Hydroxyproline O-Arabinosylation in Pollen Fertility

Sexual reproduction in flowering plants relies on the successful delivery and fusion of the male and female gametes. Ovules contain the female gametes and are located in the ovaries, which are embedded in the female reproductive tissue- collectively referred to as the pistil. The male gametes are transported through a specialized cell called the pollen tube, which must penetrate and traverse through the transmitting tract, which can be dense with carbohydrates secreted from the surrounding cells, or open- depending on the species. Therefore, successful fertilization relies on the pollen tube’s ability to grow, successfully navigate to the ovules, and burst to release the sperm cells once inside the ovule. While these physiological events are well-understood, the molecular mechanisms underlying key processes are still unclear. Sperm delivery relies on the pollen tube’s ability to maintain the structural integrity of its cell wall throughout the growth process; it must be rigid enough to penetrate the female tissue and prevent premature rupture, but also extensible at the tip to allow for elongation. This is regulated by a multitude of factors that control the structural integrity of the cell wall, which include the synthesis and trafficking of cell wall materials, secretion, and the proper assembly, organization, and modification of these materials once they are deposited into the extracellular space. Many factors have been identified, but many key players and their roles have yet to be discovered. This dissertation describes how we identified new factors that regulate pollen tube growth by influencing the structural and mechanical properties of the cell wall, by using a combination of genetic, molecular, and bioinformatic approaches. We characterized the cell wall structural defects caused by loss of protein O-arabinosylation in Arabidopsis pollen tubes which primarily included loss of cell polarity, increased bursting frequency, and decreased elongation rates which, in combination, caused poor fertility. We examined the pollen tube cell wall structure and discovered that loss of protein O-arabinosylation was associated with changes in the composition of the cell wall and the organization of its components which have important roles in regulating the mechanical properties of the cell wall. Through a forward genetics screen, we identified mutations in key secretory genes- including members of the exocyst complex- that suppressed the effects caused by loss of protein O-arabinosylation and improved pollen tube growth and fertility, and we interrogated the link between the secretory pathway and cell wall structure. We also identified a mutation in a gene involved in phosphoinositide (PI) signaling, which appears to be suppressing the effects caused by loss of O-arabinosylation through another pathway. By characterizing the effects of this suppressor mutation and learning more about the gene involved, we have identified another novel factor that regulates tip growth and cell wall structure in Arabidopsis pollen tubes. Our findings described herein demonstrate how we have contributed to the overall knowledge of the plant development and reproduction field by addressing how protein O-arabinosylation, secretion, and PI signaling pathways combine to influence the structural and mechanical properties of the cell wall.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169686/1/sbeuder_1.pd

Notch activation is required for downregulation of HoxA3-dependent endothelial cell phenotype during blood formation.

Hemogenic endothelium (HE) undergoes endothelial-to-hematopoietic transition (EHT) to generate blood, a process that requires progressive down-regulation of endothelial genes and induction of hematopoietic ones. Previously, we have shown that the transcription factor HoxA3 prevents blood formation by inhibiting Runx1 expression, maintaining endothelial gene expression and thus blocking EHT. In the present study, we show that HoxA3 also prevents blood formation by inhibiting Notch pathway. HoxA3 induced upregulation of Jag1 ligand in endothelial cells, which led to cis-inhibition of the Notch pathway, rendering the HE nonresponsive to Notch signals. While Notch activation alone was insufficient to promote blood formation in the presence of HoxA3, activation of Notch or downregulation of Jag1 resulted in a loss of the endothelial phenotype which is a prerequisite for EHT. Taken together, these results demonstrate that Notch pathway activation is necessary to downregulate endothelial markers during EHT

Exocyst mutants suppress pollen tube growth and cell wall structural defects of hydroxyproline O‐arabinosyltransferase mutants

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156472/1/tpj14808-sup-0003-FigS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156472/9/tpj14808.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156472/8/tpj14808-sup-0001-FigS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156472/7/tpj14808-sup-0004-FigS4.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156472/6/tpj14808-sup-0005-FigS5.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156472/5/tpj14808-sup-0007-FigS7.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156472/4/tpj14808-sup-0006-FigS6.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156472/3/tpj14808-sup-0002-FigS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156472/2/tpj14808_am.pd

