Proliferation activity in the polyps of Cassiopea xamachana: where the planuloid buds grow

Polyps of the Cassiopeidae family possess a unique type of asexual reproduction by producing free-swimming buds — planuloids. The process of planuloid development and transformation to polyp has been described earlier, however, the source of tissue formation is still poorly studied. Using the method of EdU incorporation we have analyzed DNA synthesis activity during planuloid formation and growth in Cassiopea xamachana. We revealed the active proliferation zone at the early stage of bud formation. This zone continued to function during planuloid growth, providing the formation of polyp structures, and preserved in polyp calyx after metamorphosis. Its proliferation activity varied at different growth stages, whereas the localization remained relatively the same.

ABA represses TOR and root meristem activity through nuclear exit of the SnRK1 kinase

The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses like drought, partly by modulating plant growth and development. However, the underlying mechanisms are poorly understood. Here, we show that cell proliferation in the Arabidopsis thaliana root meristem is controlled by the interplay between three kinases, SNF1-RELATED KINASE 2 (SnRK2), the main driver of ABA signaling, the SnRK1 energy sensor, and the growth-promoting TARGET OF RAPAMYCIN (TOR) kinase. Under favorable conditions, the SnRK1α1 catalytic subunit is enriched in the nuclei of root meristematic cells and this is accompanied by normal cell proliferation and meristem size. Depletion of SnRK2s in a snrk2.2 snrk2.3 double mutant causes constitutive cytoplasmic localization of SnRK1α1 and a reduction in meristem size, suggesting that, under non-stress conditions, SnRK2s enable growth by retaining SnRK1α1 in the nucleus. In response to elevated ABA levels, SnRK1α1 translocates to the cytoplasm and this is accompanied by inhibition of TOR, decreased cell proliferation and meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK1α1 relocalization to the cytoplasm and the inhibition of TOR. Fusion of SnRK1α1 to an SV40 nuclear localization signal leads to defective TOR repression in response to ABA, demonstrating that SnRK1α1 nuclear exit is a premise for this repression. Finally, the SnRK2-dependent changes in SnRK1α1 subcellular localization are specific to the proliferation zone of the meristem, underscoring the relevance of this mechanism for growth regulation.

PREVENTATIVE EFFECTS OF PROBIOTIC (MIACLOST) ON EXPERIMENTALLY INDUCED HYPOCALCEMIC RICKETS IN BROILER CHICKS.

A trial was conducted to study the effects of probiotic (Miaclost) supplement on experimentally induced hypocalcemic rickets in broiler chicks, a total of 180 one-day-old broiler chicks (Ross 308) were randomly divided into three equal groups 60 chicks per group with 3 replicates (20 birds /replicate) the dietary treatments consisted of a normal ration for G1, calcium-deficient ration 5% for G2 and calcium-deficient ration with addition of probiotics in drinking water for G3.Initial signs of rickets have been observed at 35-day of age in G2.while, in G1 and G3 no clinical signs observed, the gross lesions appeared enlargement of parathyroid gland, costochondral junction and increase in the width of growth plate of tibial bone of G2 whereas no gross lesions recorded in G1 and G3, the histopathological examination of parathyroid gland in G2 there were a focal parathyroid hyperplasia and increasing in numbers of syncytial cells and normal in G1 and G3, no intestinal histopathological changes in G1 and G2 and increase in height and width of the intestinal villi in probiotic group G3. Marked increase in the thickness of proliferation zone within growth plate of tibia bone in G2 and normal thickness in G1 and G3. the serum biochemical analysis of calcium of G2 recorded significantly low level in G2 and high level in G3 comparatively with G1, finally the serum alkaline phosphatase values were high significantly in G2 and normal in G3, it is concluded that probiotic (MiaClost) can be used as prophylaxis to prevent hypocalcemic rickets in broiler chicks

The annotation and analysis of complex 3D plant organs using 3DCoordX

A fundamental question in biology concerns how molecular and cellular processes become integrated during morphogenesis. In plants, characterization of 3D digital representations of organs at single-cell resolution represents a promising approach to addressing this problem. A major challenge is to provide organ-centric spatial context to cells of an organ. We developed several general rules for the annotation of cell position and embodied them in 3DCoordX, a user-interactive computer toolbox implemented in the open-source software MorphoGraphX. It enables rapid spatial annotation of cells even in highly curved biological shapes. With the help of 3DCoordX we obtained new insight by analyzing cellular growth patterns in organs of several species. For example, the data indicated the presence of a basal cell proliferation zone in the ovule primordium of Arabidopsis thaliana. Proof-of-concept analyses suggested a preferential increase in cell length associated with neck elongation in the archegonium of Marchantia polymorpha and variations in cell volume linked to central morphogenetic features of a trap of the carnivorous plant Utricularia gibba. Our work demonstrates the broad applicability of the developed strategies as they provide organ-centric spatial context to cellular features in plant organs of diverse shape complexity.

