The role of Sevenless in Drosophila R7 photoreceptor specification

Abstract

Sevenless (Sev) is a Receptor Tyrosine Kinase (RTK) that is required for the specification of the Drosophila R7 photoreceptor. Other Drosophila photoreceptors are specified by the action of another RTK; the Drosophila EGF Receptor (DER). Why Sev is required specifically in the R7 precursor, and the exact role it plays in the cell's fate assignment have long remained unclear. Notch (N) signaling plays many roles in R7 specification, one of which is to prevent DER activity from establishing the photoreceptor fate. Our current model of Sev function is that it hyperactivates the RTK pathway in the R7 precursor to overcome the N-imposed block on photoreceptor specification. From this perspective DER and Sev are viewed as engaging the same transduction machinery, the only difference between them being the level of pathway activation that they induce. To test this model, we generated a Sev/DER chimera in which the intracellular domain of Sev is replaced with that of DER. This chimerical receptor acts indistinguishably from Sev itself; a result that is entirely consistent with the two RTKs sharing identical transduction abilities. A long-standing question in regard to Sev is the function of a hydrophobic domain some 60 amino acids from the initiating Methionine. If this represents a transmembrane domain, it would endow Sev with N-terminal intracellular sequences through which it could engage internal transduction pathways. However, we find that this domain acts as an internal signal peptide, and that there is no Sev N-terminal intracellular domain. phyllopod (phyl) is the target gene of the RTK pathway, and we show that R7 precursors are selectively lost when phyl gene function is mildly compromised, and that other photoreceptors are removed when the gene function is further reduced. This result adds a key piece of evidence for the hyperactivation of the RTK pathway in the R7 precursor. To facilitate the hyperactivation of the RTK pathway, Sev is expressed at high levels. However, when we express DER at the levels at which Sev is expressed, strong gain-of-function effects result, consistent with ligand-independent activation of the receptor. This highlights another key feature of Sev; that it is expressed at high levels yet remains strictly ligand dependent. Finally, we find that activated Sev can rescue R3/4 photoreceptors when their DER function is abrogated. These results are collectively consistent with Sev and DER activating the same transduction machinery, with Sev generating a pathway hyperactivation to overcome the N-imposed block to photoreceptor specification in R7 precursors.

Figure 1. Rescue of sev° by Sev expression, and the effects of DER expressed at Sev levels.

(A) Phase contrast image through a wild type retina in which the rhabdomeres of six outer photoreceptors are arrayed in an asymmetric trapezoid shape. In the center of the array lies the small central rhabdomere of R7 (red arrows). (B) R7 is absent from every ommatidium in sev° eyes. (C) sev expressed under sev transcriptional control (sev.sev) restores R7s (red arrows) to all sev° ommatidia. (D) An antibody raised against the C-terminus of Sev highlights the typical expression pattern in wild type eye discs. (E) α-Sev.ICD staining of sev°; sev.sev eye discs show the normal Sev expression pattern. (F) In sev°; sev.sev eye discs, normal pattern formation is evident with R7 specified at the correct time and place. At the two-cone-cell-stage, R7s (highlighted by Elav (blue) and Runt (red) expression) are seen on the other side of the ommatidium from R3/4 labeled by Elav and Svp (green). (G) In sev.DER adult eyes, many R7-like cells (red arrows) are evident in each ommatidiaatidium. (H) A sev.DER eye disc labeled with an antibody raised against the C-terminal region of DER. Strong DER staining is observed in the Sev-expression cells superimposed upon the normal DER expression pattern (see Fig.2D). (I) At the two-cone-cell-stage, sev.DER eye discs show cells in cone cell positions (c) differentiating as R7-like photoreceptors (expressing Runt and Elav).

Figure 2. The signaling abilities of Sev/DER chimerical receptors

(A) The sev.SSD chimera (Sev ICD replaced with that of DER and expressed under sev transcriptional control) rescues R7s in all sev° ommatidiaatidia in adult eyes. (B) When the DER TM domain was additionally substituted and expressed under sev transcriptional control (sev.SDD) in two copies in sev°, occasional supernumerary R7s are detected (horizontal red arrow). (C) sev.SSD rescues R7s specification at the correct time and place in sev° eye discs. (D) A wild type eye disc stained with anti-DER.ICD shows staining in all cells with an upregulation in cells in and behind the morphogentic furrow. (E) sev°; sev.SSD eye discs stained with α-DER.ICD show strong upregulation in the Sev-expressing cells. (F) High magnification from image in E showing DER expression in the normal Sev pattern.

