Progress 09/15/06 to 09/14/10
Outputs
OUTPUTS: Our work indicates an important role for small RNAs in leaf polarity in maize. Interestingly, we found that organ polarity is established through a cascade of opposing small RNAs; miR390 triggers the biogenesis of ta-siRNAs on the adaxial side of developing leaf primordia. The ta-siRNAs specify adaxial fate by repressing abaxial determinants including miR166, which in turn restricts activity of the adaxializing HD-ZIPIII factors to the adaxial side. We thereby identified a novel patterning mechanism. We further used in situ hybridization to show that miR390 is expressed on the adaxial side of developing leaf primordia. This polarized expression persists in the abaxialized leaves of leafbladeless1 mutants, indicating that the adaxial restricted expression of miR390 is established independent of ta-siRNA pathway and its downstream polarity determinants, such as the hd-zipIII and yabby genes. Together, these findings suggest that miR390 is an upstream component in the genetic pathway specifying leaf polarity whose localized accumulation is regulated by positional information inherent within the SAM. Based on this data we proposed a model for the initial polarization of the incipient, which as a key outstanding question in the field. Moreover, our expression analyses of mature small RNAs and their precursors suggest that miRNA accumulation is regulated at the level of biogenesis and/or stability as miRNA precursors are expressed in cells in which the miRNA does not accumulate. We also found evidence suggesting that miRNAs may act as local mobile signals in development. This observation has potential implications for development biologists in both plants as well as animals, as miRNAs would no longer be considered as cell-autonomous regulators of gene expression but as morphogen-like signals. Our research has thereby made contributions to other disciplines. To inform the scientific community of these findings, we have presented this work in numerous departmental seminars and scientific meetings in the USA, Europe, and Africa. In addition, this grant has resulted in three papers, five reviews and two book chapter. PARTICIPANTS: Fabio Nogueira, Dan Chitwood, and Katie Petsch have both gained experience in plant developmental genetics and gene regulation by small regulatory RNAs. They have established a protocol for small RNA in situ hybridization using LNA probes. They have gained expertise in maize developmental genetics, microscopy, histology, laser capture microdissection and other techniques. In addition, Fabio Nogueira and Dan Chitwood have each been first author on a review and have published a paper. They have also presented seminars for a plant specific and a general audience and have thereby obtained important training in presenting data to diverse audiences. Through this project we have also trained members of other labs how to use the laser capture microscope and how to perform in situ hybridizations to analyze small RNAs. Fabio Nogueira and Katie Petsch have supervised high school and undergraduate students through this project. Sarah Samson (Ohio State University) characterized double mutants between lbl1 and other polarity mutants, and Ricky Knight (Locust Valley HS) and Devin Tark (Rockville Center HS) helped genotype EMS -induced alleles of ta-siRNA biogenesis components. In addition, we have hosted Priscilla Manzotti, a graduate student from Dr Giuseppe Gavazzi (University of Milan, Italy), for four month to molecularly characterize and perform in situ hybridization analysis on a dcl4 mutant, and Ryan Douglas, a graduate student from Mike Scalon's group (Cornell University), to learn small RNA in situ hybridization. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We showed that miR390 and tasiR-ARF are expressed adaxially in incipient and young leaf primordia, consistent with a role in specifying adaxial fate. As expected, expression of tasiR-ARF is lost in lbl1, but interestingly, miR390 remains polarly expressed in lbl1. Thus, the adaxial expression of miR390 is established and maintained independently of the ta-siRNA pathway, and because this pathway acts upstream of other known polarity factors, miR390 is an upstream polarity determinant. We further showed that expression of tasiR-ARF target arf3a is abaxially restricted in leaf primordia but in lbl1, expression is expanded adaxially. Preliminary analysis of a arf3a loss of function mutation suggests redundancy between arf3a and other family members, as these mutants are phenotypically normal. We used LCM with RT-PCR to determined expression profiles for nine mir166, two mir390 and four tas3 precursors in specific domains of the SAM (Aim 2). These experiments have let to several key observations. 