Adriaan Dorresteijn

Arthropod-like Expression Patterns of engrailed and wingless in the Annelid Platynereis dumerilii Suggest a Role in Segment Formation

The origin of animal segmentation, the periodic repetition of anatomical structures along the anteroposterior axis, is a long-standing issue that has been recently revived by comparative developmental genetics. In particular, a similar extensive morphological segmentation (or metamerism) is commonly recognized in annelids and arthropods. Mostly based on this supposedly homologous segmentation, these phyla have been united for a long time into the clade Articulata. However, recent phylogenetic analysis dismissed the Articulata and thus challenged the segmentation homology hypothesis. Here, we report the expression patterns of genes orthologous to the arthropod segmentation genes engrailed and wingless in the annelid Platynereis dumerilii. In Platynereis, engrailed and wingless are expressed in continuous ectodermal stripes on either side of the segmental boundary before, during, and after its formation; this expression pattern suggests that these genes are involved in segment formation. The striking similarities of engrailed and wingless expressions in Platynereis and arthropods may be due to evolutionary convergence or common heritage. In agreement with similarities in segment ontogeny and morphological organization in arthropods and annelids, we interpret our results as molecular evidence of a segmented ancestor of protostomes.

Intervertebral disc regeneration: Influence of growth factors on differentiation of human mesenchymal stem cells (hMSC)

INTRODUCTION One common cause of disability in modern society is low back pain. The main reason for this pain is the degeneration of the intervertebral disc (IVD), particularly of the nucleus pulposus (NP). For the early degeneration stage, a cell-based therapy could constitute a minimally invasive method of treatment. Therefore, adequate cells are needed. As the usage of NP cells is limited because of their insufficient amount or vitality, a promising alternative is the application of human mesenchymal stem cells (hMSCs) OBJECTIVE To investigate the potential of various growth factors to induce the differentiation of hMSCs into NP cells and thereby to obtain an alternative cell source for the treatment of IVD degeneration. METHODS hMSC-TERT were cultivated three-dimensionally in a hydrogel for 21 days to form NP cells. Cell survival and proliferation were determined using SybrGreen/propidium iodide double staining and the WST-test. To investigate the ability of several growth factors to differentiate hMSCs into NP cells, fluorescence immunostaining of NP-specific marker proteins (e.g., chondroadherin (CHAD) and the recently discovered cytokeratin 19) were performed. RESULTS Following the procedure described above, cells are able to maintain their viability and proliferation capacity throughout the cultivation time. By using a previously established immunofluorescence protocol, we were able to indicate the ability of three different growth factors for differentiating hMSCs into NP-like cells. CONCLUSION The expression of several marker proteins in all differentiation experiments indicates the ability of IGF-1, FGF-2 and PDGF-BB to differentiate hMSCs into NP-like cells apart from the usually applied TGF-beta3. Furthermore, our findings preclude the application of Cytokeratin 19 as a specific marker protein for NP cells. Further experiments have to be done to find real specific NP marker proteins to indisputably verify the differentiation of hMSCs into NP cells. If so, application of these three growth factors would possibly be an option to obtain sufficient NP cells for minimally invasive IVD regeneration.

Activation of Hox genes during caudal regeneration of the polychaete annelid Platynereis dumerilii

The capability of regenerating posterior segments and pygidial structures is ancestral for annelids and has been lost only a few times within this phylum. As one of the three major segmented taxa, annelids enable us to monitor reconstruction of lost tissues and organs. During regeneration, regional identities have to be imprinted onto the newly formed segments. In this study, we show spatial and temporal localization of expression of nine Hox genes during caudal regeneration of the polychaete annelid Platynereis dumerilii. Hox genes are homeodomain genes encoding transcriptional regulators of axial patterning in bilaterian animals during development. We demonstrate that five Platynereis Hox genes belonging to paralog groups (PG) 1, 4, 5, 6, and 9–14 are expressed in domains of the regenerating nervous system consistent with providing positional information along the anteroposterior axis of the regenerate. We report that expression in regenerating neuromeres is limited to varying subsets of perikarya, called gangliosomes. Four of nine genes analyzed do not appear to be involved in axial patterning. Two genes, Pdu-Hox2 and Pdu-Hox3, are predominantly expressed in the growth zone region. For some Hox genes expression in newly formed coelomic epithelia can be observed. Platynereis Hox genes do not exhibit temporal or spatial colinearity. Although there are some similarities to previously reported expression patterns during larval and postlarval development in Nereididae (Kulakova et al. 2007), expression patterns observed during caudal regeneration also show unique patterns.

