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Small RNAs Turn Over a New Leaf as Morphogens
Developmental Cell
Previews Small RNAs Turn Over a New Leaf as Morphogens Dana O. Robinson1 and Adrienne H.K. Roeder1,* 1Weill Institute for Cell and Molecular Biology and School of Integrative Plant Science, Section of Plant Biology, Cornell University, Ithaca, NY, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2017.10.025
In this issue of Developmental Cell, Skopelitis et al. (2017) demonstrate that sharp boundaries of gene expression can be created by threshold-based readout of mobile small RNA gradients. Support for this hypothesis comes from manipulation of small RNAs involved in top-bottom leaf patterning and from a novel synthetic biology approach. Multicellular organisms must communicate positional information across distances of several cell layers to establish tissue-level patterning. A fundamental question in developmental biology is how such intercellular communication occurs. Morphogen gradients fill this role in animal development; however, in plant development, no signaling molecules meeting the definition of ‘‘morphogen’’ have been identified. Recently, Skopelitis et al. hypothesized that small RNAs can act as morphogens in plants (Skopelitis et al., 2012). In this issue of Developmental Cell, the same authors test this hypothesis: their results show that small RNAs in plants can act in a concentration-dependent manner similar to that of animal morphogens to generate sharply delineated domains of gene expression (Skopelitis et al., 2017). Canonical morphogens have been described exclusively in animal systems. The concept was predicted and named by Alan Turing in 1952, and the first morphogen (Bicoid) was discovered in the Drosophila embryo in 1988 (Driever €sslein-Volhard, 1988). Several and Nu more morphogens have since been discovered in animal systems; proposed mammalian morphogens include bone morphogenetic protein (BMP) and sonic hedgehog (SHH) (Gurdon and Bourillot, 2001). To be a morphogen, a signal must be mobile and diffusible, create a gradient, and directly affect cells in a concentration-dependent manner (Rogers and Schier, 2011). In plants, no perfect analogs to these morphogens have been found. Several other mechanisms of long-range signaling have been described: these include peptide ligands (e.g., CLV3), cell-to-cell movement of transcription factors through small pores called plasmo-
desmata (e.g., SHORTROOT), and plant hormones (e.g., auxin) (Benkovics and Timmermans, 2014). Here, the authors test the extent to which small RNAs can behave like morphogens. Small RNAs are known to act as mobile signals in plants. When loaded into the vasculature, small RNAs can travel long distances to induce systemic silencing throughout the plant (Benkovics and Timmermans, 2014). Additionally, small RNAs can move or diffuse from cell to cell through plasmodesmata (pores connecting cells) for distances of about 10 to 15 cell lengths, and this type of movement can establish a gradient in small RNA concentration across cell layers (de Felippes et al., 2011). Some small RNAs involved in development (e.g., miR166 and miR394) establish gradients over smaller distances of three to six cells (Benkovics and Timmermans, 2014). Interestingly, animal microRNAs may also move in an analogous cell-to-cell manner via exosomes (Benkovics and Timmermans, 2014). In this issue, Skopelitis et al. (2017) describe two opposing small RNA gradients that establish the patterns of gene expression that differentiate the top (adaxial) and bottom (abaxial) faces of the leaf. Remarkably, this system robustly establishes a stable boundary between top and bottom that spans the large, flat leaf. Top fate is directed by class III homeodomain leucine zipper (HD-ZIP) transcription factors, including PHABULOSA (PHB). Bottom fate is directed by the auxin response factors ARF3 and ARF4. Both PHB and the ARFs are expressed in sharply delineated domains of the leaf, and both are negatively regulated by small RNAs originating from the opposite face of the leaf (Figure 1) (Chitwood et al., 2009; Skopelitis et al., 2017). PHB
and other top determinants are negatively regulated by microRNA166 (miR166), which is expressed on the bottom; ARF3 and other bottom determinants are inhibited by the top-expressed trans-acting short interfering (tasi) RNA tasiARF (Kuhlemeier and Timmermans, 2016). These two small RNAs diffuse in counterposed gradients across the developing leaf. The question of how continuous gradients of small RNAs are translated into sharp on-off boundaries of PHB and ARF3 expression is addressed in Skopelitis et al. (2017). One explanation for how a small RNA gradient is translated into a sharp target gene expression boundary posits that extrinsic factors fine-tune the small RNA concentration at the nascent boundary. These factors would likely be prepatterned by other components in the top-bottom signaling system. Skopelitis et al. (2017) explored this possibility by reversing the concentration gradient of tasiARF, removing the interactions from the established top-bottom context. To achieve this, the authors expressed an artificial microRNA identical to tasiARF (miRARF) in a background with no endogenous tasiARF expression. When expressed from the ‘‘correct’’ top face, miRARF rescued the wild-type bottom expression pattern of ARF3. When miRARF was instead expressed from the bottom face, ARF3 expression switched to the top face and retained the sharp expression boundary. The fact that ARF3-tasiARF interactions can be ‘‘flipped’’ relative to the leaf’s overall polarity indicates that pre-patterned polarity factors are not necessary for boundary formation and instead suggests that the formation of a sharp boundary is intrinsic to the miRNA-target pair.