1) the mir166 genes are under intricate transcriptional control. mir166a, c, f, and i are expressed in or immediately below the incipient leaf and could contribute to adaxial/abaxial patterning of the leaf. 2) miRNA accumulation is regulated at the level of biogenesis or stability, because mir166a and the mir390 precursors are expressed at the tip of the SAM where the mature miRNAs do not accumulate but their targets are expressed. 3) most interestingly, we found that the mir390 precursors are expressed specifically in the L1 whereas miR390 accumulates in both the L1 and L2 layers of the incipient leaf. This incongruence in expression patterns suggests that miRNAs may be able to traffic from the L1 into the underlying L2. 4) we found that altered expression of mir166c and mir166i in the incipient leaf underlies the leaf polarity defects in lbl1. We have begun to characterize mutants defective for other ta-siRNA pathway components (Aim 3). We obtained four rdr6 alleles, including two null alleles. These condition an embryo lethal phenotype and fail to establish a shoot meristem. Expression of kn1 is altered in rdr6 embryos and absent from the initiating SAM. A similar phenotype is observed when severe lbl1 alleles are introgressed into the maize inbred W22, suggesting that this phenotype resembles the ta-siRNA pathway loss-of-function phenotype. We are analyzing the expression patterns of ta-siRNA components during embryogenesis. miR390 is an early marker for proembryo fate and is expressed prior to the onset of hd-zipIII and kn1 expression. As in the shoot, miR166 is misexpressed in mutant embryos, which is correlated with reduced rld1 expression. In collaboration with Dr. Mike Scanlon's group (Cornell University, Ithaca, NY) we characterized an ago7 mutant. We have also shown that rgd766 results from mutation of DCL4. Two additional DCL4 alleles that are null alleles. These alleles give rise to phenotypes that resemble those of weak lbl1 alleles, indicating partial redundancy of DCL4 with another small RNA biogenesis component. Interaction of the dcl4 mutants with mutations in remaining maize DCL family members is ongoing.
Publications
- Kidner, C.A., and Timmermans, M.C.P. (2010) Signaling sides: Adaxial-abaxial petterning in leaves. In: M.C.P. Timmermans (Ed.) Current Topics in Developmental Biology - Plant Development. Elsevier, Amsterdam, pp 141-168.
- Douglas, R.N., Wiley, D., Sarkar, A., Springer, N., Timmermans, M.C.P., and Scanlon, M.J. (2010) ragged seedling2 encodes an ARGONAUTE7-like protein required for mediolateral expansion, but not dorsiventrality, of maize leaves. Plant Cell 22, May 7. [Epub ahead of print]
- Husbands, A.Y., Chitwood, D.H., Plavskin, Y. and Timmermans, M.C.P. (2009) Signals and prepatterns: new insights into organ polarity in plants. Genes & Dev. 23, 1986-1997.
- Chitwood, D.H., Guo, M., Nogueira, F.T.S., and Timmermans, M.C.P. (2007) Establishing leaf polarity: the role of small RNAs and positional signals in the shoot apex. Development 134, 813-823.
- Kidner, C., and Timmermans, M.C.P. (2007) Mixing and matching pathways in leaf polarity. Curr. Op. Plant Sci. 10, 13-20.
- Nogueira F.T.S, Sarkar, A., Chitwood, D.H., and Timmermans, M.C.P. (2007) Distinct small regulatory RNAs interact to specify organ polarity in plants. Symp. Quant. Biol. 71, 157-164.
- Nogueira, F.T.S., Chitwood, D.H., Madi, S., Kazuhiro Ohtsu, K., Schnable, P.S., Scanlon, M.J., and Timmermans, M.C.P. (2009) Regulation of small RNA accumulation in the maize shoot apex. PLoS Genetics 5, e1000320.
- Foster, T.M., and Timmermans, M.C.P. (2009) Axial patterning of the maize leaf. In: J.L. Bennetzen and S.C. Hake (Eds.) Handbook of Maize: its biology. Springer USA, pp 161-178.
- Nogueira, F.T.S., and Timmermans, M.C.P. (2007) An interplay between small RNAs patterns leaves. Plant Sign. & Behavior 2, 519-521.
- Nogueira, F.T.S., Madi, S., Chitwood, D.H., Juarez, M.T., and Timmermans, M.C.P. (2007) Two small regulatory RNAs establish opposing fates of a developmental axis. Genes & Dev. 21, 750-755.
