A molecular mechanism that confines the activity pattern of miR165 in Arabidopsis leaf primordia

In Arabidopsis leaf primordia, the expression of HD‐Zip III, which promotes tissue differentiation on the adaxial side of the leaf primordia, is repressed by miRNA165/166 (miR165/166). Small RNAs, including miRNAs, can move from cell to cell. In this study, HD‐Zip III expression was strikingly repressed by miR165/166 in the epidermis and parenchyma cells on the abaxial side of the leaf primordia compared with those on the adaxial side. We also found that the MIR165A locus, which was expressed in the abaxial epidermis, was sufficient to establish the rigid repression pattern of HD‐Zip III expression in the leaf primordia. Ectopic expression analyses of MIR165A showed that the abaxial‐biased miR165 activity in the leaf primordia was formed neither by a polarized distribution of factors affecting miR165 activity nor by a physical boundary inhibiting the cell‐to‐cell movement of miRNA between the adaxial and abaxial sides. We revealed that cis‐acting factors, including the promoter, backbone, and mature miRNA sequence of MIR165A, are necessary for the abaxial‐biased activity of miR165 in the leaf primordia. We also found that the abaxial‐determining genes YABBYs are trans‐acting factors that are necessary for the miR165 activity pattern, resulting in the rigid determination of the adaxial–abaxial boundary in leaf primordia. Thus, we proposed a molecular mechanism in which the abaxial‐biased patterning of miR165 activity is confined.     

<Emphasis Type="Italic">MIR166</Emphasis>/<Emphasis Type="Italic">165</Emphasis> genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The miR166/165 group and its target genes regulate diverse aspects of plant development, including apical and lateral meristem formation, leaf polarity, and vascular development. We demonstrate here that MIR166/165 genes are dynamically controlled in regulating shoot apical meristem (SAM) and floral development in parallel to the WUSCHEL (WUS)-CLAVATA (CLV) pathway. Although miR166 and miR165 cleave same target mRNAs, individual MIR166/165 genes exhibit distinct expression domains in different plant tissues. The MIR166/165 expression is also temporarily regulated. Consistent with the dynamic expression patterns, an array of alterations in SAM activities and floral architectures was observed in the miR166/165-overproducing plants. In addition, when a MIR166a-overexpressing mutant was genetically crossed with mutants defective in the WUS-CLV pathway, the resultant crosses exhibited additive phenotypic effects, suggesting that the miR166/165-mediated signal exerts its role via a distinct signaling pathway.     

HYL1 regulates the balance between adaxial and abaxial identity for leaf flattening via miRNA-mediated pathways

HYPONASTIC LEAVES1 (HYL1) is an important regulator of microRNA (miRNA) biogenesis. Incurvature of rosette leaves in loss-of-function mutants of HYL1 implicates the regulation of leaf flatness by HYL1 via miRNA pathways. Recent studies have identified jba-1D, jaw-1D, and oe-160c, the dominant mutants of MIR166g, MIR319a, and MIR160c genes, respectively, which display three types of leaf curvature. However, it remains unclear whether or how HYL1 controls leaf flatness through the pathways mediated by these miRNAs. To define which miRNAs and target genes are relevant to the hyl1 phenotype in terms of leaf incurvature, the effects of three mutated MIRNA genes and their targets on the direction and extent of leaf curvature in hyl1 mutants were examined. The genetic analysis shows that the hyl1 phenotype is strongly rescued by jba-1D, but not by jaw-1D or oe-160c, whereas the mutant phenotypes of jba-1D, jaw-1D, or oe-160c leaves are compromised by the hyl1 allele. Expression analysis indicates that reduced accumulation of miR166, rather than of miR319a or miR160, causes incurvature of hyl1 leaves, and that miR319a-targeted TCP3 positively regulates the adaxial identity gene PHABULOSA while miR160-targeted ARF16 negatively regulates the abaxial identity gene FILAMENTOUS FLOWER. In these cases, the direction and extent of leaf incurvature are associated with the expression ratio of adaxial to abaxial genes (adaxial to abaxial ratio). HYL1 regulates the balance between adaxial and abaxial identity and modulates leaf flatness by preventing leaf incurvature, wavy margins, and downward curvature. It is concluded that HYL1 monitors the roles of miR165/166, miR319a, and miR160 in leaf flattening through the relative activities of adaxial and abaxial identity genes, thus playing an essential role in leaf development.     