CTNI-43. CXCL12 INHIBITION IN MGMT UNMETHYLATED GLIOBLASTOMA – RESULTS OF AN EARLY PROOF-OF-CONCEPT ASSESSMENT IN THE MULTICENTRIC PHASE I/II GLORIA TRIAL (NCT04121455)

BACKGROUND Preclinical studies showed that CXCL12-mediated influx of highly angiogenic monocytes/macrophages is a key driver of tumor re-vascularization and re-growth after radiotherapy (RT) of glioblastoma (GBM). We report findings from a phase I/II proof-of concept (PoC) study on CXCL12 inhibition during and after RT of GBM. METHODS Patients ≥18years with incompletely or unresected GBM without MGMT promoter hypermethylation and ECOG≤2 were eligible to participate. Patients received continuous (24/7) i.v. infusions of 200mg/week (n=3), 400mg/week (n=3) or 600mg/week (n=3) of the CXCL12 inhibitor olaptesed pegol (OLA) for 26 weeks during and after normo- or hypofractionated RT (60Gy/40.05Gy). The primary endpoint was safety as per the incidence of treatment-related adverse events. The study was accompanied by PoC-research including multiparametric MRI biomarkers (relative cerebral blood volume, rCBV; fractional tumor burden with high perfusion, FTBhigh; apparent diffusion coefficient, ADC) and of multiplexed immunofluorescence imaging (CODEX®) of reference and patient samples. Initial results of these analyses are reported for the first six patients enrolled. RESULTS Five of six (83%) patients assessed with advanced MRI showed response under OLA in rCBV/FTBhigh and ADC. Maximum reduction in perfusion (rCBV) from baseline was 55%, maximum reduction of FTBhigh was 55% and maximum increase in ADC was 77%. Furthermore, five of six (83%) patients analyzed showed reduction of enhancing tissue volumes in at least one scan under OLA therapy. In both one patient and two reference samples CXCL12 co-localized with endothelial cells of the microvascular proliferation zone. In a paired sample (before/during OLA) of one patient, endothelial cells stained positive for CXCL12 before but not during treatment and almost all GBM cells were negative in Ki67 staining in the sample obtained under OLA therapy. CONCLUSIONS Advanced MRI and multiplexed immunofluorescence suggest efficacy of combined radiotherapy and CXCL12 inhibition in unmethylated GBM. Funded by NOXXON Pharma AG; ClinicalTrials.gov number, NCT04121455.

Spatiotemporal control of cell cycle acceleration during axolotl spinal cord regeneration

Axolotls are uniquely able to resolve spinal cord injuries, but little is known about the mechanisms underlying spinal cord regeneration. We previously found that tail amputation leads to reactivation of a developmental-like program in spinal cord ependymal cells (Rodrigo Albors et al., 2015), characterized by a high-proliferation zone emerging 4 days post-amputation (Rost et al., 2016). What underlies this spatiotemporal pattern of cell proliferation, however, remained unknown. Here, we use modelling, tightly linked to experimental data, to demonstrate that this regenerative response is consistent with a signal that recruits ependymal cells during ~85 hours after amputation within ~830mm of the injury. We adapted FUCCI technology to axolotls (AxFUCCI) to visualize cell cycles in vivo. AxFUCCI axolotls confirmed the predicted appearance time and size of the injury-induced recruitment zone and revealed cell cycle synchrony between ependymal cells. Our modeling and imaging move us closer to understanding bona fide spinal cord regeneration.

Modeling the spatiotemporal control of cell cycle acceleration during axolotl spinal cord regeneration

Axolotls are uniquely able to resolve spinal cord injuries, but little is known about the mechanisms underlying spinal cord regeneration. We found that tail amputation leads to reactivation of a developmental-like program in spinal cord ependymal cells (Rodrigo Albors et al., 2015). We also identified a high-proliferation zone and demonstrated that cell cycle acceleration is the major driver of regenerative growth (Rost et al., 2016). What underlies this spatiotemporal pattern of cell proliferation, however, remained unknown. Here, using a modelling approach supported by experimental data, we show that the proliferative response in the regenerating spinal cord is consistent with a signal that starts recruiting cells 24 hours after amputation and spreads about one millimeter from the injury. Finally, our model predicts that the observed shorter S phase can explain spinal cord outgrowth in the first four days of regeneration but after, G1 shortening is also necessary to explain outgrowth dynamics.