|
Progress 09/15/08 to 09/14/09
Outputs
OUTPUTS: Our work indicates an important role for small RNAs in leaf polarity in maize. Interestingly, we found that organ polarity is established through a cascade of opposing small RNAs; miR390 triggers the biogenesis of ta-siRNAs on the adaxial side of developing leaf primordia. The ta-siRNAs specify adaxial fate by repressing abaxial determinants including miR166, which in turn restricts activity of the adaxializing HD-ZIPIII factors to the adaxial side. We thereby identified a novel patterning mechanism. We further used in situ hybridization to show that miR390 is expressed on the adaxial side of developing leaf primordia. This polarized expression persists in the abaxialized leaves of leafbladeless1 mutants, indicating that the adaxial restricted expression of miR390 is established independent of ta-siRNA pathway and its downstream polarity determinants, such as the hd-zipIII and yabby genes. Together, these findings suggest that miR390 is an upstream component in the genetic pathway specifying leaf polarity whose localized accumulation is regulated by positional information inherent within the SAM. Based on this data we proposed a model for the initial polarization of the incipient, which as a key outstanding question in the field. Moreover, our expression analyses of mature small RNAs and their precursors suggest that miRNA accumulation is regulated at the level of biogenesis and/or stability as miRNA precursors are expressed in cells in which the miRNA does not accumulate. We also found some evidence suggesting that miRNAs may act as local mobile signals in development. This observation has potential implications for development biologists in both plants as well as animals, as miRNAs would no longer be considered as cell-autonomous regulators of gene expression but as morphogen-like signals. Our research has thereby made contributions to other disciplines. To inform the scientific community of these findings, we have presented this work in numerous departmental seminars and scientific meetings in the USA, Europe, and Africa. In addition, this grant has resulted in three papers, four reviews and one book chapter. PARTICIPANTS: Marja Timmermans, PI Fabio Nogueira, Postdoctoral Fellow Katie Petsch, Postdoctoral Fellow Daniel Chitwood, Graduate student at the CSHL Watson School for Biological Sciences Shahinez Madi, technician Fabio Nogueria and Shahinez Madi worked on the cloning and characterization of lbl1, they determined its role in ta-siRNA biogenesis, and identified arf3 genes as potential ta-siRNA targets. Fabio Nogueira and Daniel Chitwood performed the small RNA in situ hybridization experiments. All three worked jointly on the LCM-RT-PCR analysis of the mir166, mir390 and tas3 precursor transcripts. Katie Petsch has mainly been working on Aim3 and has characterized the new rdr6 and dcl4 alleles. Marja Timmermans is involved in the genetic analysis of lbl1 and other ta-siRNA pathway components, advices the other personnel on the project and helped writing the reviews and manuscripts. Training and Development: Fabio Nogueira, Dan Chitwood, and Katie Petsch have both gained experience in plant developmental genetics and gene regulation by small regulatory RNAs. They have established a protocol for small RNA in situ hybridization using LNA probes. They have gained expertise in maize developmental genetics, microscopy, histology, laser capture microdissection and other techniques. In addition, Fabio Nogueira and Dan Chitwood have each been first author on a review and have published a paper. They have also presented seminars for a plant specific and a general audience and have thereby obtained important training in presenting data to diverse audiences. Through this project we have also trained members of other labs how to use the laser capture microscope and how to perform in situ hybridizations to analyze small RNAs. Fabio Nogueira and Katie Petsch have supervised high school and undergraduate students through this project. Sarah Samson (Ohio State University) characterized double mutants between lbl1 and other polarity mutants, and Ricky Knight (Locust Valley HS) and Devin Tark (Rockville Center HS) helped genotype EMS -induced alleles of ta-siRNA biogenesis components. In addition, we have hosted Priscilla Manzotti, a graduate student from Dr Giuseppe Gavazzi (University of Milan, Italy), for four month to molecularly characterize and perform in situ hybridization analysis on a dcl4 mutant, and Ryan Douglas, a graduate student from Mike Scalon's group (Cornell University), to learn small RNA in situ hybridization. Collaborators and contacts: Our observation that small RNAs may act as mobile signals has resulted in collaborations with Dr. Jim Carrington's lab at Oregon State University. We are collaborating with Drs Michael Scanlon (Cornell University, Ithaca, NY) and Giuseppe Gavazzi (University of Milan, Italy) on the characterization of mutants defective for AGO7 and DCL4, other components in the ta-siRNA biogenesis pathway. In addition, I have written a review on adaxial-abaxial patterning in plants together with Catherine Kidner from the Royal Botanic Garden in Edinburgh, UK, and a book chapter on axial patterning of the maize leaf with Toshi Foster from The Horticulture and Food Research Institute of New Zealand. TARGET AUDIENCES: Contributions within and