^x1 double mutant clones. (C) This is in contrast to the ommatidial cluster development and the ability of the ommatidial cells to maintain proper identity (shown by Elav expression in wts^x1 single mutant clones). (D) Quantification of the number of Elav positive cells within a mature photoreceptor cluster in four genotypes (wild-type, rbf1^120a, wts^x1 and rbf1^120a wts^x1) (see Materials and Methods for details). Counting was done from the third Elav positive column behind the MF to the posterior edge, an area in a wild-type eye disc that will have developed a range of either 5–7 detectable Elav positive cells per cluster. Error bars are standard deviations from the mean of each category per genotype. A Student's t-test between each mutant genotype and the wild-type population revealed that no statistical difference between the rbf mutant and wild-type tissue existed. A p-value <0.05 existed between the wts mutant and wild-type tissue for the number of ommatidial clusters with 7 Elav positive cells. A p-value <0.05 existed between the rbf wts double mutant an wild-type for the number of ommatidial clusters with 1, 2, or 3 Elav positive cells and a p-value <0.01 for the number of ommatidial clusters with 5, 6, or 7 Elav positive cells. We note that the distribution of the defects in differentiation are not directly due to developmental recruitment as we can find complete and incomplete ommatidial clusters in the rbf wts double mutant eye disc (see distribution of percentages). (E,F) Expression of the late neuronal marker Neuroglian (Nrg) (red) in a wild-type disc (E). rbf^120a wts^x1 double mutant cells initially express the late neuronal marker Neuroglian (Nrg) (red), but then lose expression of Nrg in the posterior regions of the disc (F). (G,H) Wild-type expression of Elav in pupal retinas (44–48hr APF) reveals that Elav expression is not recovered later in development of rbf^120a wts^x1 double mutant photoreceptor cells (mutant tissue is separated from wild-type tissue by green outline on gray scale). A variable number of Elav positive cells, from normal (yellow arrow) to highly reduced (red arrows), can be found amongst each rbf wts double mutant ommatidium in (H).

Figure 4. Expression of the eye specification factor Eyes Absent (Eya) in dedifferentiating rbf wts double mutant photoreceptors.

Clones of mutant cells were induced with the FLP-FRT system and distinguished by the lack of GFP (green). (A–Ai) Expression of the eye specification factor Eyes Absent (Eya) (red) and the neuronal specific protein Elav (blue) in a wild-type disc. Eya expression is highest in mature, differentiated cells, marked by Elav (blue). (B–Bi) Loss of wts does not affect the expression pattern of Eya. The reduced expression of Eya (red) in population of the unspecified interommatidial cells between ommatidial clusters (marked by Elav (blue)) can be seen more easily in a wts single mutant background (Bi). (C) Eya is expressed in rbf^120a wts^x1 double mutant cells. (Ci) Examples of rbf^120a wts^x1 double mutant cells adjacent to ommatidial clusters can frequently be found where cells no longer express Elav but have a high level of Eya (pointed by arrows). (D) rbf^120a wts^x1 double mutant cells do not transdifferentiate into cone cells (Cut expression is in red) later in development (44–48 hr APF).

Figure 5. rbf wts double mutant cells fail to exit the cell cycle while undergoing photoreceptor differentiation.

S phase cells in the eye disc were revealed by BrdU labeling. (A) No BrdU positive cells were found posterior to the second mitotic wave (SMW) in a wild-type disc. (B) Inappropriate S phases posterior to the SMW in the rbf¹⁴ wts^x1 double mutant tissue. (C) Differentiated rbf¹⁴ wts^x1 double mutant cells (Elav) (blue) were undergoing S phases (pointed by arrows). (D) rbf¹⁴ wts^x1 double mutant cells expressed the R8 marker Sens (blue) even during mitosis (marked by pH3) (red). Examples are pointed by arrows. (E) rbf¹⁴ wts^x1 double mutant photoreceptors (Elav) (blue) continued to undergo mitosis (marked by pH3) (red) in pupal eye discs.

Figure 6. Cells lacking rbf and wts fail to maintain the differentiated state even in the absence of inappropriate proliferation.

(A) rbf^120a wts^x1 de2f1⁷²⁹ triple mutant cells failed to proliferate posterior to the SMW as revealed by BrdU labeling. (B) The number of Sens positive cells remained reduced in the posterior of the rbf^120a wts^x1 de2f1⁷²⁹ triple mutant tissue.

Figure 7. Aberrant Yki activity is not sufficient to trigger dedifferentiation of rbf mutant cells.

(A) In the posterior of the wild-type eye imaginal disc, Yki (magenta) is mainly present in interommatidial cells and excluded from differentiating photoreceptors (marked by Senseless (green)). (B,C) Yki is localized in the nuclei (marked by DAPI (blue)) of rbf^120a wts^X1 double mutant cells (B) and rbf^120a e2f1⁷²⁹ wts^X1 triple mutant cells (C). Clones of mutant cells are marked by the lack of GFP and outlined. (D,E) Expression of a constitutively active Yki^S168A in the posterior compartment of a wild-type eye disc using a GMR-Gal4 driver drives inappropriate cell divisions of interommatidial cells (pH3 (green)), but has no effect upon differentiated ommatidial cells (Elav (blue), Senseless (red)) (D). In contrast, expression of a constitutively active Yki^S168A in the posterior compartment of a rbf^120a hemizygous disc using a GMR-Gal4 does drive postmitotic photoreceptors into the cell cycle, as seen by co-localization of pH3 with Sens and Elav positive cells (white). However, this does not cause dedifferentiation of rbf mutant cells since cells still retain expression of both Sens and Elav in the most posterior regions (E).