Nodal points and complexity of Notch-Ras signal integration

Abstract

Metazoans use a handful of highly conserved signaling pathways to create a signaling backbone that governs development. How these few signals have such a versatile action likely depends upon the larger-scale network they form through integration, as exemplified by cross-talk between the Notch and receptor tyrosine kinase (RTK) pathways. We examined the transcriptional output of Notch-RTK cross-talk during Drosophila development and present in vivo data supporting a role for selected mutually regulated genes in signal integration. Interestingly, Notch-RTK integration did not lead to general antagonism of either pathway, as is commonly believed. Instead, integration had a combinatorial effect on specific cross-regulated targets, which unexpectedly included numerous core components of the RTK and other major signaling pathways (TGF-beta, Hh, Jak/Stat, nuclear receptor and Wnt). We find the majority of Ras-responsive genes are also Notch-responsive, suggesting Notch may function to specify the response to Ras activation.

Fig. 1.

An overview of identified transcriptional targets. (A) Venn diagram of probe sets responding to Notch^act (Notch), Ras1^V12 (Ras), or both transgenes in combination (Notch + Ras) in comparisons with w¹¹¹⁸; arm-GAL4 controls. Number of probe sets within each category is listed. A total of 138 probe sets representing 131 Class A mutually Notch- and Ras-responsive genes are indicated. Patterns of response to Notch and Ras are indicated in the dashed box by arrows representing up- or down-regulation. The number of genes with each response is shown. (B) Venn diagrams showing Notch (N-W) and/or Ras (R-W) targets identified in comparisons either with w¹¹¹⁸; arm-GAL4 controls (W) or in dual-transgenic Notch^act-Ras1^V12 (NR) samples compared with Ras1^V12 (NR-R) or Notch^act (NR-N) expressed singly (Materials and Methods). A total of 107 probe sets representing 106 Class B genes with a response to both Notch and Ras are indicated. Patterns of response to Notch and Ras are indicated as in A.

Fig. 2.

The Class A genes Gap1, Mkp3, and fringe are Notch-Ras responsive. (A) Signal levels for Gap1 (probe set 151669_at) in controls, Notch^act (N), Notch^act/Ras1^V12 dual transgenics (NR), and Ras1^V12 (R) at both time points. Error bars indicate 95% confidence intervals. Additive Notch-Ras transcriptional influence on Gap1 is observed. (B) Signal levels for Mkp3 probe set 141660_at. (C) Signal levels for Mkp3 probe set 149030_at. In both B and C, an additive Notch-Ras transcriptional influence on Mkp3 was observed during time point 2. No response was detected during the first time point. (D) Signal levels for fringe (probe set 143664_at) show an additive Notch-Ras transcriptional influence during the second time point only. In E–J, increased levels of Gap1, Mkp3, and fringe phenocopy both Notch activation and decreased RTK signaling. (E) The notum of a wild-type fly. (F) Notum of a sca-GAL4, UAS-Ras1^V12 fly reared at 18 °C. Supernumerary machrochaete are observed in individuals with nearly complete penetrance for thoracic and posterior alar bristles, indicated by dotted lines. (G) Notch^Abruptex16 notum reared at 25 °C. Loss of thoracic machrochaete is seen (blue arrowheads) at high penetrance. (H) Notum of a sca-GAL4, UAS-Gap1 fly reared at 25 °C. Missing thoracic machrochaete are observed (blue arrowhead) with a penetrance of 14.9% at 25 °C and 87.3% at 29 °C. Missing posterior alar bristles are also observed at high penetrance (red arrowhead). Neither sca-GAL4 nor UAS-Gap1 parental lines displayed abnormalities at either temperature. (I) Notum of a sca-GAL4, UAS-fng fly reared at 29 °C. Missing thoracic machrochaete are observed (blue arrowheads) with a penetrance of 98%. UAS-fng parental lines are wild type in the absence of GAL4. (J) Notum of a sca-GAL4, UAS-Mkp3 fly reared at 29 °C. Missing thoracic machrochaete are observed (blue arrowheads) with a penetrance of 100%. Missing posterior alar bristles also are observed at high penetrance (red arrowheads). UAS-Mkp3 parental lines are wild type in the absence of GAL4. (K–T) Mkp3 interacts genetically with Notch pathway components. (K) A control wing heterozygous for the null Notch allele N^54L9 (N^54L9/+). (L) N^54L9/+; Mkp3^e01514/+ transheterozygotes show a weak enhancement of the Notch wing margin defect. (M) A control wing heterozygous for the null Notch allele N^55e11 (N^55e11/+). (N) N^55e11/+; Mkp3^e01514/+: arrow points to region of enhancement. In panels O–R, tests were performed in a C96-GAL4 background. (O) A wing from a Notch gain-of-function allele Notch^Abruptex16/Y male. (P) In Notch^Abruptex16/Y; Mkp3^e01514/+, arrow indicates suppression of the L4 vein loss. (Q) deltex¹⁵²/Y. (R) deltex¹⁵²/Y; Mkp3^e01514/+: enhanced deltex phenotype. (S) Delta^9P. (T) Delta^9P; Mkp3^e01514/+: enhanced wing deltas are observed. Mkp3^e01514/+ wings were wild type (data not shown).