outside the plant development field: We have developed an LNA in situ hybridization protocol as well as LCM-RT-PCR for expression analysis of small RNAs and their precursors. These will be valuable techniques within the plant community. Our expression analyses suggest that miRNA accumulation is regulated at the level of biogenesis and/or stability. Moreover, our data suggests that miRNAs may be mobile. This observation has potential implications for development biologists in both plants as well as animals, as miRNAs would no longer be considered as cell-autonomous regulators of gene expression but as morphogen-like signals. In addition, whether small RNAs are mobile over few cell distances will also need to be considered when small RNAs are used as tools for gene silencing or therapies. Outreach activities: We assist in the educational efforts of the DNA Learning Center at CSHL, which is visited by numerous high school students and other members from the general public. One of their research labs is aimed at characterizing the behavior of the Ac transposable element from maize. Students that participate in the lab isolate maize genomic DNA, use PCR and sequence analysis to characterize new transposon excision alleles. Via CSHL and the DNA Learning Center I have given lectures for the lay audience on RNAi and small RNAs in plants. In addition, I lectured in the Plant Genetics Course at Cold Spring Harbor lab on the role of small RNAs in development. PROJECT MODIFICATIONS: none
Impacts
Aim1 was largely completed last year, so please refer to the progress report of 2008. With the increased availability of reverse genetic resources in maize, we have now identified a loss of function allele of arf3a and this is currently being analyzed phenotypically. Also, based on the now available genomic sequence for arf3a, we have generated constructs that allow the expression of ARF3-GFP fusion proteins that are either regulated or no longer under control of tasiR-ARF regulation. These are in the pipeline for transformation into corn. Aim2 was completed last year, so please refer to the progress report of 2008. We have begun to characterize mutants defective for other components in the ta-siRNA pathway (Aim 3). We have obtained four rdr6 alleles: two weak alleles resulting from amino acid substitutions in conserved protein domains, and two null allele resulting from mutation into a premature stop codon or insertion of a Mutator element. Both rdr6 null alleles condition an embryo lethal phenotype with embryos failing to establish a shoot apical meristem. Expression of kn1 is altered in these embryos and fails to initiate in the initiating SAM. A similar phenotype is observed when severe lbl1 alleles are introgressed into the maize inbred W22, suggesting that this phenotype resembles the tas3 ta-siRNA pathway loss-of-function phenotype. We are currently analyzing the expression patterns of ta-siRNA components during embryogenesis. This revealed that miR390 is an early marker for proembryo fate. Its expression marks the developing proembryo, and is observed prior to the onset of rld1 (an hd-zipIII family member) expression and kn1 expression. In collaboration with Dr. Mike Scanlon's group (Cornell University, Ithaca, NY) we are characterizing a mutant defective for AGO7. We have also shown that rgd766 results from an single amino acid change in DCL4. From additional EMS screens we have obtained two additional DCL4 alleles, which are null alleles due to a premature stop codon. Interestingly, neither dcl4 null allele gives rise to an embryo lethal phenotype, instead their mutant phenotypes resemble those of weak lbl1 alleles. dcl4 mutants develop abaxialized leaves that have altered expression of miR166, hd-zipIII genes, and arf3a.This finding indicates partial redundancy of DCL4 with another small RNA biogenesis component. Considering data from Arabidopsis, it is envisioned that DCL4 acts redundant with another DCL family member. Giuseppe Gavazzi's group (University of Milan, Italy) has identified a potential dominant enhancer of dcl4, and we are currently testing whether this enhancer maps to one of the remaining maize DCL family members. Finally, we have identified two new polarity mutants in maize which are being mapped using bulked segregant analysis.
Publications
- Husbands, A.Y., Chitwood, D.H., Plavskin, Y. and Timmermans, M.C.P. (2009) Signals and prepatterns: new insights into organ polarity in plants. Genes & Dev. 23, 1986-1997.
- Nogueira, F.T.S., Chitwood, D.H., Madi, S., Kazuhiro Ohtsu, K., Schnable, P.S., Scanlon, M.J., and Timmermans, M.C.P. (2009) Regulation of small RNA accumulation in the maize shoot apex. PLoS Genetics 5, e1000320.
- Foster, T.M., and Timmermans, M.C.P. (2009) Axial patterning of the maize leaf. In: J.L. Bennetzen and S.C. Hake (Eds.) Handbook of Maize: its biology. Springer USA, pp 161-178.
- Nogueira, F.T.S., and Timmermans, M.C.P. (2007) An interplay between small RNAs patterns leaves. Plant Sign. & Behavior 2, 519-521.
- Nogueira, F.T.S., Madi, S., Chitwood, D.H., Juarez, M.T., and Timmermans, M.C.P. (2007) Two small regulatory RNAs establish opposing fates of a developmental axis. Genes & Dev. 21, 750-755.
- Chitwood, D.H., Guo, M., Nogueira, F.T.S., and Timmermans, M.C.P. (2007) Establishing leaf polarity: the role of small RNAs and positional signals in the shoot apex. Development 134, 813-823.