Developmental Cell 43, November 6, 2017 ª 2017 Elsevier Inc. 253
Developmental Cell
Previews A
adaxial leaf face (top)
B tasiARF
ARF3 transcript
ARF3
tasiARF
HD-ZIP class III (PHB)
silencing
binary threshold ARF3, ARF4
buffering
miR166
abaxial leaf face (bottom)
Figure 1. Mobile Small RNA Gradients Generate Sharp, Stable Developmental Boundaries (A) Schematic of factors establishing top-bottom polarity in the leaf. Left: factors establishing adaxial (top) fate. Class III HD-ZIPs (dark green nuclei), which promote top fate, are expressed in the two uppermost cell layers. Their expression is limited to these cell layers by the action of miR166, a microRNA expressed in the bottom-most cell layer (light green nuclei) and diffusing upward (green gradient). Right: factors establishing abaxial (bottom) fate. The auxin response factors ARF3 and ARF4 (dark red nuclei) promote bottom fate and are expressed in the two bottom-most leaf layers. tasiARF, a small RNA, diffuses (pink gradient) from its expression domain in the two uppermost leaf layers (pink nuclei) to limit ARF3/ARF4 expression. (B) The diffusion gradient of tasiARF creates a binary threshold in the expression of ARF3. tasiARF levels are high in the two top-most cell layers, where it is produced, and decrease as tasiARF diffuses toward the leaf’s bottom face. ARF3 is transcribed throughout the leaf. In the upper and middle leaf cells, where tasiARF levels are high relative to ARF3, tasiARF silences ARF3. When the ratio of tasiARF to ARF3 drops below a binary threshold, ARF3 is expressed. tasiARF acts to buffer ARF3 expression below this binary threshold.
dual behaviors of blocking expression above a threshold concentration and equalizing target protein expression below that threshold. This dual behavior allows small RNAs to produce a sharp, stable boundary over a large expanse of tissue. How common is this threshold behavior of microRNAs? Do microRNAs forming opposing proximal-distal (outerinner) gradients in the leaf, such as miR319 and miR396, also create sharp boundaries of their targets? The same microRNA can have different behaviors in different contexts: whereas miR166 directs a sharp boundary of its target PHB in the leaf, PHB expression has a more graded response to miR166 in the root (Benkovics and Timmermans, 2014). In the future, it will be important to further explore the behavior of small RNA gradients and to identify the factors that determine whether a microRNA produces a threshold-boundary response or a gradient in target expression. REFERENCES
The authors next used an elegant synthetic biology approach to create a ‘‘general case’’ demonstrating that small RNA gradients can create sharp boundaries. The authors ubiquitously expressed GFP and expressed an artificial microRNA (miRGFP) against it. Driving miRGFP under a top promoter created a sharp onoff boundary of GFP expression on the leaf’s bottom face, confirming that the formation of such boundaries is a general capability of small RNA gradients. These experiments also revealed a buffering effect of the microRNA on the target in cell layers beyond those in which silencing occurred. The authors describe these results as a ‘‘dose-dependent binary readout’’ in which target expression is silenced in cells above a given miRNAto-target ratio and buffered in cells below that ratio. This buffering effect is similar to that described for microRNAs in animal cells (Schmiedel et al., 2015), but the dual action is novel. Importantly, these properties are intrinsic to the microRNA-
target pair and do not require the action of downstream network components, given that these properties occur in a synthetic GFP system. Finally, the authors tested the sensitivity of the binary readout threshold. They created an estradiol-inducible miRGFP construct: induction of this construct generates a time series of increasing miRGFP expression, altering the miRNA-to-target ratio. As miRGFP expression increases, the position of the GFP silencing threshold moves. This demonstrates that the relative ratio of miRGFP to target tunes the position of the boundary. This result illustrates how the boundary-forming mechanism might be used in varying developmental contexts, e.g., thicker or thinner leaves. Small RNAs thus meet the criteria for a morphogen: they are mobile and diffusible, create a gradient, and directly affect cells in a concentration-dependent manner. However, their behavior extends beyond that of a morphogen in their novel
