Technology and provenience of the oldest pottery in the northern Pannonian Basin indicates its affiliation to hunter-gatherers

Consensus holds that pottery technology came to Central Europe from the Northern Balkans with independent pottery traditions existing concurrently in Eastern Europe. An unusual grass-tempered pottery dating back to around 5800 cal BC found in lake sediments at Santovka, Slovakia, predated the earliest known Neolithic pottery in the region (~ 5500 cal BC), suggesting unexplored narratives of pottery introduction. Analyses of the pottery’s technology, origin, and grass temper shedding light on ceramic traditions' spread can unveil mobility patterns and community lifestyles. Our findings indicate a non-local provenance, low temperature firing, Festugc sp. grass temper and unique rectangular or cylindrical vessel shapes which align with Eastern European hunter-gatherer practices. Moreover, the pottery style and technology have no analogies in the contemporary Danubian pottery traditions and have more similarities to those of the Eastern traditions. The pottery's raw materials likely originated from distant areas, indicating extensive territorial access for its creators. Our findings imply late Mesolithic hunter-gatherers as the probable artisans and with implications for the site's significance in the late Mesolithic landscape

Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here, we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage.

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale

Serotonin limits generation of chromaffin cells during adrenal organ development

Adrenal glands are the major organs releasing catecholamines and regulating our stress response. The mechanisms balancing generation of adrenergic chromaffin cells and protecting against neuroblastoma tumors are still enigmatic. Here we revealed that serotonin (5HT) controls the numbers of chromaffin cells by acting upon their immediate progenitor "bridge" cells via 5-hydroxytryptamine receptor 3A (HTR3A), and the aggressive HTR3Ahigh human neuroblastoma cell lines reduce proliferation in response to HTR3A-specific agonists. In embryos (in vivo), the physiological increase of 5HT caused a prolongation of the cell cycle in "bridge" progenitors leading to a smaller chromaffin population and changing the balance of hormones and behavioral patterns in adulthood. These behavioral effects and smaller adrenals were mirrored in the progeny of pregnant female mice subjected to experimental stress, suggesting a maternal-fetal link that controls developmental adaptations. Finally, these results corresponded to a size-distribution of adrenals found in wild rodents with different coping strategies

CTCF/SMARCA5 are recruited to <i>SPI1</i> locus in myeloid cells and upon AZA treatment in AML.

<p><b>A:</b> Sequence conservation of human <i>SPI1</i> locus (VISTA) generated by aligning with murine DNA. Regulatory regions and positions of ChIP amplicons are numbered in respect to human <i>SPI1</i> TSS. <b>B:</b> ChIP of CTCF and SMARCA5 in mixed myeloid cells. <b>C:</b> ChIP of CTCF and <b>D:</b> SMARCA5 in OCI-M2 without (OCI-M2) or with AZA (OCI-M2 AZA) treatment. Y-axis: ChIP enrichment. X-axis: amplicons (distance relative to <i>SPI1</i> TSS). URE, Upstream Regulatory Element of <i>SPI1</i> gene; ENH, enhancer; ELE, element. Error bars: the standard errors (SE). Asterisks: p-values (t-test, 0.05–0.005).</p

Model of epigenetic regulation of <i>SPI1</i> gene by CTCF and SMARCA5.

<p>CTCF binding site (−14.4 kb) becomes occupied by CTCF and SMARCA5 upon AZA-mediated DNA demethylation in AML blasts. Cohesin member’s recruitment partially overlaps with CTCF/SMARCA5 and display spreading over −11.0 kb and URE of <i>SPI1</i>. More diffuse occupancy of both CTCF and SMARCA5 at <i>SPI1</i> gene that was observed in mixed myeloid cells was not achieved in AML blasts, however the AZA treatment partially restored CTCF/SMARCA5 occupancy. Nevertheless, SMARCA5/CTCF is unable to potentiate <i>SPI1</i> derepression in AML blasts and instead, inhibits <i>SPI1</i> transcription possibly through the enhancer-blocking effect at the −14.4 Enhancer.</p

DNA methylation of CTCF binding site in <i>SPI1</i> locus.

<p><b>A:</b> DNA sequence of the CTCF binding site at −14.4 kb Enhancer region within the <i>SPI1</i> locus (CGs are numbered on the top). <b>B:</b> % of DNA unmethylation identified by sequencing of bisulphite-treated DNA isolated from CD34+ cells of AML/MDS patients (N = 3, information in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087448#pone.0087448.s007" target="_blank">Table S1</a>) and control CD34+ cell donors (N = 1) and mixed myeloid cells (N = 1) was performed at the region −14.4 kb of <i>SPI1</i> locus. The primer sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087448#pone.0087448.s008" target="_blank">Table S2</a>. <b>C:</b> % of DNA unmethylation, data in CD34+ cells of MDS patient without AZA therapy (N = 1) and MDS patient treated by AZA (N = 1). <b>D:</b> % of DNA unmethylation in untreated OCI-M2 and AZA-treated OCI-M2. Y-axis: % of unmethylated CpGs; x-axis: number of CpG; error bars indicate standard errors.</p

Ctcf and Smarca5 interact in AML cells.

<p><b>A</b> Ctcf occupancy at ICR. ChIP of Ctcf-siRNA-treated MEL cells (Ctcf siRNA, black bars) or non-specific siRNA (Ctrl siRNA, white) at 72 hrs. Y-axis: specific IP DNA fragment enrichment over control IP (standard error, SE). X-axis: ICR amplicons (relative to H19-TSS). Asterisks: p-values (t test, 0.05–0.005). B Smarca5 occupancy at ICR. MEL-shSmarca5 treated 48 hrs with doxycycline (+DOX, black bars) or untreated (-DOX, white). C Ctcf occupancy is decreased at the ICR upon Smarca5 knockdown. Occupancy of Ctcf (lysates from 1B) determined by ChIP. D Smarca5 at ICR upon Ctcf knockdown. Occupancy of Smarca5 (lysates from 1A) by ChIP. E Knock-down of Smarca5 and Ctcf. Protein lysates from samples 1A and 1B were analyzed by Immunoblotting. Migration of Ctcf, Smarca5, and β-actin bands are indicated. Level of downregulation (bellow blots) was determined by densitometry. F Co-IP of Smarca5 and Ctcf in MEL cells. Antibodies for IP and detection are indicated; asterisk indicates nonspecific signal.</p

Smarca5 regulates Ctcf target genes.

<p><b>A:</b> H19 and Igf2 mRNA expression upon Ctcf knockdown (Ctcf siRNA) or Ctrl siRNA. <b>B:</b> Smarca5 knockdown (+DOX) compared to untreatment (-DOX). RT-PCR analyses (A&B) were done at 72hrs. Y-axis: specific mRNAs relative to Hprt1 levels. <b>C:</b> mRNA levels of PU.1 and Cebpa at 96 hrs (4 days) upon Smarca5 knockdown. Y-axis: specific mRNA relative to average of Hprt1 and Gapdh was normalized on negative control (non-specific siRNA). Error bars: the standard errors (SE). Asterisks: p-values (t-test, 0.05–0.005). D: PU.1 and β-actin expression determined by Immunoblotting at 144 hrs (6 days) upon Smarca5 knockdown. Level of downregulation (bellow blots) was determined by densitometry.</p