M2WISH: An easy and efficient protocol for whole-mount mRNA in situ hybridization that allows 3D cell resolution of gene expression in Arabidopsis thaliana.

Gene expression analysis is essential for understanding the mechanisms involved in plant development. Here, we developed M2WISH, a protocol based on MicroWave treatment for Wholemount mRNA In Situ Hybridization in Arabidopsis. By permeabilizing tissues without damaging cellular organization this protocol results in high and homogeneous hybridization yields that enable systematic analysis of gene expression dynamics. Moreover, when combined with cellular histochemical staining, M2WISH successfully provides a cellular resolution of gene expression. Thus, we demonstrate the robustness of M2WISH with 10 genes on roots, aerial meristems, leaves, and embryos in the seed. We applied M2WISH to study the spatial dynamics of WUSCHEL (WUS) and CLAVATA3 (CLV3) expression during in vitro meristematic conversion of roots into shoot apical meristems. Thus, we showed that shoot apical meristems could arise from two different types of root structures that differed by their CLV3 gene expression patterns. We constructed 3D cellular representations of WUS and CLV3 gene co-expression pattern and stressed the variability inherent to meristem conversion. Thus, this protocol generates a large amount of data on the localization of gene expression, which can be used to model complex systems.

The NGATHA-like Genes DPA4 and SOD7 Are Not Required for Stem Cell Specification during Embryo Development in Arabidopsis thaliana.

In plants, stem cells are embedded in structures called meristems. Meristems can be formed either during embryogenesis or during the plant's life such as, for instance, axillary meristems. While the regulation of the stem cell population in an established meristem is well described, how it is initiated in newly formed meristems is less well understood. Recently, two transcription factors of the NGATHA-like family, DEVELOPMENT-RELATED PcG TARGET IN THE APEX4 (DPA4)/NGAL3 and SUPPRESSOR OF DA1-1 7 (SOD7)/NGAL2 have been shown to facilitate de novo stem cell initiation in Arabidopsis thaliana axillary meristems. Here, we tested whether the DPA4 and SOD7 genes had a similar role during stem cell formation in embryo shoot apical meristems. Using DPA4 and SOD7 reporter lines, we characterized the expression pattern of these genes during embryo development, revealing only a partial overlap with the stem cell population. In addition, we showed that the expression of a stem cell reporter was not modified in dpa4-2 sod7-2 double mutant embryos compared to the wild type. Together, these observations suggest that DPA4 and SOD7 are not required for stem cell specification during embryo shoot apical meristem initiation. This work stresses the difference in the regulatory network leading to meristem formation during the embryonic and post-embryonic phases.

De novo stem cell establishment in meristems requires repression of organ boundary cell fate.

Stem cells play important roles in animal and plant biology, as they sustain morphogenesis and tissue replenishment following aging or injury. In plants, stem cells are embedded in multicellular structures called meristems. The formation of new meristems is essential for the plastic expansion of the highly branched shoot and root systems. In particular, axillary meristems (AMs) that produce lateral shoots arise from the division of boundary domain cells at the leaf base. The CUP-SHAPED COTYLEDON (CUC) genes are major determinants of the boundary domain and are required for AM initiation. However, how AMs get structured and how stem cells become established de novo remain elusive. Here, we show that two NGATHA-LIKE (NGAL) transcription factors, DEVELOPMENT-RELATED PcG TARGET IN THE APEX4 (DPA4)/NGAL3 and SUPPRESSOR OF DA1-1 7 (SOD7)/NGAL2, redundantly repress CUC expression in initiating AMs of Arabidopsis thaliana. Ectopic boundary fate leads to abnormal growth and organization of the AM and prevents de novo stem cell establishment. Floral meristems of the dpa4 sod7 double mutant show a similar delay in de novo stem cell establishment. Altogether, while boundary fate is required for the initiation of AMs, our work reveals how it is later repressed to allow proper meristem establishment and de novo stem cell niche formation.

Meristem Initiation and de novo Stem Cell Formation.