Maternal inheritance of twist and analysis of MAPK activation in embryos of the polychaete annelid Platynereis dumerilii.

In this study, we aimed to identify molecular mechanisms involved in the specification of the 4d (mesentoblast) lineage in Platynereis dumerilii. We employ RT-PCR and in situ hybridization against the Platynereis dumerilii twist homolog (Pdu-twist) to reveal mesodermal specification within this lineage. We show that Pdu-twist mRNA is already maternally distributed. After fertilization, ooplasmatic segregation leads to relocation of Pdu-twist transcripts into the somatoblast (2d) lineage and 4d, indicating that the maternal component of Pdu-twist might be an important prerequisite for further mesoderm specification but does not represent a defining characteristic of the mesentoblast. However, after the primordial germ cells have separated from the 4d lineage, zygotic transcription of Pdu-twist is exclusively observed in the myogenic progenitors, suggesting that mesodermal specification occurs after the 4d stage. Previous studies on spiral cleaving embryos revealed a spatio-temporal correlation between the 4d lineage and the activity of an embryonic organizer that is capable to induce the developmental fates of certain micromeres. This has raised the question if specification of the 4d lineage could be connected to the organizer activity. Therefore, we aimed to reveal the existence of such a proposed conserved organizer in Platynereis employing antibody staining against dpERK. In contrast to former observations in other spiralian embryos, activation of MAPK signaling during 2d and 4d formation cannot be detected which questions the existence of a conserved connection between organizer function and specification of the 4d lineage. However, our experiments unveil robust MAPK activation in the prospective nephroblasts as well as in the macromeres and some micromeres at the blastopore in gastrulating embryos. Inhibition of MAPK activation leads to larvae with a shortened body axis, defects in trunk muscle spreading and improper nervous system condensation, indicating a critical function for MAPK signaling for the reorganization of embryonic tissues during the gastrulation process.

Identification and characterization of a twist ortholog in the polychaete annelid Platynereis dumerilii reveals mesodermal expression of Pdu-twist

The basic helix-loop-helix transcription factor twist plays a key role during mesoderm development in Bilateria. In this study, we identified a twist ortholog in the polychaete annelid Platynereis dumerilii and analyze its expression during larval development, postlarval growth up to the adult stage, and caudal regeneration after amputation of posterior segments. At late larval stages, Pdu-twist is expressed in the mesodermal anlagen and in developing muscles. During adulthood and caudal regeneration, Pdu-twist is expressed in the posterior growth zone, in mesodermal cells within the newly forming segments and budding parapodia. Our results indicate that Pdu-twist is involved in mesoderm formation during larval development, posterior growth, and caudal regeneration.

Cholinergic urethral brush cells are widespread throughout placental mammals

We previously identified a population of cholinergic epithelial cells in murine, human and rat urethrae that exhibits a structural marker of brush cells (villin) and expresses components of the canonical taste transduction signaling cascade (α-gustducin, phospholipase Cβ2 (PLCβ2), transient receptor potential cation channel melanostatin 5 (TRPM5)). These cells serve as sentinels, monitoring the chemical composition of the luminal content for potentially hazardous compounds such as bacteria, and initiate protective reflexes counteracting further ingression. In order to elucidate cross-species conservation of the urethral chemosensory pathway we investigated the occurrence and molecular make-up of urethral brush cells in placental mammals. We screened 11 additional species, at least one in each of the five mammalian taxonomic units primates, carnivora, perissodactyla, artiodactyla and rodentia, for immunohistochemical labeling of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), villin, and taste cascade components (α-gustducin, PLCβ2, TRPM5). Corresponding to findings in previously investigated species, urethral epithelial cells with brush cell shape were immunolabeled in all 11 mammals. In 8 species, immunoreactivities against all marker proteins and ChAT were observed, and double-labeling immunofluorescence confirmed the cholinergic nature of villin-positive and chemosensory (TRPM5-positive) cells. In cat and horse, these cells were not labeled by the ChAT antiserum used in this study, and unspecific reactions of the secondary antiserum precluded conclusions about ChAT-expression in the bovine epithelium. These data indicate that urethral brush cells are widespread throughout the mammalian kingdom and evolved not later than about 64.5millionyears ago.