- Kidner, C., and Timmermans, M.C.P. (2007) Mixing and matching pathways in leaf polarity. Curr. Op. Plant Sci. 10, 13-20.
- Nogueira F.T.S, Sarkar, A., Chitwood, D.H., and Timmermans, M.C.P. (2007) Distinct small regulatory RNAs interact to specify organ polarity in plants. Symp. Quant. Biol. 71, 157-164.
|
Progress 09/15/07 to 09/14/08
Outputs
OUTPUTS: Our work indicates an important role for small RNAs in leaf polarity in maize. Interestingly, we found that organ polarity is established through a cascade of opposing small RNAs; miR390 triggers the biogenesis of ta-siRNAs on the adaxial side of developing leaf primordia. The ta-siRNAs specify adaxial fate by repressing abaxial determinants including miR166, which in turn restricts activity of the adaxializing HD-ZIPIII factors to the adaxial side. We thereby identified a novel patterning mechanism. We further used in situ hybridization to show that miR390 is expressed on the adaxial side of developing leaf primordia. This polarized expression persists in the abaxialized leaves of leafbladeless1 mutants, indicating that the adaxial restricted expression of miR390 is established independent of ta-siRNA pathway and its downstream polarity determinants, such as the hd-zipIII and yabby genes. Together, these findings suggest that miR390 is an upstream component in the genetic pathway specifying leaf polarity. Moreover, our expression analyses of mature small RNAs and their precursors suggest that miRNA accumulation is regulated at the level of biogenesis and/or stability as miRNA precursors are expressed in cells in which the miRNA does not accumulate. We also found some evidence suggesting that miRNAs may act as local mobile signals in development. This observation has potential implications for development biologists in both plants as well as animals, as miRNAs would no longer be considered as cell-autonomous regulators of gene expression but as morphogen-like signals. Our research has thereby made contributions to other disciplines. To inform the scientific community of these findings, we have presented this work in four departmental seminars and four scientific meetings in the USA, Holland, Morocco, Switzerland, and Austria. In addition, this grant has resulted in three papers, three reviews and one book chapter. PARTICIPANTS: Marja Timmermans, PI Fabio Nogueira, Postdoctoral Fellow Daniel Chitwood, Graduate student at the CSHL Watson School for Biological Sciences Shahinez Madi, technician Fabio Nogueria and Shahinez Madi worked on the cloning and characterization of lbl1, they determined its role in ta-siRNA biogenesis, and identified arf3 genes as potential ta-siRNA targets. Fabio Nogueira and Daniel Chitwood performed the small RNA in situ hybridization experiments. All three worked jointly on the LCM-RT-PCR analysis of the mir166, mir390 and tas3 precursor transcripts. Marja Timmermans is involved in the genetic analysis of lbl1 and other ta-siRNA pathway components, advises the other personnel on the project and helped writing the reviews and manuscripts. Training and Development: Fabio Nogueira and Dan Chitwood have both gained experience in plant developmental genetics and gene regulation by small regulatory RNAs. They have established a protocol for small RNA in situ hybridization using LNA probes. They have gained expertise in microscopy, histology, laser capture microdissection and other techniques. In addition, they have each been first author on a review and have published three papers. Both Fabio Nogueira and Dan Chitwood have also presented seminars for plant specific and general audiences and have thereby obtained important training in presenting their data to diverse audiences. Priscilla Manzotti from Dr Giuseppe Gavazzi (University of Milan, Italy) worked in my lab for four month to molecularly characterize and perform in situ hybridization analysis on a dcl4 mutant. Collaborators and contacts: Our observation that small RNAs may act as mobile signals has resulted in collaborations with Dr. Jim Carrington's lab at Oregon State University. We are collaborating with Drs Michael Scanlon (Cornell University, Ithaca, NY) and Giuseppe Gavazzi (University of Milan, Italy) on the characterization of mutants defective for AGO7 and DCL4, other components in the ta-siRNA biogenesis pathwayy. In addition, I have written a review on adaxial-abaxial patterning in plants together with Catherine Kidner from the Royal Botanic Garden in Edinburgh, UK, and a book chapter on axial patterning of the maize leaf with Toshi Foster from The Horticulture and Food Research Institute of New Zealand. TARGET AUDIENCES: Contributions within and outside the plant development field: We have developed an LNA in situ hybridization protocol as well as LCM-RT-PCR for expression analysis of small RNAs and their precursors. These will be valuable techniques within the plant community. Our expression analyses suggest that miRNA accumulation is regulated at the level of biogenesis and/or stability. Moreover, our