The ascl1a and dlx genes have a regulatory role in the development of GABAergic interneurons in the zebrafish diencephalon

During development of the mouse forebrain interneurons, the Dlx genes play a key role in a gene regulatory network (GRN) that leads to the GABAergic phenotype. Here, we have examined the regulatory relationships between the ascl1a, dlx, and gad1b genes in the zebrafish forebrain. Expression of ascl1a overlaps with dlx1a in the telencephalon and diencephalon during early forebrain development. The loss of Ascl1a function results in a loss of dlx expression, and subsequent losses of dlx5a and gad1b expression in the diencephalic prethalamus and hypothalamus. Loss of Dlx1a and Dlx2a function, and, to a lesser extent, of Dlx5a and Dlx6a, impairs gad1b expression in the prethalamus and hypothalamus. We conclude that dlx1a/2a act downstream of ascl1a but upstream of dlx5a/dlx6a and gad1b to activate GABAergic specification. This pathway is conserved in the diencephalon, but has diverged between mammals and teleosts in the telencephalon.     

Pattern Dynamics in Adaxial-Abaxial Specific Gene Expression Are Modulated by a Plastid Retrograde Signal during Arabidopsis thaliana Leaf Development

The maintenance and reformation of gene expression domains are the basis for the morphogenic processes of multicellular systems. In a leaf primordium of Arabidopsis thaliana, the expression of FILAMENTOUS FLOWER (FIL) and the activity of the microRNA miR165/166 are specific to the abaxial side. This miR165/166 activity restricts the target gene expression to the adaxial side. The adaxial and abaxial specific gene expressions are crucial for the wide expansion of leaf lamina. The FIL-expression and the miR165/166-free domains are almost mutually exclusive, and they have been considered to be maintained during leaf development. However, we found here that the position of the boundary between the two domains gradually shifts from the adaxial side to the abaxial side. The cell lineage analysis revealed that this boundary shifting was associated with a sequential gene expression switch from the FIL-expressing (miR165/166 active) to the miR165/166-free (non-FIL-expressing) states. Our genetic analyses using the enlarged fil expression domain2 (enf2) mutant and chemical treatment experiments revealed that impairment in the plastid (chloroplast) gene expression machinery retards this boundary shifting and inhibits the lamina expansion. Furthermore, these developmental effects caused by the abnormal plastids were not observed in the genomes uncoupled1 (gun1) mutant background. This study characterizes the dynamic nature of the adaxial-abaxial specification process in leaf primordia and reveals that the dynamic process is affected by the GUN1-dependent retrograde signal in response to the failure of plastid gene expression. These findings advance our understanding on the molecular mechanism linking the plastid function to the leaf morphogenic processes.     

Virus-induced gene silencing of PEAM4 affects floral morphology by altering the expression pattern of PsSOC1a and PsPVP in pea

pea-MADS4 (PEAM4) regulates floral morphology in Pisum sativum L., however, its molecular mechanisms still remain unclear. Virus-induced gene silencing (VIGS) is a recently developed reverse genetic approach that facilities an easier and more rapid study of gene functions. In this study, the PEAM4 gene was effectively silenced by VIGS using a pea early browning virus (PEBV) in wild type pea JI992. The infected plants showed abnormal phenotypes, as the floral organs, especially the sepals and petals changed in both size and shape, which made the corolla less closed. The petals changed in morphology and internal symmetry with, the stamens reduced and carpel dehisced. Larger sepals and longer tendrils with small cauline leaves appeared, with some sepals turning into bracts, and secondary inflorescences with fused floral organs were formed, indicating a flower-to-inflorescence change. The infected plants also displayed a delayed and prolonged flowering time. The PEAM4-VIGS plants with altered floral morphology were similar to the pim (proliferating inflorescence meristem) mutant and also mimicked the phenotypes of ap1 mutants in Arabidopsis. The expression pattern of the homologous genes PsSOC1a and PsSVP, which were involved in flowering time and florescence morphological control downstream of PEAM4, were analyzed by real-time RT-PCR and mRNA in situ hybridization. PsSOC1a and PsSVP were ectopically expressed and enhanced in the floral meristems from PEAM4-silenced plants. Our data suggests that PEAM4 may have a similar molecular mechanism as AtAP1, which inhibits the expression of PsSOC1a and PsSVP in the floral meristem from the early stages of flower development. As such, in this way PEAM4 plays a crucial role in maintaining floral organ identity and flower development in pea.     