Fig. 3.

Notch and Ras have an impact on RTK signaling through their mutual regulation of RTK core components. (A) The RTK signal transduction mechanism is shown. Proteins depicted with orange filled symbols mediate RTK activation and signal transduction. Arrows indicate a positive effect on signaling. Proteins in depicted with gray filled symbols oppose RTK signaling. Blunted arrows indicate points of antagonism. Proteins outlined in red responded to Ras activation in our study, those outlined in blue responded to Notch, and those in purple responded to both Notch and Ras. Known or novel genetic interactors of both pathways are indicated by underlined red text. In B and C, Notch signaling can help specify RTK transcriptional output. (B) In the absence of Notch signaling, activation of Ras leads to transcription effect A, as indicted. (C) Upon coactivation with Notch, transcriptional output downstream of Ras activation is partially respecified. Although some canonical targets remain uninfluenced (transcription effect A′), other targets show an altered response (transcription effect B). Thus, Notch activation may play an important role in specifying transcriptional effects downstream of Ras activation.

Fig. 4.

Core targets of Notch-Ras coregulation. (A) Complete linkage cluster of Class A or B Notch-Ras targets mutually regulated at both time points. Transcriptional response is indicated as in Fig. 3. Ras pathway genetic interactors are underlined, and known (anterior open) and novel N genetic interactors are italicized. (B) Confirmation by RT-PCR of differential expression for CG1942, CG9119, and knot in Ras1^V12 (R), Notch^act (N), dual Notch^act-Ras1^V12 (NR), and nontransgenic GAL4 only (W) samples. Rp49 was used in parallel as a control. All RNA were from time point 1. Cycles of PCR amplification are indicated. (C) Control wings heterozygous for a null Notch allele, N^54L9 (N^54L9/+), display a 38% penetrance. In D–F, wings are heterozygous for both N^54L9 and the listed gene. The penetrance of the Notch wing nicking phenotype is shown in parentheses. For calculations, wings displaying any margin defect are scored as mutant. (D) knot¹/+: enhanced (82%). (H) twist¹: enhanced (77%). (I) heartless^AB42: enhanced (100%). Knot, twist, and heartless are known genetic interactors of Ras or EGFR (red asterisks). knot functions in a Notch-Ras-responsive cell fate switch. In G–J, dashed lines indicate regions of affected machrochaete specification. (G) Notum of a wild-type fly. (H) Notum of a sca-GAL4, UAS-Ras1^V12 fly reared at 18 °C. Supernumerary machrochaete observed with nearly complete penetrance for thoracic and posterior alar bristles. (I) Notum of a sca-GAL4, UAS-knot fly reared at 25 °C. Supernumerary machrochaete are observed at 58.5% penetrance for thoracic bristles and 100% for anterior postalar bristles. Both sca-GAL4 and UAS-knot parental lines had rare bristle abnormalities (18). (J) hs-flp122; FRT42D kn^KN4 pwn/FRT 42D ubi-gfp flies after 30 min at 38 °C at 48–72 h AEL (Materials and Methods). Loss-of-function knot clones show bristle defects at <5% penetrance. Clone boundary is marked with dashed line. Bristles marked with pawn (pwn) are indicated by blue arrows. Black arrows indicate adjacent machrochaete showing incomplete development where instead of bristle, a small, empty socket formed. This phenotype was not seen in control pwn homozygotes (data not shown). (K) Overall, these results suggest a model wherein knot acts a nodal point downstream of Notch-Ras integration to influence the outcome of cell fate decisions.