HoxA3 Controls Notch Pathway to Repress Blood Development

Hematopoietic stem cells (HSC) are generated from a specialized subset of endothelial cells, hemogenic endothelium. Previous studies performed by our group showed that HoxA3 restrains the cell at the hemogenic endothelium stage, inhibiting further differentiation toward blood by direct repression of Runx1. Building on our previous work, we show here that overexpression of HoxA3 affects the Notch pathway. Upon HoxA3 upregulation in endothelial cells, Jag1 is induced, Mfng (Manic) and Lfng (Lunatic) fringes are downregulated, and there is a trend towards Notch target gene repression. These data suggest that in the presence of HoxA3, endothelial cells are blocked from receiving Notch signal through ligand cis-inhibition with resulting blood inhibition. In order to test this hypothesis, we evaluated the effect of activation or inhibition of the Notch pathway during blood development. We show here that the number of blood progenitors originating from the hemogenic endothelium is decreased when the Notch pathway is inhibited. Conversely, induction of the pathway by upregulation of NICD (Notch1 Intra Cellular Domain) promotes an increase in the number of blood progenitors originating from hemogenic endothelium. Furthermore, inhibition of the pathway when HoxA3 is upregulated has little or no effect in blood while induction of the pathway in HoxA3 presence in part promotes blood development. Taken together, these results demonstrate that Notch pathway is both sufficient and essential for blood development. Specifically HoxA3 inhibits Notch signal reception in two ways: 1) HoxA3 increases Jag1 ligand expression that acts in cis-inhibition; 2) represses Manic and Lunatic fringes both necessary to increase the affinity of Notch receptors for the Delta ligands. When this blockage is bypassed by NICD upregulation, blood is formed, demonstrating that HoxA3-dependent Notch inhibition results in blood suppression

Forced down-regulation of Jag1 in endothelial cells removes Notch pathway cis inhibition by HoxA3 and initiates EHT.

<p><b>A)</b> Jag1 RNA expression level in Bend3 cells infected with empty vector (Con) or Jag1 shRNA (Jag1 KD). <b>B)</b> Western blot using specific antibody against Jag1 and GAPDH in Bend3 cells infected with empty vector (pGIPZ) or Jag1 shRNA (Plko.1 Jag1). <b>C)</b> Frequency of Flk1+/VE-cadherin+ cells obtained from day 6 EBs, transduced with empty vector (CON) or with shRNA-Jag1-GFP (JKD) and co-cultured on OP9 for 5 days in Control (Con) or HoxA3 overexpression. <b>D)</b> Gene expression levels of Notch pathway components Hes1, Hey1, Hey2 from purified control (white bar) or HoxA3-overexpressing cells (black bars), transduced with empty vector (CON) or with shRNA-Jag1-GFP (JKD) and co-cultured on OP9-DLL1 for 5 days. Graphs in panel D show one representative experiment with triplicate measurements. *: p<0.05; **: p<0.01; ***: p<0.001. Statistical analysis is reported on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s011" target="_blank">S6 Table</a></b>.</p

Proposed model.

<p><b>A)</b> Illustration of ligand <i>cis</i> inhibition in the hemogenic endothelium. HoxA3 dependent Jag1 overexpression in <i>cis</i> interacts with Notch receptors, inhibiting Notch ligand in <i>trans</i> to interact with the receptor. When HoxA3 is withdrawal the pathway is activated in <i>trans</i> and Runx1 can be induced in the hemogenic endothelium. <b>B)</b> Effect of HoxA3 on Notch pathway. HoxA3 upregulates Jag1 to inhibit the Notch pathway. The pathway in turn will promote downregulation of endothelial specific transcripts, and initiate the EHT.</p

Notch signaling in <i>trans</i> does not rescue HoxA3 mediated inhibition of Notch.