Plant aerial development relies on meristem activity which ensures main body plant axis development during plant life. While the shoot apical meristem (SAM) formed in the embryo only contributes to the main stem, the branched structure observed in many plants relies on axillary meristems (AMs) formed post-embryonically. These AMs initiate from a few cells of the leaf axil that retain meristematic characteristics, increase in number, and finally organize into a structure similar to the SAM. In this review, we will discuss recent findings on de novo establishment of a stem cell population and its regulatory niche, a key step essential for the indeterminate fate of AMs. We stress that de novo stem cell formation is a progressive process, which starts with a transient regulatory network promoting stem cell formation and that is different from the one acting in functional meristems. This transient stage can be called premeristems and we discuss whether this concept can be extended to the formation of meristems other than AMs.

Beneath a hairy problem: Phylogeny, morphology, and biogeography circumscribe the new Miconia supersection Discolores (Melastomataceae: Miconieae).

Miconia is among the largest plant genera in the Neotropics and a taxonomically complex lineage. Indeed, molecular phylogenetic data shows that none of its traditionally accepted sections are monophyletic, preventing taxonomic advances within the genus. Miconia is the largest plant genus in the Brazilian Atlantic Forest, including three main lineages, the Leandra s.s. clade (ca. 215 spp.), the Miconia sect. Chaenanthera (24 spp.), and the Miconia discolor clade (estimated 77 spp.). Out of these lineages, the Miconia discolor clade is the only currently lacking phylogenetic data, complicating its taxonomy. In this study, we reconstruct the phylogeny of the Miconia discolor clade, using three plastid (atpF-H, psbK-I, and psaI-accD) and two nuclear (ETS and ITS) markers. We sampled 60 out of the 77 species of the group, representing 78% of its diversity. Taxa were selected considering their distribution, morphology, and previous phylogenetic knowledge. We used the newly reconstructed phylogeny to better understand phylogenetic relationships among Atlantic Forest species and morphologically similar taxa, and to propose a new infrageneric classification for the Miconia discolor clade: the Miconia supersection Discolores. We further studied the evolution of seven morphological characters using a Maximum Likelihood approach, and estimated the ancestral range distribution of various lineages in order to understand the biogeographic history of this clade. We found that dichasial inflorescences represent the ancestral condition within Miconia, subsequently giving rise to scorpioid and glomerulate inflorescences in the studied group. We describe Miconia supersect. Discolores, originated in the Amazon region, which is recognized by a dense layer of branched tricomes covering young branches and non-dichasial inflorescences, including three main lineages: (i) Miconia sect. Albicantes, characterized by persistent bracts and arachnoid indument on the abaxial surface of leaves, mainly distributed in the Amazon basin; (ii) Miconia sect. Discolores, characterized by caducous calyx lobes and glomerulate inflorescences, centered in the Atlantic Forest; and (iii) Miconia sect. Multispicatae, characterized by leaves not completely covered with indument, and capitate stigma, mainly distributed in the Atlantic Forest. All three sections and the supersection originated in the Neogene, between the Late Miocene and the Early Pliocene.

Maxillary Third Molar Tooth Accidentally Displaced in Buccal Space: Report of Two Cases.

The extraction of retained and completely impacted third molars is one of the most common surgical procedures performed by dental practitioners with low rates of complications. The accidental displacement during the surgeries of the maxillary third molar into adjacent anatomical spaces is one of the most critical problems that can arise. The most common sites of migration during surgical interventions are the infratemporal fossa, the pterygomandibular space, the maxillary sinus, the buccal space, and the lateral pharyngeal space. In this paper, two cases in which a maxillary third molar accidentally was displaced into the buccal space are presented, the retrieval of the tooth via intra-oral approach is explained, and the anatomical spaces implications are discussed.

A higher-level nuclear phylogenomic study of the carrot family (Apiaceae).