Thm2 interacts with paralog, Thm1, and sensitizes to Hedgehog signaling in postnatal skeletogenesis

Ciliopathies are genetic syndromes that link osteochondrodysplasias to dysfunction of primary cilia. Primary cilia extend from the surface of bone and cartilage cells, to receive extracellular cues and mediate signaling pathways. Mutations in several genes that encode components of the intraflagellar transport-A ciliary protein complex have been identified in skeletal ciliopathies, including THM1. Here, we report a role for genetic interaction between Thm1 and its paralog, Thm2, in skeletogenesis. THM2 localizes to the ciliary axoneme, but unlike its paralog, Thm2 deficiency does not affect ciliogenesis and Thm2-null mice survive into adulthood. Since paralogs often have redundant functions, we crossed a Thm1 null (aln) allele into the Thm2 colony. After 5 generations of backcrossing the colony onto a C57BL6/J background, we observed that by postnatal day 14, Thm2-/-; Thm1aln/+ mice are smaller than control littermates. Thm2-/-; Thm1aln/+ mice exhibit shortened long bones, narrow ribcage, shortened cranium and mandibular defects. Mutant mice also show aberrant architecture of the tibial growth plate, with an expanded proliferation zone and diminished hypertrophic zone, indicating impaired chondrocyte differentiation. Using microcomputed tomography, Thm2-/-; Thm1aln/+ tibia were revealed to have reduced cortical and trabecular bone mineral density. Deletion of one allele of Gli2, a major transcriptional activator of the Hedgehog (Hh) pathway, exacerbated the small phenotype of Thm2-/-; Thm1aln/+ mice and caused small stature in Thm2-null mice. Together, these data reveal Thm2 as a novel locus that sensitizes to Hh signaling in skeletal development. Further, Thm2-/-; Thm1aln/+ mice present a new postnatal ciliopathy model of osteochondrodysplasia.

Cellular Composition and Differentiation Signaling in Chicken Small Intestinal Epithelium

The small intestine plays an important role for animals to digest and absorb nutrients. The epithelial lining of the intestine develops from the embryonic endoderm of the embryo. The mature intestinal epithelium is composed of different types of functional epithelial cells that are derived from stem cells, which are located in the crypts. Chickens have been widely used as an animal model for researching vertebrate embryonic development. However, little is known about the molecular basis of development and differentiation within the chicken small intestinal epithelium. This review introduces processes of development and growth in the chicken gut, and compares the cellular characteristics and signaling pathways between chicken and mammals, including Notch and Wnt signaling that control the differentiation in the small intestinal epithelium. There is evidence that the chicken intestinal epithelium has a distinct cellular architecture and proliferation zone compared to mammals. The establishment of an in vitro cell culture model for chickens will provide a novel tool to explore molecular regulation of the chicken intestinal development and differentiation.

Mitotic granule cell precursors undergo highly dynamic morphological transitions throughout the external germinal layer of the chick cerebellum

The developing cerebellum of amniotes is characterised by a unique, transient, secondary proliferation zone: the external germinal layer (EGL). The EGL is comprised solely of granule cell precursors, whose progeny migrate inwardly to form the internal granule cell layer. While a range of cell morphologies in the EGL has long been known, how they reflect the cells’ differentiation status has previously only been inferred. Observations have suggested a deterministic maturation from outer to inner EGL that we wished to test experimentally. To do this, we electroporated granule cell precursors in chick with plasmids encoding fluorescent proteins and probed labelled cells with markers of both proliferation (phosphohistone H3) and differentiation (Axonin1/TAG1 and NeuroD1). We show that granule cell precursors can display a range of complex forms throughout the EGL while mitotically active. Overexpression of full length NeuroD1 within granule cell precursors does not abolish proliferation, but biases granule cells towards precocious differentiation, alters their migration path and results in a smaller and less foliated cerebellum. Our results show that granule cells show a greater flexibility in differentiation than previously assumed. We speculate that this allows the EGL to regulate its proliferative activity in response to overall patterns of cerebellar growth.