<p><b>A)</b> Experimental procedure <b>B)</b> Representative flow cytometric profile of endothelial surface markers Flk-1/Ve-Cadherin and hematopoietic surface markers c-Kit/CD41, and c-Kit/CD45 obtained from 200,000 EB-derived Flk1⁺/VE-cadherin⁺ cells and co-cultured on OP9 control (CON) or OP9 overexpressing Dll1 (OP9-Dll1) for 5 days in Control or HoxA3-overexpressing HE cells. <b>C)</b> Quantification of frequencies of hematopoietic surface markers (CD41, CD45) of the same cell as in <b>B</b>. <b>D)</b> Gene expression levels of the Notch pathway target genes (Hes1, Hey1, Hey2) and hematopoietic gene markers (PU.1, Runx1, Gata1) on control (white bar) or HoxA3-overexpressing (black bars) HE cells co-cultured on OP9 controls (CON) or OP9-DLL1 for 5 days (Flk1⁺/VE-cadherin⁺ and CD41⁺/c-Kit⁺ cells were pulled together). <b>E)</b> Iimmunofluorescence staining for activated Notch1 (NICD-red), VE-Cadherin (VECad-green) and Hoechst (blue) showing adherent endothelial clusters growing in Control (Con) or HoxA3 overexpression (HoxA3), derived from endothelial cells (Flk1⁺/VE-cadherin⁺) co-cultured on OP9-DLL1 cells *: p<0.05. Statistical analysis is reported on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s010" target="_blank">S5 Table</a></b>.</p

Repression of EHT by HoxA3 is not affected by inhibition Notch pathway.

<p><b>A)</b> Experimental procedure, Endothelial Derived Cells (ENDO). <b>B)</b> Representative flow cytometric profile of endothelial surface markers Flk-1/Ve-Cadherin and hematopoietic surface markers c-Kit/CD41, and c-Kit/CD45 on 200,000 EB-derived Flk1⁺/VE-cadherin⁺ cells without or with HoxA3 overexpression and co-cultured on OP9 for 5 days in the presence or absence of the Notch inhibitor DAPT (20 μM). <b>C)</b> Frequency of endothelial surface markers Flk-1⁺/Ve-Cadherin⁺ in EB-derived cells. *: p<0.05; **: p<0.01. Two way ANOVA analyses of Flk-1⁺/Ve-Cadherin⁺, CD41⁺ and CD45⁺ frequencies are reported on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s008" target="_blank">S3 Table</a></b>.</p

HoxA3 up-regulates the Notch ligand Jag1 but does not activate the Notch pathway.

<p><b>A)</b> Experimental procedure (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#sec002" target="_blank">methods</a> for details). <b>B)</b> Gene expression levels of the Notch pathway components in purified endothelial cells (Flk1⁺/VE-Cadherin⁺) derived from day 6 control EBs (white bars) or upon 6 hours of HoxA3 up-regulation (black bars). <b>C)</b> Western blot analysis using Jag1 specific antibody in purified endothelial cells (Flk1⁺/VE-Cadherin⁺) derived from day 6 control EBs (white bars) or upon 2 days of HoxA3 up-regulation (black bars) <b>D)</b> Experimental procedure (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#sec002" target="_blank">methods</a> for details). <b>E)</b> Immunofluorescence staining for Jag1 (red), VE-Cadherin (green) and Hoechst (blue) showing adherent endothelial clusters growing in Control (Con) or HoxA3 overexpression (HoxA3), derived from endothelial cells (Flk1⁺/VE-cadherin⁺) and co-cultured for 5 days on OP9 cells. Bar: 100 μM. <b>F)</b> Gene expression levels of Notch ligands Jag1/Jag2/Dll1/Dll3/Dll4, Notch receptors Notch1 to Notch4, cofactors Lfng (lunatic fringe) Mfng (manic fringe) and target genes Hes1/Hey2/Hey1 on purified control cells (white bars) or cells overexpressing HoxA3 (black bars) derived from endothelial cells (Flk1⁺/VE-cadherin⁺) co-cultured for 5 days on OP9 cells. *: p<0.05. Detailed statistical analysis is reported in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s007" target="_blank">S2 Table</a></b>.</p