MIR166基因家族在陆生植物中的进化模式分析

MicroRNA（miRNA）是一类广泛存在于真核生物中的具有转录后水平调控功能的内源非编码小分子RNA。在植物中．miRNA通过对靶基因的剪切或沉默来实现对植物生命活动的调控,它是基因表达调控网络的重要组成部分。miR165／166（miR166）是陆生植物中最为古老的MIRNA家族之一,它通过对3型同源异域型-亮氨酸拉链（1id—ZIPⅢ）等靶标的调控,在植物的众多发育时期起着关键的调控作用。本文分析了MIR166基因在陆生植物中的进化关系,并对MIR166在基部陆生植物小立碗藓（Physcomitrella patens）中的复制及进化进行了研究。此外,HD—ZIPⅢ蛋白是植物中重要的一类转录因子,miR166对HD-ZIP Ⅲ基因的调控作用在陆地植物保守的存在,本文对HD—ZIP Ⅲ基因和miR166在进化中的相互作用进行了初步的探讨。     

GUS activity for miR165a/166b, REV, and WUS/CLV3 in in vitro direct Arabidopsis thaliana shoot regeneration

In Arabidopsis thaliana, the process of shoot regeneration in vitro requires the presence of specific miRNAs. We describe here the β-glucuronidase (GUS) expression domains for miR165a/166b, REV, and WUS/CLV3 during direct shoot regeneration. Increased GUS activity of miR166b and REV were first detected within the shoot apical meristem of explants, while no pmiR165a::GUS activity appeared there. The zone of pWUS::GUS activity covered the inner sides of developing protuberances, while that of pCLV3::GUS was more restricted. Once the primary shoot had emerged from the protuberance, pREV::GUS activity was turned on throughout the protuberance. pmiR165a::GUS activity was detected in a small number of protuberance surface cells, while pWUS::GUS activity was restricted to within a few cells beneath the protuberance surface. After the differentiation of leaf-like structures, GUS activity for miR165a and miR166b appeared largely on their abaxial surface, while pWUS::GUS activity was concentrated at the apex of the primary shoot, and no pCLV3::GUS activity was detectable. Following the formation of secondary shoots, pmiR165a::GUS activity was detected on their abaxial surface. GUS activity for miR166b, REV, and WUS/CLV3 were concentrated in the stem apical meristem. The observations suggested that each member of this set of genes might play a distinct role in both primary and secondary shoot regeneration.     

Sexually dimorphic expression of the sex chromosome-linked genes cntfa and pdlim3a in the medaka brain

In vertebrates, sex differences in the brain have been attributed to differences in gonadal hormone secretion; however, recent evidence in mammals and birds shows that sex chromosome-linked genes, independent of gonadal hormones, also mediate sex differences in the brain. In this study, we searched for genes that were differentially expressed between the sexes in the brain of a teleost fish, medaka (Oryzias latipes), and identified two sex chromosome genes with male-biased expression, cntfa (encoding ciliary neurotrophic factor a) and pdlim3a (encoding PDZ and LIM domain 3 a). These genes were found to be located 3–4 Mb from and on opposite sides of the Y chromosome-specific region containing the sex-determining gene (the medaka X and Y chromosomes are genetically identical, differing only in this region). The male-biased expression of both genes was evident prior to the onset of sexual maturity. Sex-reversed XY females, as well as wild-type XY males, had more pronounced expression of these genes than XX males and XX females, indicating that the Y allele confers higher expression than the X allele for both genes. In addition, their expression was affected to some extent by sex steroid hormones, thereby possibly serving as focal points of the crosstalk between the genetic and hormonal pathways underlying brain sex differences. Given that sex chromosomes of lower vertebrates, including teleost fish, have evolved independently in different genera or species, sex chromosome genes with sexually dimorphic expression in the brain may contribute to genus- or species-specific sex differences in a variety of traits.     