PREMISE: The carrot family (Apiaceae) comprises 466 genera, which include many well-known crops (e.g., aniseed, caraway, carrots, celery, coriander, cumin, dill, fennel, parsley, and parsnips). Higher-level phylogenetic relationships among subfamilies, tribes, and other major clades of Apiaceae are not fully resolved. This study aims to address this important knowledge gap. METHODS: Target sequence capture with the universal Angiosperms353 probe set was used to examine phylogenetic relationships in 234 genera of Apiaceae, representing all four currently recognized subfamilies (Apioideae, Azorelloideae, Mackinlayoideae, and Saniculoideae). Recovered nuclear genes were analyzed using both multispecies coalescent and concatenation approaches. RESULTS: We recovered hundreds of nuclear genes even from old and poor-quality herbarium specimens. Of particular note, we placed with strong support three incertae sedis genera (Platysace, Klotzchia, and Hermas); all three occupy isolated positions, with Platysace resolved as sister to all remaining Apiaceae. We placed nine genera (Apodicarpum, Bonannia, Grafia, Haplosciadium, Microsciadium, Physotrichia, Ptychotis, Tricholaser, Xatardia) that have never previously been included in any molecular phylogenetic study. CONCLUSIONS: We provide support for the maintenance of the four existing subfamilies of Apiaceae, while recognizing that Hermas, Klotzschia, and the Platysace clade may each need to be accommodated in additional subfamilies (pending improved sampling). The placement of the currently apioid genus Phlyctidocarpa can be accommodated by the expansion of subfamily Saniculoideae, although adequate morphological synapomorphies for this grouping are yet to be defined. This is the first phylogenetic study of the Apiaceae using high-throughput sequencing methods and represents an unprecedented evolutionary framework for the group.

Vesicular monoamine transporter-1 (VMAT-1) mRNA and immunoreactive proteins in mouse brain.

OBJECTIVE: Vesicular monoamine transporter 1 (VMAT-1) mRNA and protein were examined (1) to determine whether adult mouse brain expresses full-length VMAT-1 mRNA that can be translated to functional transporter protein and (2) to compare immunoreactive VMAT-1 proteins in brain and adrenal. METHODS: VMAT-1 mRNA was detected in mouse brain with RT-PCR. The cDNA was sequenced, cloned into an expression vector, transfected into COS-1 cells, and cell protein was assayed for VMAT-1 activity. Immunoreactive proteins were examined on western blots probed with four different antibodies to VMAT-1. RESULTS: Sequencing confirmed identity of the entire coding sequences of VMAT-1 cDNA from mouse medulla oblongata/pons and adrenal to a Gen-Bank reference sequence. Transfection of the brain cDNA into COS-1 cells resulted in transporter activity that was blocked by the VMAT inhibitor reserpine and a proton ionophore, but not by tetrabenazine, which has a high affinity for VMAT-2. Antibodies to either the C- or N- terminus of VMAT-1 detected two proteins (73 and 55 kD) in transfected COS-1 cells. The C-terminal antibodies detected both proteins in extracts of mouse medulla/pons, cortex, hypothalamus, and cerebellum but only the 73 kD protein and higher molecular weight immunoreactive proteins in mouse adrenal and rat PC12 cells, which are positive controls for rodent VMAT-1. CONCLUSIONS: These findings demonstrate that a functional VMAT-1 mRNA coding sequence is expressed in mouse brain and suggest processing of VMAT-1 protein differs in mouse adrenal and brain.

The demise of subfamily Hydrocotyloideae (Apiaceae) and the re-alignment of its genera across the entire order Apiales.

As circumscribed by Drude, the umbellifer subfamily Hydrocotyloideae posed a major hindrance to resolving the phylogeny of order Apiales. Previous studies have suggested its polyphyly, but have not had sufficient sampling to address the issue fully. To put an end to the out-dated concept of Hydrocotyloideae, we investigated the placement of 40 of the 42 genera once placed in the subfamily, using extensive taxon sampling across the entire order. Molecular phylogenies were constructed using plastid sequences of the rpl16 intron and the trnD-trnT regions and revealed at least six hydrocotyloid lineages dispersed across both families Apiaceae and Araliaceae. The most speciose of these clades corresponds to the recently erected subfamily Azorelloideae. Another lineage includes genera grouped in Mackinlayoideae, where relationships are well resolved. Platysace appears paraphyletic with respect to Homalosciadium, and their placement is well supported as a basal lineage in Apiaceae. The type genus, Hydrocotyle, belongs to a supported clade in Araliaceae. The placements of Hermas as sister to a clade consisting of Apiaceae subfamilies Apioideae and Saniculoideae, and of Choritaenia as sister to Lichtensteinia in a clade with affinities to both Apioideae and Saniculoideae, calls into question the circumscriptions of the two subfamilies. Finally, plastid data suggest that many former hydrocotyloid genera are non-monophyletic (e.g., Azorella, Schizeilema, and Eremocharis) and are in dire need of additional phylogenetic and taxonomic studies.