Diversity of Culex torrentium Martini, 1925 — a potential vector of arboviruses and filaria in Europe

Culex torrentium is one of the most common mosquito species in Germany. Due to its sympatric occurrence as well as its similar morphological and ecological characteristics, it has often been confused with another common species, Culex pipiens. Both species are known to be potential vectors for different arboviruses (not only in Germany) with C. torrentium being a possible vector for Sindbis or Ockelbo virus. In our study, we analyzed the genetic variability in a 658 bp fragment of the cytochrome c oxidase subunit I gene (coxI) of C. torrentium, from nine localities in the Frankfurt/Rhine-Main Metropolitan Region. The results of our genetic survey indicate a higher genetic diversity in this gene region for C. torrentium than for the morphologically similar C. pipiens. Our findings may explain the difficulties in the past to find morphological characteristics that apply to all populations of C. torrentium, when attempting to separate them clearly from C. pipiens, by any other criteria than male genitalia. Being ornithophilic, possible hybrids between C. torrentium and the humanophilic C. pipiens biotype molestus, could potentially serve as important vectors for zoonotic diseases. Therefore, we recommend that greater emphasis is placed on the ecological characteristics, population structure, and the taxonomy of this often neglected species, in the future.

Formation of body appendages during caudal regeneration in Platynereis dumerilii : adaptation of conserved molecular toolsets

BackgroundPlatynereis and other polychaete annelids with homonomous segmentation are regarded to closely resemble ancestral forms of bilateria. The head region comprises the prostomium, the peristomium, a variable number of cephalized body segments and several appendages, like cirri, antennae and palps. The trunk of such polychaetes shows numerous, nearly identical segments. Each segment bears a parapodium with species-specific morphology on either side. The posterior end of the trunk features a segment proliferation zone and a terminal pygidium with the anus and anal cirri. The removal of a substantial part of the posterior trunk is by no means lethal. Cells at the site of injury dedifferentiate and proliferate forming a blastema to regenerate both the pygidium and the proliferation zone. The pygidium forms new anal cirri, and the proliferation zone generates new segments at a rapid pace. The formation of body appendages like the cirri and the segmental parapodia can thus be studied in the caudal regenerate of Platynereis within only a few days.ResultsThe development of body appendages in Platynereis is regulated by a network of genes common to polychaetes but also shared by distant taxa. We isolated DNA sequences from P. dumerilii of five genes known to be involved in appendage formation within other groups: Meis/homothorax, Pbx1/extradenticle, Dlx/Distal-less, decapentaplegic and specificprotein1/buttonhead. Analyses of expression patterns during caudal regeneration by in situ hybridization reveal striking similarities related to expression in arthropods and vertebrates. All genes exhibit transient expression during differentiation and growth of segments. As was shown previously in other phyla Pdu-Meis/hth and Pdu-Pbx1/exd are co-expressed, although the expression is not limited to the proximal part of the parapodia. Pdu-Dll is prominent in parapodia but upregulated in the anal cirri. No direct dependence concerning Pdu-Dll and Pdu-sp/btd expression is observed in Platynereis. Pdu-dpp shows an expression pattern not comparable to its expression in other taxa.ConclusionsThe expression patterns observed suggest conserved roles of these genes during appendage formation across different clades, but the underlying mechanisms utilizing this toolset might not be identical. Some genes show broad expression along the proximodistal axis indicating a possible role in proximodistal patterning of body appendages. Other genes exhibit expression patterns limited to specific parts and tissues of the growing parapodia, thus presumably being involved in formation of taxon-specific morphological differences.

Expression of katanin p80 in human spermatogenesis

OBJECTIVE To define the stage-by-stage expression of KATNB1 during human spermatogenesis. DESIGN Gene expression analysis, histologic and immunohistochemical evaluation. SETTING University research laboratories and andrological clinic. PATIENT(S) Eighty human testicular biopsy samples: 43 showing normal spermatogenesis, 9 with maturation arrest at level of spermatocytes, 8 with maturation arrest at level of spermatogonia, and 20 with a Sertoli cell only syndrome. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Evaluation of katanin p80 expression in normal as well as impaired spermatogenesis on mRNA (RT-PCR, RT-qPCR, and in situ hybridization) and protein level (immunohistochemistry/immunofluorescence). RESULT(S) KATNB1 messenger RNA is exclusively expressed in germ cells, and quantitatively reduced in maturation arrests at the level of spermatogonia. The KATNB1 protein was detected in type B spermatogonia entering meiosis and in the Golgi complex of pachytene spermatocytes. Immediately before the first meiotic division, it is colocalized with the cleaving centriole. It was also detected in early round spermatids in the dictyosome. CONCLUSION(S) The expression and localization of KATNB1 support a role in spindle formation. The localization of KATNB1 in early round spermatids suggests an involvement in the formation of microtubule-based structures during spermiogenesis (manchette and flagellum). These data are consistent with the demonstrated role of KATNB1 in mouse meiosis, nuclear shaping, and flagellum formation of sperm and suggest the strong conservation of function even between distantly related species.