Non-cell-autonomous inhibition of photoreceptor development by Dip3

Abstract

We show here that the Drosophila MADF/BESS domain transcription factor Dip3, which is expressed in differentiating photoreceptors, regulates neuronal differentiation in the compound eye. Loss of Dip3 activity in photoreceptors leads to an extra photoreceptor in many ommatidia, while ectopic expression of Dip3 in non-neuronal cells results in photoreceptor loss. These findings are consistent with the idea that Dip3 is required non-cell autonomously to block extra photoreceptor formation. Dip3 may mediate the spatially restricted potentiation of Notch (N) signaling since the Dip3 misexpression phenotype is suppressed by reducing N signaling and misexpression of Dip3 leads to ectopic activity of a N-responsive enhancer. Analysis of mosaic ommatidia suggests that no specific photoreceptor must be mutant to generate the mutant phenotype. Remarkably, however, mosaic pupal ommatidia with three or fewer Dip3(+) photoreceptors always differentiate an extra photoreceptor, while those with four or more Dip3(+) photoreceptors never differentiate an extra photoreceptor. These findings are consistent with the notion that Dip3 in photoreceptors activates a heretofore unsuspected diffusible ligand that may work in conjunction with the N pathway to prevent a subpopulation of undifferentiated cells from choosing a neuronal fate.

Figure 1. Dip3 localizes to speckles in the nuclei of all photoreceptors

Immunolocalization of Dip3 in mosaic (A) and wildtype (B–C) 3rd instar larval eye discs. Dip3 is localized posterior to the morphogenetic furrow (A–B). Co-localization of Dip3 and Sens shows that Dip3 is expressed in R8 photoreceptors and the expression is detected from the 4^th row posterior to the morphogenetic furrow (B, see also supplemental Fig. S1). Dip3 is localized in the nuclei of R1-R7 photoreceptors (white asterisks), but not in a non-neuronal nucleus (red asterisk) (C-C’’’).

Figure 2. Loss of Dip3 leads to ectopic photoreceptor development

(A) Schematic drawings of UAS-Dip3 (EP-Dip3) and Dip3-null alleles (Dip3¹ and Dip3^1J2). The red dot on Dip3^1J2 shows the position of a point mutation expected to disrupt splicing. Tangential sections and schematic drawings of wildtype (B) or Dip3¹ (C) eyes. At this focal plane, only the rhabdomeres of R1-7 are seen in wild-type eyes (B). In Dip3¹ eyes, there often is an extra rhabdomere with the cell body attached, located next to R7 (C, arrow) and denoted by the letter “E”. The size of the extra rhabdomere is roughly the same as those of R1-6 suggesting that the extra photoreceptor is an outer photoreceptor. The extra photoreceptor is also visible as an extra Elav positive cell in Dip3¹ mid-pupal ommatidia (compare E and E’ to D and D’). In these ommatidia, there are 9 Elav positive nuclei. One of the nuclei, most likely R7 and therefore marked with SalM, is pushed toward the center of the cluster. D’ and E’ are high magnification views of portions of D and E. Each dot in D’ and E’ represents one nucleus.

Figure 3. Misexpressing Dip3 in undifferentiated cells results in an increase in number of cone and pigment cells at the expense of photoreceptors

(A,B) Schematic drawings of the Dip3 expression pattern in the presence of each driver and the corresponding phenotypes. Expression of Dip3 in cone cells or in photoreceptors produced no visible phenotypes (A). However, when Dip3 was expressed in a cell population that included undifferentiated and pigment cells, smooth eyes were observed (B). In drawings on the left in A and B, gray stippling indicates the presence of endogenous Dip3, while gray shading indicates Dip3 misexpression. C-cone cells; P-photoreceptors; Pc-pigment cells. (C,D) Thick sections of adult wildtype (C) or sev>Dip3 eyes (D). The sev>Dip3 eye shows reduced distance between the rhabdomere clusters, and reduced numbers of rhabdomeres. (E,F) TUNEL staining of late pupal eye disc (96 hrs APF) showed increased cell death in inter-ommatidial cells but not other cells. (G–L) Antibody staining of mid-pupal eyes. In wild type eyes, each ommatidium contains eight photoreceptors, four cone cells and two primary pigment cells as marked by antibody staining for Elav, Ct and BarHI, respectively (G, I, K). However, in Dip3 misexpressing eyes, almost all ommatidia contain fewer than eight photoreceptors (H), along with five cone cells (J), and three primary pigment cells (L).

Figure 4. Dip3 enhances N signaling

(A, B) Scanning electron micrographs of adult eyes raised at 29°C. Insets show magnified views of the same eyes. Reducing the N signal suppresses the Dip3 misexpression phenotype. At 29°C, the eyes of GMR>Dip3 female flies show no clear boundary between the ommatidia (A). However, under the same conditions, when a copy of N^ts is taken away by temperature shift, more bristles and clear demarcation between the ommatidia are observed (B). (C, D) Dip3 over-expression drives ectopic expression of mδ(0.5)-lacZ , a reporter of N signaling. In 3rd instar eye discs, this reporter is expressed only in the R4 photoreceptor, giving rise to diagonal stripes of lacZ positive cells posterior to the morphogenetic furrow (C). However, when Dip3 was over-expressed, extra mδ(0.5)-lacZ positive cells were detected (D, arrows). (E,F) Mis-expression of Dip3 in the wing inhibits formation of the anterior cross-vein, the posterior cross-vein, and the distal segment of L5 (compare F to E, dotted ovals).

Figure 5. The MADF domain is essential for Dip3 function

(A) Schematic drawing of the Dip3 protein including the positions of four point mutations that almost abolish Dip3 function. These mutations were identified in a genetic screen looking for mutations in Dip3 that suppress Dip3 misexpression phenotypes (see text for details). (B,C) Scanning electron micrographs of adult eyes in which Dip3 containing the R40H mutation is misexpressed using the GMR-Gal4 driver (B) or ey-Gal4 driver (C) at 25°C. Misexpression of wild-type Dip3 with GMR-Gal4 produces smooth eyes (Fig. 2B), while misexpression Dip3^R40H produces a relatively normal eye (B). A tangential section of a homozygous Dip3^R40H adult eye shows ommatidia containing an extra photoreceptor (D).

Figure 6. A model for Dip3 function

In normal development, the presence of Dip3 in photoreceptors inhibits some cells from assuming a neuronal fate by modulating N signaling. When Dip3 expression is lost, as in Dip3¹, the neuronal fate inhibition is alleviated leading to the formation of extra photoreceptors. Overexpression of Dip3 in undifferentiated cells (or perhaps primary pigment cells) enhances N signaling, which results in the activation of non-neuronal markers such as D-Pax2 (a cone cell determinant) or Bar-H1 (a primary pigment cell determinant), and inhibition of proneural genes. This leads to loss of photoreceptors and gain of cone and pigment cells. It is not clear if the extra cone and pigment cells arise from the inhibited neuronal precursors, undifferentiated cells, or both.