data suggests that miRNAs may be mobile. This observation has potential implications for development biologists in both plants as well as animals, as miRNAs would no longer be considered as cell-autonomous regulators of gene expression but as morphogen-like signals. In addition, whether small RNAs are mobile over few cell distances will also need to be considered when small RNAs are used as tools for gene silencing or therapies. Outreach activities: We assist in the educational efforts of the DNA Learning Center at CSHL, which is visited by numerous high school students and other members from the general public. One of their research labs is aimed at characterizing the behavior of the Ac transposable element from maize. Students that participate in the lab isolate maize genomic DNA, use PCR and sequence analysis to characterize new transposon excision alleles. Via CSHL and the DNA Learning Center I have given lectures for the lay audience on RNAi and small RNAs in plants. In addition, I lectured in the Plant Genetics Course at Cold Spring Harbor lab on the role of small RNAs in development. PROJECT MODIFICATIONS: none
Impacts
We established an LNA in situ hybridization protocol to show that miR390 and tasiR-ARF are expressed adaxially in incipient and young leaf primordia, consistent with a role for the ta-siRNA pathway in specifying adaxial cell fate. As expected, no expression of tasiR-ARF was observed in lbl1 mutants, but interestingly, we observed that miR390 remains polarly expressed in lbl1. Thus, the adaxial expression of miR390 is established and maintained independently of the ta-siRNA pathway, and because this pathway acts upstream of other known polarity determinants, miR390 is an upstream polarity determinant. We further showed that expression of tasiR-ARF target arf3a is abaxially restricted in normal leaf primordia (consistent with arf3a being an abaxial determinant), but in lbl1, expression is expanded into the adaxial domain. We established laser microdissection in combination with RT-PCR to analyze the expression domains of the low abundant miRNA precursors (Aim 2). We determined expression profiles for nine mir166, two mir390 and four tas3 precursors in specific domains of the SAM where adaxial/abaxial polarity is established; the tip of the SAM, at the incipient primordium, below the incipient leaf, and in the epidermal (L1) and subepidermal (L2) layers of the SAM. These experiments have let to several key observations. First, the mir166 family members are under intricate transcriptional control. mir166a, c, f, and i are expressed in or immediately below the incipient leaf and could contribute to adaxial/abaxial patterning of the leaf. Second, miRNA accumulation is regulated at the level of biogenesis or stability, because mir166a and the mir390 precursors are expressed at the tip of the SAM where the mature miRNAs do not accumulate but their targets are abundantly expressed. Third and most interestingly, we found that the mir390 precursors are expressed specifically in the L1 whereas miR390 accumulates in both the L1 and L2 layers of the incipient leaf. This incongruence in expression patterns suggests the possibility that miRNAs may be able to traffic from the L1 into the underlying L2. By analyzing the expression levels of the mir166 precursors in lbl1 mutant apices, we = found that altered expression of mir166c and mir166i in the incipient leaf underlies the leaf polarity defects in lbl1. We have begun to characterize mutants defective for other components in the ta-siRNA pathway (Aim 3). We have obtained a null allele for RDR6. rdr6 mutant embryos fail to establish a shoot apical meristem. A similar phenotype is observed when severe lbl1 alleles are introgressed into the maize inbred W22. We are currently analyzing the expression patterns of ta-siRNA components during embryogenesis. In collaboration with Dr. Mike Scanlon's group (Cornell University, Ithaca, NY) we are characterizing a mutant defective for AGO7. Likewise, we hosted a student from Giuseppe Gavazzi's group (University of Milan, Italy) to characterize an allele of DCL4. We showed that this dcl4 mutant resembles lbl1, and develops abaxilaized leaves that have altered expression of miR166, hd-zipIII genes, and arf3a.
Publications
- Nogueira, F.T.S., Chitwood, D.H., Madi, S., Kazuhiro Ohtsu, K., Schnable, P.S., Scanlon, M.J., and Timmermans, M.C.P. (2009) Complex regulation of small RNA accumulation in the maize shoot apex. PLoS Genetics in press.
- Foster, T.M., and Timmermans, M.C.P. (2009) Axial patterning of the maize leaf. In: J.L. Bennetzen and S.C. Hake (Eds.) Handbook of Maize: its biology. Springer USA, pp 161-178.
- Nogueira, F.T.S., and Timmermans, M.C.P. (2007) An interplay between small RNAs patterns leaves. Plant Sign. & Behavior 2, 519-521.
- Nogueira, F.T.S., Madi, S., Chitwood, D.H., Juarez, M.T., and Timmermans, M.C.P. (2007) Two small regulatory RNAs establish opposing fates of a developmental axis. Genes & Dev. 21, 750-755.
