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First published online 7 January 2004
doi: 10.1242/dev.00941

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Senseless represses nuclear transduction of Egfr pathway activation

¹ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
² Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA
³ Department of Ophthalmology, Baylor College of Medicine, Houston, TX 77030, USA
⁴ Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA
⁵ Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA

View larger version (30K): [in a new window]   Fig. 1. R8 and non-R8 photoreceptors differentiate according to distinct developmental paradigms. (A) Non-R8 photoreceptors are recruited by Egfr signaling. Spitz (Spi, purple circles) is initially secreted by R8 and binds to the Egf receptor to induce photoreceptor differentiation (yellow). As the field of Spi expands outward from R8, photoreceptors differentiate in a stepwise fashion (R2/R5, R3/R4, R1/R6, R7). R8 differentiation does not require Egfr pathway activation (blue). (B) In wild-type ommatidia (top), Senseless (Sens) is expressed in the presumptive R8 (pre-R8) cell and Rough (Ro) expression is repressed. The pre-R8 cell then differentiates as an R8 photoreceptor. In sens mutant ommatidia (bottom), the pre-R8 cell expresses Ro and differentiates as a cell of the R2/R5 subtype. (C) R8 differentiation requires Ato and Sens function, while R2/R5 differentiation requires Spi/Egfr activation, which in turn induces Ro expression. Since Sens is a repressor of Ro in R8, it is possible that this prevention of Ro expression occurs via Sens-mediated repression of the Spi/Egfr pathway.

View larger version (100K): [in a new window]   Fig. 2. The presumptive R8 cell requires Spitz-mediated activation of the Egfr pathway to differentiate in sens mutants. Third instar eye imaginal discs are presented with posterior to the left in this and subsequent figures. (A-C,E-G) Mutant tissue is negatively marked by the absence of GFP (green). (A-C) Neuronal differentiation is indicated by an antibody to Elav (red). (A) Single R8 neurons (overlap with Sens, blue) are detected in rho-1 rho-3 mutant tissue, which lack all activation of the Egfr pathway. This suggests that R8 differentiation does not require Egfr activation. (B) Clusters of variable numbers of neurons are detected in sens mutant tissue. (C) Neurons are not detected in rho-1 rho-3 sens mutant tissue except at the clonal border, where non autonomous effects cause photoreceptor differentiation. This suggests that neuronal differentiation of the pre-R8 as an R2/R5 cell in sens mutants is dependent on Egfr activation. (D) spi sens double mutant tissue is identified by the absence of ß-gal (blue) and outlined with the dotted line. (D') sens mutant tissue is also marked by the absence of Sens (green). Elav (red) marks neurons. (D'') Overlay of D and D'. Tissue that lacks both spi and sens function does not contain Elav-positive cells except near the borders of the clone, where non autonomous function of spi is sufficient to induce some neuronal differentiation. Differentiation of the pre-R8 as an R2/R5 cell in sens mutant tissue is therefore also dependent on spi function. (E-F) R8 selection (pre-R8) is marked by sca-lacZ (red). (E) Pre-R8s are selected in rho-1 rho-3 mutant tissue. (F) Pre-R8s are still selected in rho-1 rho-3 sens mutant tissue, indicating that the loss of neuronal differentiation in these mutants is not secondary to a failure of R8 selection. (G) Boss (red), a marker for R8 differentiation, is absent in rho-1 rho-3 sens ro mutant tissue, suggesting that the R8 rescue seen in sens ro double mutants cannot occur when Egfr signaling is absent.

View larger version (120K): [in a new window]   Fig. 3. sens is a repressor of the Egfr pathway. (A-D) Light micrographs of adult Drosophila eyes. (A) EgfrElp homozygotes have few ommatidia as well as prominent gaps of tissue between ommatidia (arrow). (B) sens mutant clone (unpigmented). The sens homozygous mutant tissue is roughened in appearance compared to sens heterozygous tissue, which is wild type in appearance (pigmented). (C) sens mutant clone (unpigmented) induced in an EgfrElp heterozygous background. Overall the eye is smaller, suggesting a dominant interaction between sens and EgfrElp. In the sens homozygous mutant area, there are reduced numbers of ommatidia, as well as gaps of tissue between ommatidia, similar to EgfrElp homozygotes (arrow, compare with A). (D) Expression of UAS-sens with ey-GAL4 in an EgfrElp homozygote is sufficient to suppress the EgfrElp phenotype. These results suggest that sens acts as a repressor of the Egfr pathway. (E,F) Third instar expression of an enhancer trap in the nuclear effector of the Egfr pathway, pnt1277 (pnt-lacZ, red). pnt-lacZ is not expressed in Sens-expressing R8 cells (green, arrow), suggesting that the Egfr pathway is not activated to a high degree in the nucleus of R8.