254 Developmental Cell 43, November 6, 2017
Benkovics, A.H., and Timmermans, M.C. (2014). Curr. Opin. Genet. Dev. 27, 83–91. Chitwood, D.H., Nogueira, F.T., Howell, M.D., Montgomery, T.A., Carrington, J.C., and Timmermans, M.C. (2009). Genes Dev. 23, 549–554. de Felippes, F.F., Ott, F., and Weigel, D. (2011). Nucleic Acids Res. 39, 2880–2889. €sslein-Volhard, C. (1988). Cell Driever, W., and Nu 54, 83–93. Gurdon, J.B., and Bourillot, P.Y. (2001). Nature 413, 797–803. Kuhlemeier, C., and Timmermans, M.C. (2016). Development 143, 3230–3237. Rogers, K.W., and Schier, A.F. (2011). Annu. Rev. Cell Dev. Biol. 27, 377–407. Schmiedel, J.M., Klemm, S.L., Zheng, Y., Sahay, €thgen, N., Marks, D.S., and van OudenaarA., Blu den, A. (2015). Science 348, 128–132. Skopelitis, D.S., Husbands, A.Y., and Timmermans, M.C. (2012). Curr. Opin. Cell Biol. 24, 217–224. Skopelitis, D.S., Benkovics, A.H., Husbands, A., and Timmermans, M.C.P. (2017). Dev. Cell 43, this issue, 265–273.
Previews Small RNAs Turn Over a New Leaf as Morphogens Dana O. Robinson1 and Adrienne H.K. Roeder1,* 1Weill Institute for Cell and Molecular Biology and School of Integrative Plant Science, Section of Plant Biology, Cornell University, Ithaca, NY, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2017.10.025
In this issue of Developmental Cell, Skopelitis et al. (2017) demonstrate that sharp boundaries of gene expression can be created by threshold-based readout of mobile small RNA gradients. Support for this hypothesis comes from manipulation of small RNAs involved in top-bottom leaf patterning and from a novel synthetic biology approach. Multicellular organisms must communicate positional information across distances of several cell layers to establish tissue-level patterning. A fundamental question in developmental biology is how such intercellular communication occurs. Morphogen gradients fill this role in animal development; however, in plant development, no signaling molecules meeting the definition of ‘‘morphogen’’ have been identified. Recently, Skopelitis et al. hypothesized that small RNAs can act as morphogens in plants (Skopelitis et al., 2012). In this issue of Developmental Cell, the same authors test this hypothesis: their results show that small RNAs in plants can act in a concentration-dependent manner similar to that of animal morphogens to generate sharply delineated domains of gene expression (Skopelitis et al., 2017). Canonical morphogens have been described exclusively in animal systems. The concept was predicted and named by Alan Turing in 1952, and the first morphogen (Bicoid) was discovered in the Drosophila embryo in 1988 (Driever €sslein-Volhard, 1988). Several and Nu more morphogens have since been discovered in animal systems; proposed mammalian morphogens include bone morphogenetic protein (BMP) and sonic hedgehog (SHH) (Gurdon and Bourillot, 2001). To be a morphogen, a signal must be mobile and diffusible, create a gradient, and directly affect cells in a concentration-dependent manner (Rogers and Schier, 2011). In plants, no perfect analogs to these morphogens have been found. Several other mechanisms of long-range signaling have been described: these include peptide ligands (e.g., CLV3), cell-to-cell movement of transcription factors through small pores called plasmo-
desmata (e.g., SHORTROOT), and plant hormones (e.g., auxin) (Benkovics and Timmermans, 2014). Here, the authors test the extent to which small RNAs can behave like morphogens. Small RNAs are known to act as mobile signals in plants. When loaded into the vasculature, small RNAs can travel long distances to induce systemic silencing throughout the plant (Benkovics and Timmermans, 2014). Additionally, small RNAs can move or diffuse from cell to cell through plasmodesmata (pores connecting cells) for distances of about 10 to 15 cell lengths, and this type of movement can establish a gradient in small RNA concentration across cell layers (de Felippes et al., 2011). Some small RNAs involved in development (e.g., miR166 and miR394) establish