- Chitwood, D.H., Guo, M., Nogueira, F.T.S., and Timmermans, M.C.P. (2007) Establishing leaf polarity: the role of small RNAs and positional signals in the shoot apex. Development 134, 813-823.
- Kidner, C., and Timmermans, M.C.P. (2007) Mixing and matching pathways in leaf polarity. Curr. Op. Plant Sci. 10, 13-20.
- Nogueira F.T.S, Sarkar, A., Chitwood, D.H., and Timmermans, M.C.P. (2007) Distinct small regulatory RNAs interact to specify organ polarity in plants. Symp. Quant. Biol. 71, 157-164.
|
Progress 09/15/06 to 09/14/07
Outputs
OUTPUTS: Our work indicates an important role for small RNAs in leaf polarity in plants. Interestingly we found that the opposing activity between two distinct small RNAs sets up organ polarity; ta-siRNAs specify adaxial fate by repressing abaxial determinants including miR166, which in turn restricts activity of the adaxializing HD-ZIPIII factors to the adaxial side. We thereby identified a novel patterning mechanism. Moreover, our expression analyses of mature microRNAs and their precursors suggest that microRNAs may act as local mobile signals in development. This observation has potential implications for development biologists in both plants as well as animals, as miRNAs would no longer be considered as cell-autonomous regulators of gene expression but as morphogen-like signals. Our research has thereby made contributions to other disciplines. To inform the scientific community of these findings, we have presented this work in five departmental seminars and three scientific
meetings in the USA, Holland and France. Outreach activities: We assist in the educational efforts of the DNA Learning Center at CSHL, which is visited by numerous high school students and other members from the general public. One of their research labs is aimed at characterizing the behavior of the Ac transposable element from maize. Students that participate in the lab isolate maize genomic DNA, use PCR and sequence analysis to characterize new transposon excision alleles. Via CSHL and the DNA Learning Center I have given lectures for the lay audience on RNAi and small RNAs in plants. Training and Development: Fabio Nogueira and Dan Chitwood have both gained experience in plant developmental genetics and gene regulation by small regulatory RNAs. They have established a protocol for small RNA in situ hybridization using LNA probes. They have gained expertise in microscopy, histology, laser capture microdissection and other techniques. In addition, they have each been first author on
a review and have published a paper. Fabio Nogueira has also presented seminars for a plant specific and a general audience and has thereby obtained important training in presenting his data to diverse audiences.Through this project we have also trained members of other labs how to the laser capture microscope and how to perform in situ hybridizations to analyze small RNAs.
PARTICIPANTS: Marja Timmermans, PI Fabio Nogueira, Postdoctoral Fellow Daniel Chitwood, Graduate student at the CSHL Watson School for Biological Sciences Shahinez Madi, technician Fabio Nogueria and Shahinez Madi worked on the cloning and characterization of lbl1, they determined its role in ta-siRNA biogenesis, and identified arf3 genes as potential ta-siRNA targets. Fabio Nogueira and Daniel Chitwood performed the small RNA in situ hybridization experiments. All three worked jointly on the LCM-RT-PCR analysis of the mir166, mir390 and tas3 precursor transcripts. Marja Timmermans is involved in the genetic analysis of lbl1, advises the other personnel on the project and helped writing the reviews and manuscripts. Training and Development: Fabio Nogueira and Dan Chitwood have both gained experience in plant developmental genetics and gene regulation by small regulatory RNAs. They have established a protocol for small RNA in situ hybridization using LNA probes. They have gained
expertise in microscopy, histology, laser capture microdissection and other techniques. In addition, they have each been first author on a review and have published a paper. Fabio Nogueira has also presented seminars for a plant specific and a general audience and has thereby obtained important training in presenting his data to diverse audiences. Collaborators and contacts: Our observation that small RNAs may act as mobile signals has resulted in collaborations with Jim Carrington's lab at Oregon State University as well as with Robert Williams, DuPont/Pioneer Hybrid. In addition, I have written a review on adaxial-abaxial patterning in plants together with Catherine Kidner from the Royal Botanic Garden in Edinburgh, UK.