Requirement for frzb and fzd7a in cranial neural crest convergence and extension mechanisms during zebrafish palate and jaw morphogenesis

Regulation of convergence and extension by wnt-frizzled signaling is a common theme in embryogenesis. This study examines the functional requirements of frzb and fzd7a in convergence and extension mechanisms during craniofacial development. Using a morpholino knockdown approach, we found that frzb and fzd7a are dispensable for directed migration of the bilateral trabeculae, but necessary for the convergence and extension of the palatal elements, where the extension process is mediated by chondrocyte proliferation, morphologic change and intercalation. In contrast, frzb and fzd7a are required for convergence of the mandibular prominences, where knockdown of either frzb or fzd7a resulted in complete loss of lower jaw structures. Further, we found that bapx1 was specifically downregulated in the wnt9a/frzb/fzd7a morphants, while general neural crest markers were unaffected. In addition, expression of wnt9a and frzb was also absent in the edn−/− mutant. Notably, over-expression of bapx1 was sufficient to partially rescue mandibular elements in the wnt9a/frzb/fzd7a morphants, demonstrating genetic epistasis of bapx1 acting downstream of edn1 and wnt9a/frzb/fzd7a in lower jaw development. This study underscores the important role of wnt-frizzled signaling in convergence and extension in palate and craniofacial morphogenesis, distinct regulation of upper vs. lower jaw structures, and integration of wnt-frizzled with endothelin signaling to coordinate shaping of the facial form.     

Bmpr1a is required for proper migration of the AVE through regulation of Dkk1 expression in the pre-streak mouse embryo

Here, we report a novel mechanism regulating migration of the anterior visceral endoderm (AVE) by BMP signaling through BMPRIA. In Bmpr1a-deficient (Bmpr-null) embryos, the AVE does not migrate at all. In embryos with an epiblast-specific deletion of Bmpr1a (Bmpr1a^null/flox; Sox2Cre embryos), the AVE cells migrate randomly from the distal end of embryos, resulting in an expansion of the AVE. Dkk1, which is normally expressed in the anterior proximal visceral endoderm (PxVE), is downregulated in Bmpr-null embryos, whereas it is circumferentially expressed in Bmpr1a^null/flox; Sox2Cre embryos at E5.75-6.5. These results demonstrate an association of the position of Dkk1 expressing cells with direction of the migration of AVE. In Bmpr1a^null/flox; Sox2Cre embryos, a drastic decrease of WNT signaling is observed at E6.0. Addition of WNT3A to the culture of Bmpr1a^null/flox; Sox2Cre embryos at E5.5 restores expression patterns of Dkk1 and Cer1. These data indicate that BMP signaling in the epiblast induces Wnt3 and Wnt3a expression to maintain WNT signaling in the VE, resulting in downregulation of Dkk1 to establish the anterior expression domain. Thus, our results suggest that BMP signaling regulates the expression patterns of Dkk1 for anterior migration of the AVE.     

SHORT-ROOT regulates vascular patterning,but not apical meristematic activity in the Arabidopsis root through cytokinin homeostasis

SHORT-ROOT (SHR) is a key regulator of radial patterning and stem-cell renewal in the Arabidopsis root. Although SHR is expressed in the stele, its function in the vascular tissue was not recognized until recently. In shr, the protoxylem is missing due to the loss of expression of microRNA165A (miR165A) and microRNA166B (miR165B). shr is also defective in lateral root formation, but the mechanism remains unclear. To dissect the SHR developmental pathway, we recently have identified its direct targets at the genome scale by chromatin immunoprecipitation followed by microarray analysis (ChIP-chip). In further studies, we have shown that SHR regulates cytokinin homeostasis through cytokinin oxidase 3 and that this role of SHR is critical to vascular patterning in the root. In this communication we report that SHR also regulates miR165A and miR166B indirectly through its effect on cytokinin homeostasis. Although cytokinin is inhibitory to root growth, the root-apical-meristem defect in shr was not alleviated by reduction of endogenous cytokinin. These results together suggest that SHR regulates vascular patterning, but not root apical meristematic activity, through cytokinin homeostasis.