The Targeted Deletion of Genes Responsible for Expression of the Mth60 Fimbriae Leads to Loss of Cell-Cell Connections in Methanothermobacter thermautotrophicus ΔH.

This study is a continuation by the Environmental Biotechnology Group of the University of Tübingen in memoriam to Reinhard Wirth, who initiated the work on Mth60 fimbriae at the University of Regensburg. Growth in biofilms or biofilm-like structures is the prevailing lifestyle for most microbes in nature. The first crucial step to initiate biofilms is the adherence of microbes to biotic and abiotic surfaces. Therefore, it is crucial to elucidate the initial step of biofilm formation, which is generally established through cell-surface structures (i.e., cell appendages), such as fimbriae or pili, that adhere to biotic and abiotic surfaces. The Mth60 fimbriae of Methanothermobacter thermautotrophicus ΔH are one of only a few known archaeal cell appendages that do not assemble via the type IV pili assembly mechanism. Here, we report the constitutive expression of Mth60 fimbria-encoding genes from a shuttle-vector construct and the deletion of the Mth60 fimbria-encoding genes from the genomic DNA of M. thermautotrophicus ΔH. For this, we expanded our system for genetic modification of M. thermautotrophicus ΔH using an allelic-exchange method. While overexpression of the respective genes increased the number of Mth60 fimbriae, deletion of the Mth60 fimbria-encoding genes led to a loss of Mth60 fimbriae in planktonic cells of M. thermautotrophicus ΔH compared to the wild-type strain. This, either increased or decreased, number of Mth60 fimbriae correlated with a significant increase or decrease of biotic cell-cell connections in the respective M. thermautotrophicus ΔH strains compared to the wild-type strain. IMPORTANCE Methanothermobacter spp. have been studied for the biochemistry of hydrogenotrophic methanogenesis for many years. However, a detailed investigation of certain aspects, such as regulatory processes, was impossible due to the lack of genetic tools. Here, we amend our genetic toolbox for M. thermautotrophicus ΔH with an allelic exchange method. We report the deletion of genes that encode the Mth60 fimbriae. Our findings provide the first genetic evidence of whether the expression of these genes underlies regulation and reveal a role of the Mth60 fimbriae in the formation of cell-cell connections of M. thermautotrophicus ΔH.

Ciliomotor circuitry underlying whole-body coordination of ciliary activity in the Platynereis larva.

Ciliated surfaces harbouring synchronously beating cilia can generate fluid flow or drive locomotion. In ciliary swimmers, ciliary beating, arrests, and changes in beat frequency are often coordinated across extended or discontinuous surfaces. To understand how such coordination is achieved, we studied the ciliated larvae of Platynereis dumerilii, a marine annelid. Platynereis larvae have segmental multiciliated cells that regularly display spontaneous coordinated ciliary arrests. We used whole-body connectomics, activity imaging, transgenesis, and neuron ablation to characterize the ciliomotor circuitry. We identified cholinergic, serotonergic, and catecholaminergic ciliomotor neurons. The synchronous rhythmic activation of cholinergic cells drives the coordinated arrests of all cilia. The serotonergic cells are active when cilia are beating. Serotonin inhibits the cholinergic rhythm, and increases ciliary beat frequency. Based on their connectivity and alternating activity, the catecholaminergic cells may generate the rhythm. The ciliomotor circuitry thus constitutes a stop-and-go pacemaker system for the whole-body coordination of ciliary locomotion.

Object-based representation and analysis of light and electron microscopic volume data using Blender.

BACKGROUND: Rapid improvements in light and electron microscopy imaging techniques and the development of 3D anatomical atlases necessitate new approaches for the visualization and analysis of image data. Pixel-based representations of raw light microscopy data suffer from limitations in the number of channels that can be visualized simultaneously. Complex electron microscopic reconstructions from large tissue volumes are also challenging to visualize and analyze. RESULTS: Here we exploit the advanced visualization capabilities and flexibility of the open-source platform Blender to visualize and analyze anatomical atlases. We use light-microscopy-based gene expression atlases and electron microscopy connectome volume data from larval stages of the marine annelid Platynereis dumerilii. We build object-based larval gene expression atlases in Blender and develop tools for annotation and coexpression analysis. We also represent and analyze connectome data including neuronal reconstructions and underlying synaptic connectivity. CONCLUSIONS: We demonstrate the power and flexibility of Blender for visualizing and exploring complex anatomical atlases. The resources we have developed for Platynereis will facilitate data sharing and the standardization of anatomical atlases for this species. The flexibility of Blender, particularly its embedded Python application programming interface, means that our methods can be easily extended to other organisms.

Whole-body gene expression pattern registration in Platynereis larvae.

BACKGROUND: Digital anatomical atlases are increasingly used in order to depict different gene expression patterns and neuronal morphologies within a standardized reference template. In evo-devo, a discipline in which the comparison of gene expression patterns is a widely used approach, such standardized anatomical atlases would allow a more rigorous assessment of the conservation of and changes in gene expression patterns during micro- and macroevolutionary time scales. Due to its small size and invariant early development, the annelid Platynereis dumerilii is particularly well suited for such studies. Recently a reference template with registered gene expression patterns has been generated for the anterior part (episphere) of the Platynereis trochophore larva and used for the detailed study of neuronal development. RESULTS: Here we introduce and evaluate a method for whole-body gene expression pattern registration for Platynereis trochophore and nectochaete larvae based on whole-mount in situ hybridization, confocal microscopy, and image registration. We achieved high-resolution whole-body scanning using the mounting medium 2,2'-thiodiethanol (TDE), which allows the matching of the refractive index of the sample to that of glass and immersion oil thereby reducing spherical aberration and improving depth penetration. This approach allowed us to scan entire whole-mount larvae stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) in situ hybridization and counterstained fluorescently with an acetylated-tubulin antibody and the nuclear stain 4'6-diamidino-2-phenylindole (DAPI). Due to the submicron isotropic voxel size whole-mount larvae could be scanned in any orientation. Based on the whole-body scans, we generated four different reference templates by the iterative registration and averaging of 40 individual image stacks using either the acetylated-tubulin or the nuclear-stain signal for each developmental stage. We then registered to these templates the expression patterns of cell-type specific genes. In order to evaluate the gene expression pattern registration, we analyzed the absolute deviation of cell-center positions. Both the acetylated-tubulin- and the nuclear-stain-based templates allowed near-cellular-resolution gene expression registration. Nuclear-stain-based templates often performed significantly better than acetylated-tubulin-based templates. We provide detailed guidelines and scripts for the use and further expansion of the Platynereis gene expression atlas. CONCLUSIONS: We established whole-body reference templates for the generation of gene expression atlases for Platynereis trochophore and nectochaete larvae. We anticipate that nuclear-staining-based image registration will be applicable for whole-body alignment of the embryonic and larval stages of other organisms in a similar size range.