TARGET AUDIENCES: Contributions within and outside the plant development field: We have developed an LNA in situ hybridization protocol as well as LCM-RT-PCR for expression analysis of small RNAs and their precursors. These will be valuable techniques within the plant community. Our expression analyses suggest that miRNA accumulation is regulated at the level of biogenesis and/or stability. Moreover, our data suggests that miRNAs may be mobile. This observation has potential implications for development biologists in both plants as well as animals, as miRNAs would no longer be considered as cell-autonomous regulators of gene expression but as morphogen-like signals. In addition, whether small RNAs are mobile over few cell distances will also need to be considered when small RNAs are used as tools for gene silencing or therapies. Outreach activities: We assist in the educational efforts of the DNA Learning Center at CSHL, which is visited by numerous high school students and other
members from the general public. One of their research labs is aimed at characterizing the behavior of the Ac transposable element from maize. Students that participate in the lab isolate maize genomic DNA, use PCR and sequence analysis to characterize new transposon excision alleles. Via CSHL and the DNA Learning Center I have given lectures for the lay audience on RNAi and small RNAs in plants. In addition, I lectured in the Plant Genetics Course at Cold Spring Harbor lab on the role of small RNAs in development.
Impacts
We have established an LNA in situ hybridization protocol for expression analysis in maize tissues, and determined the spatiotemporal patterns of expression for miR390 and tasiR-ARF in leaves (Aim 1). We have shown that although this pathway is conserved between monocot and dicot plant species, mutations affecting this pathway in maize and Arabidopsis give rise to very distinct leaf defects. Whereas mutations blocking the accumulation of ta-siRNAs in Arabidopsis develop no obvious leaf polarity defects, similar mutations in maize develop leaves that are abaxialized. Consistent with a role in adaxial cell fate, our expression analysis indicates that miR390 and tasiR-ARF are expressed adaxially in incipient and young leaf primordia. Also, lbl1 which we showed is required for ta-siRNA biogenesis is expressed on the adaxial side of developing leaf primordia, but perhaps consistent with a role in viral resistance is expressed also broadly in the SAM. We also established the
use of laser capture microdissection in combination with RT-PCR analysis to analyze the expression domains of the unusually low abundant miRNA precursor transcripts (Aim 2). We have determined expression profiles for the nine mir166, two mir390 and four tas3 precursors in specific domains of the SAM where the pattern of accumulation of these small RNAs sets up adaxial/abaxial polarity. Expression of the small RNA genes was analyzed in the following SAM regions; the tip of the SAM, at the incipient primordium, below the incipient leaf, and in the epidermal (L1) and subepidermal (L2) layers of the SAM. These experiments have let to several exciting observations. First, the mir166 family members exhibit distinct spatial and temporal expression patterns, suggesting that the dynamic pattern of miR166 accumulation reported previously results in part from the intricate transcriptional regulation of its precursors. mir166a, c, f, and i are expressed in or immediately below the incipient leaf
and could contribute to adaxial/abaxial patterning of the new leaf. Second, miRNA accumulation is regulated at the level of biogenesis or stability, because mir166a and the mir390 precursors are expressed at the tip of the SAM where the mature miRNAs do not accumulate but their targets (hd-zipIII and tas3) are abundantly expressed. Third and most interestingly, we found that the mir390 precursors are expressed specifically in the L1 whereas miR390 accumulates in both the L1 and L2 layers of the incipient leaf. This incongruence in expression patterns suggests the intriguing possibility that miRNAs may be able to traffic from the L1 into 1-2 cell layers of the underlying L2. By analyzing the expression levels of the mir166 precursors in lbl1 mutant apices, we have found that altered expression of mir166c and mir166i in the incipient leaf underlies the ectopic expression of miR166 and associated leaf polarity defects in lbl1 (Aim 2). The mechanism by which the ta-siRNA pathway regulated
the expression domain of these miR166 precursors is still under investigation.
Publications
- Nogueira, F.T.S., and Timmermans, M.C.P. (2007) An interplay between small RNAs patterns leaves. Plant Sign. & Behavior in press.
- Nogueira, F.T.S., Madi, S., Chitwood, D.H., Juarez, M.T., and Timmermans, M.C.P. (2007) Two small regulatory RNAs establish opposing fates of a developmental axis. Genes & Dev. 21, 750-755.
- Chitwood, D.H., Guo, M., Nogueira, F.T.S., and Timmermans, M.C.P. (2007) Establishing leaf polarity: the role of small RNAs and positional signals in the shoot apex. Development 134, 813-823.
- Kidner, C., and Timmermans, M.C.P. (2007) Mixing and matching pathways in leaf polarity. Curr. Op. Plant Sci. 10, 13-20.
- Nogueira F.T.S, Sarkar, A., Chitwood, D.H., and Timmermans, M.C.P. (2007) Distinct small regulatory RNAs interact to specify organ polarity in plants. Symp. Quant. Biol. 71, 157-164.
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