View larger version (105K): [in a new window]   Fig. 4. Sens prevents Egfr pathway activation in the nucleus. UAS constructs were ubiquitously expressed in clones using flpout-GAL4. (A-A''') Co-misexpression of UAS-Egfract and UAS-lacZ posterior to the morphogenetic furrow (MF). Elav (red) is expressed in almost all cells within the clone (blue). Sens (green) is not detected within the clone. (B,C) Co-misexpression of UAS-Egfract and UAS-lacZ anterior to and within the MF. (B-B''') Elav (red) is expressed within and surrounding the clone (blue). Sens (green) is not expressed within the clone but is ectopically induced non autonomously. (C-C''') dpERK (red) and Sens (green) are expressed non-autonomously. Together, B and C are consistent with the presence of ectopic MFs surrounding areas of Egfr activation. (D-D''') Misexpression of UAS-Egfract anterior to the MF. pointed (pnt) transcription (pnt-lacZ, blue) occurs in most ectopic Elav-positive (red) cells. (E,F) Co-misexpression of UAS-Egfract and UAS-sens anterior to the MF. (E-E''') pnt transcription (pnt-lacZ, blue) does not occur and numbers of Elav-positive cells (red) are greatly reduced in the clone, which is marked by the Sens expressing cells (green). (F-F''') dpERK (red) is expressed autonomously at a high level within the clone, which is marked by Sens-expressing cells (green). Thus, sens is sufficient to block Egfr-induced pnt transcription, photoreceptor differentiation, and ectopic MF generation, but does not prevent dpERK induction.

View larger version (120K): [in a new window]   Fig. 5. Expression of nuclear effectors of the Egfr pathway prevents R8 differentiation. (A-H) Misexpression clones. sca-GAL4 was used to induce expression of UAS-Egfract, UAS-pnt-P1 or UAS-ro in R8. Third instar clones (A-E,G) are negatively marked by the absence of ßgal (green). (A-C) Misexpression of UAS-Egfract in R8. Expression of Sens (A), Ro (B) and Boss (C), are not disrupted, suggesting that robust activation of the Egfr pathway at the level of the cell membrane is not sufficient to perturb R8 differentiation. UAS-Egfract induced predicted phenotypes in other tissues (not shown), indicating that the transgene was active in this assay. (D-F) Misexpression of UAS-pnt-P1 in R8. (D,E) Apical (D) and basal (E) expression of Sens (red). Sens-expressing nuclei are not evenly spaced and are apically displaced. (F) Adult retinal sections at the level of R8 show ommatidia with a variably reduced number of photoreceptors and a lack of small rhabdomeres, consistent with a disruption of R8 differentiation and similar to sens loss-of-function phenotypes (arrow). Compare area of clone (below solid line) to neighboring wild-type tissue (above solid line). (G-H) Misexpression of UAS-ro in R8. (G) Expression of Sens (red) is initially wild type in appearance but is reduced by the fourth column of R8 differentiation and absent by the sixth column. (H) At the level of R8, no small rhabdomeres are detected within the clone (below solid line), phenocopying sens loss of function.

View larger version (16K): [in a new window]   Fig. 6. Model for Sens action in R8. Spi induces Egfr activation and the signal transduction cascade is induced normally. However, Sens prevents transcription of pnt-P1, thereby blocking the pathway at the final step. This relationship is likely to specifically mediate cell differentiation in R8 (see text). Sens also represses ro, an early target of the Egfr pathway. This second relationship regulates the cell fate decision in the founder photoreceptor between R8 and R2/R5 (see text).