gradients over smaller distances of three to six cells (Benkovics and Timmermans, 2014). Interestingly, animal microRNAs may also move in an analogous cell-to-cell manner via exosomes (Benkovics and Timmermans, 2014). In this issue, Skopelitis et al. (2017) describe two opposing small RNA gradients that establish the patterns of gene expression that differentiate the top (adaxial) and bottom (abaxial) faces of the leaf. Remarkably, this system robustly establishes a stable boundary between top and bottom that spans the large, flat leaf. Top fate is directed by class III homeodomain leucine zipper (HD-ZIP) transcription factors, including PHABULOSA (PHB). Bottom fate is directed by the auxin response factors ARF3 and ARF4. Both PHB and the ARFs are expressed in sharply delineated domains of the leaf, and both are negatively regulated by small RNAs originating from the opposite face of the leaf (Figure 1) (Chitwood et al., 2009; Skopelitis et al., 2017). PHB
and other top determinants are negatively regulated by microRNA166 (miR166), which is expressed on the bottom; ARF3 and other bottom determinants are inhibited by the top-expressed trans-acting short interfering (tasi) RNA tasiARF (Kuhlemeier and Timmermans, 2016). These two small RNAs diffuse in counterposed gradients across the developing leaf. The question of how continuous gradients of small RNAs are translated into sharp on-off boundaries of PHB and ARF3 expression is addressed in Skopelitis et al. (2017). One explanation for how a small RNA gradient is translated into a sharp target gene expression boundary posits that extrinsic factors fine-tune the small RNA concentration at the nascent boundary. These factors would likely be prepatterned by other components in the top-bottom signaling system. Skopelitis et al. (2017) explored this possibility by reversing the concentration gradient of tasiARF, removing the interactions from the established top-bottom context. To achieve this, the authors expressed an artificial microRNA identical to tasiARF (miRARF) in a background with no endogenous tasiARF expression. When expressed from the ‘‘correct’’ top face, miRARF rescued the wild-type bottom expression pattern of ARF3. When miRARF was instead expressed from the bottom face, ARF3 expression switched to the top face and retained the sharp expression boundary. The fact that ARF3-tasiARF interactions can be ‘‘flipped’’ relative to the leaf’s overall polarity indicates that pre-patterned polarity factors are not necessary for boundary formation and instead suggests that the formation of a sharp boundary is intrinsic to the miRNA-target pair.
Developmental Cell 43, November 6, 2017 ª 2017 Elsevier Inc. 253
Developmental Cell
Previews A
adaxial leaf face (top)
B tasiARF
ARF3 transcript
ARF3
tasiARF
HD-ZIP class III (PHB)
silencing
binary threshold ARF3, ARF4
buffering
miR166
abaxial leaf face (bottom)
Figure 1. Mobile Small RNA Gradients Generate Sharp, Stable Developmental Boundaries (A) Schematic of factors establishing top-bottom polarity in the leaf. Left: factors establishing adaxial (top) fate. Class III HD-ZIPs (dark green nuclei), which promote top fate, are expressed in the two uppermost cell layers. Their expression is limited to these cell layers by the action of miR166, a microRNA expressed in the bottom-most cell layer (light green nuclei) and diffusing upward (green gradient). Right: factors establishing abaxial (bottom) fate. The auxin response factors ARF3 and ARF4 (dark red nuclei) promote bottom fate and are expressed in the two bottom-most leaf layers. tasiARF, a small RNA, diffuses (pink gradient) from its expression domain in the two uppermost leaf layers (pink nuclei) to limit ARF3/ARF4 expression. (B) The diffusion gradient of tasiARF creates a binary threshold in the expression of ARF3. tasiARF levels are high in the two top-most cell layers, where it is produced, and decrease as tasiARF diffuses toward the leaf’s bottom face. ARF3 is transcribed throughout the leaf. In the upper and middle leaf cells, where tasiARF levels are high relative to ARF3, tasiARF silences ARF3. When the ratio of tasiARF to ARF3 drops below a binary threshold, ARF3 is expressed. tasiARF acts to buffer ARF3 expression below this binary threshold.