Figure 3. Schematic summary of the molecular features of Sev

(A) A Kyte-Doolittle hydrophobicity plot of Sev reveals two salient hydrophobic domains (red arrows). The one to the right indicates the conventional TM domain. The one to the left may be an N-terminal TM or an internal signal peptide. (B) Shows the Sev primary peptide carrying the two highlighted hydrophobic domains. (C) The primary peptide is cleaved into α and β subunits. (D) Two topological arrangements of the α /β dimer: (i) if the N-terminal hydrophobic domain is a TM, there is an N-terminal ICD; (ii) if it represents and internal signal peptide, all a subunit sequences are exclusively extracellular. (E) Visual description of the N-terminal architecture of three transgenes. In sev. sev, ~60 residues (black) lie N-terminal to the hydrophobic domain (HD – red). In sev.G.sev the N-terminal ~60 amino acids are replaced by GFP (green). In sev.M.sev the N-terminal residues are removed, and the HD domain is replaced with a conventional signal sequence (blue) followed by a Myc tag (yellow). (F) Schematic description of DER and Sev and the two chimeras (SSD and SDD).

Figure 4. The effects of substituting the Sev N-terminal ~60 residues with GFP

(A) When the Sev N-terminal ~60 amino acids are replaced with GFP and expressed under sev transcriptional control (sev-G:sev) one copy of the transgene fails to rescue all R7s of sev^° ommatidia. Red arrow indicates a rescued R7, and the green circles highlight ommatidia lacking an R7. (B) Two copies of sev-G:sev rescue R7s in all sev° ommatidia. (C) Two copies of sev-G:sev rescue R7s at the correct time and place in sev⁰eye discs as evidenced by Runt and Elav positive nuclei in the cells occupying the R7 positions. (D) GFP staining of sev ; sev-G:sev eye discs shows expression in the Sev-expressing cells. (E) α-Sev.ICD staining of sev°; sev-G:sev shows only weak staining. (F) The staining level of α-Sev.ICD in sev° sev.sev eye discs co-incubated with sev°; sev-G:sev eye discs shown in D.

Figure 5. A heterologous signal sequence substitutes for the Sev N-terminal hydrophobic domain

All panels show images from sev°; sev.M:sev animals in which the N-terminal hydrophobic domain and all residues N-terminal to it are replaced with a conventional signal peptide followed by the Myc tag. (A) In adult eyes all R7s are rescued (red arrows). (B–F) Eye discs stainings. (B, C) show that pattern formation is normal as monitored by Elav, Runt and Svp expressions, and R7s are specified at the correct time and position. (D) α-Myc staining highlights the normal Sev expression pattern. (E) α-Sev.ICD shows the normal Sev expression pattern. (F) Superimposition of the Myc and Sev staining show their coincident expression in the Sev expressing cells.

Figure 6. A chimera with the DER ICD and a heterologous signal sequence rescues sev°

All panels are from sev°; sev.M:sev animals in which the N-terminal hydrophobic domain and all residues N-terminal to it are replaced with a conventional signal peptide followed by the Myc tag, and the Sev ICD is replaced with that of DER. (A) In adult eyes all R7s are rescued (red arrows). (B–F) Eye discs stainings. (B, C) show that pattern formation is normal as monitored by Elav, Runt and Svp expressions, and R7s are specified at the correct time and position. (D) α-Myc staining highlights the normal Sev expression pattern. (E) α-DER.ICD shows the Sev expression pattern superimposed on the normal DER expression. (F) Superimposition of the Myc and DER staining show their coincident expression in the Sev expressing cells.

Figure 7. Selective R7 sensitivity to phyl gene function reduction, and Sev* rescue of R3/4 DER function

(A) Section through a GMR.GaU; UAS.phyl adult eye shows the typical sev° phenotype with each ommatidium specifically lacking R7. (B) When phyl gene function is further compromised (GMR.GaU; 2XUAS.phyl.RNAi) only 4 large rhabdomere photoreceptors are evident in most ommatidia. (C) sev.Gal4; UAS.derDN eye discs stained for Elav (blue) Svp (green) and Runt (red). Only R2/8/5 clusters are evident indicating that photoreceptor recruitment ceases at this stage. (D) When UAS.sev* is introduced into the sev.Gal4; UAS.derDN background, the recruitment of the R3/4 photoreceptors is rescued. (E) α-Sev staining (green) indicates normal expression in R3/4 precursors in the early ommatidia, but is degenerate in later ommatidiaatidia. (F) Co-staining of sev.Gal4; UAS.derDN with Sev and Elav highlights the normal R3/4 Sev expression before photoreceptor differentiation begins.