dual behaviors of blocking expression above a threshold concentration and equalizing target protein expression below that threshold. This dual behavior allows small RNAs to produce a sharp, stable boundary over a large expanse of tissue. How common is this threshold behavior of microRNAs? Do microRNAs forming opposing proximal-distal (outerinner) gradients in the leaf, such as miR319 and miR396, also create sharp boundaries of their targets? The same microRNA can have different behaviors in different contexts: whereas miR166 directs a sharp boundary of its target PHB in the leaf, PHB expression has a more graded response to miR166 in the root (Benkovics and Timmermans, 2014). In the future, it will be important to further explore the behavior of small RNA gradients and to identify the factors that determine whether a microRNA produces a threshold-boundary response or a gradient in target expression. REFERENCES
The authors next used an elegant synthetic biology approach to create a ‘‘general case’’ demonstrating that small RNA gradients can create sharp boundaries. The authors ubiquitously expressed GFP and expressed an artificial microRNA (miRGFP) against it. Driving miRGFP under a top promoter created a sharp onoff boundary of GFP expression on the leaf’s bottom face, confirming that the formation of such boundaries is a general capability of small RNA gradients. These experiments also revealed a buffering effect of the microRNA on the target in cell layers beyond those in which silencing occurred. The authors describe these results as a ‘‘dose-dependent binary readout’’ in which target expression is silenced in cells above a given miRNAto-target ratio and buffered in cells below that ratio. This buffering effect is similar to that described for microRNAs in animal cells (Schmiedel et al., 2015), but the dual action is novel. Importantly, these properties are intrinsic to the microRNA-
target pair and do not require the action of downstream network components, given that these properties occur in a synthetic GFP system. Finally, the authors tested the sensitivity of the binary readout threshold. They created an estradiol-inducible miRGFP construct: induction of this construct generates a time series of increasing miRGFP expression, altering the miRNA-to-target ratio. As miRGFP expression increases, the position of the GFP silencing threshold moves. This demonstrates that the relative ratio of miRGFP to target tunes the position of the boundary. This result illustrates how the boundary-forming mechanism might be used in varying developmental contexts, e.g., thicker or thinner leaves. Small RNAs thus meet the criteria for a morphogen: they are mobile and diffusible, create a gradient, and directly affect cells in a concentration-dependent manner. However, their behavior extends beyond that of a morphogen in their novel
254 Developmental Cell 43, November 6, 2017
Benkovics, A.H., and Timmermans, M.C. (2014). Curr. Opin. Genet. Dev. 27, 83–91. Chitwood, D.H., Nogueira, F.T., Howell, M.D., Montgomery, T.A., Carrington, J.C., and Timmermans, M.C. (2009). Genes Dev. 23, 549–554. de Felippes, F.F., Ott, F., and Weigel, D. (2011). Nucleic Acids Res. 39, 2880–2889. €sslein-Volhard, C. (1988). Cell Driever, W., and Nu 54, 83–93. Gurdon, J.B., and Bourillot, P.Y. (2001). Nature 413, 797–803. Kuhlemeier, C., and Timmermans, M.C. (2016). Development 143, 3230–3237. Rogers, K.W., and Schier, A.F. (2011). Annu. Rev. Cell Dev. Biol. 27, 377–407. Schmiedel, J.M., Klemm, S.L., Zheng, Y., Sahay, €thgen, N., Marks, D.S., and van OudenaarA., Blu den, A. (2015). Science 348, 128–132. Skopelitis, D.S., Husbands, A.Y., and Timmermans, M.C. (2012). Curr. Opin. Cell Biol. 24, 217–224. Skopelitis, D.S., Benkovics, A.H., Husbands, A., and Timmermans, M.C.P. (2017). Dev. Cell 43, this issue, 265–273.