Human spinal cord in vitro differentiation pace is initially maintained in heterologous embryonic environments

Species-specific differentiation pace in vitro indicates that some aspects of neural differentiation are governed by cell intrinsic properties. Here we describe a novel in vitro human neural-rosette assay that recapitulates dorsal spinal cord differentiation but proceeds more rapidly than in the human embryo, suggesting that it lacks endogenous signalling dynamics. To test whether in vitro conditions represent an intrinsic differentiation pace, human iPSC-derived neural rosettes were challenged by grafting into the faster differentiating chicken embryonic neural tube iso-chronically, or hetero-chronically into older embryos. In both contexts in vitro differentiation pace was initially unchanged, while long-term analysis revealed iso-chronic slowed and hetero-chronic conditions promoted human neural differentiation. Moreover, hetero-chronic conditions did not alter the human neural differentiation programme, which progressed to neurogenesis, while the host embryo advanced into gliogenesis. This study demonstrates that intrinsic properties limit human differentiation pace, and that timely extrinsic signals are required for progression through an intrinsic human neural differentiation programme

FGF Signalling Regulates Chromatin Organisation during Neural Differentiation via Mechanisms that Can Be Uncoupled from Transcription

Changes in higher order chromatin organisation have been linked to transcriptional regulation; however, little is known about how such organisation alters during embryonic development or how it is regulated by extrinsic signals. Here we analyse changes in chromatin organisation as neural differentiation progresses, exploiting the clear spatial separation of the temporal events of differentiation along the elongating body axis of the mouse embryo. Combining fluorescence in situ hybridisation with super-resolution structured illumination microscopy, we show that chromatin around key differentiation gene loci Pax6 and Irx3 undergoes both decompaction and displacement towards the nuclear centre coincident with transcriptional onset. Conversely, down-regulation of Fgf8 as neural differentiation commences correlates with a more peripheral nuclear position of this locus. During normal neural differentiation, fibroblast growth factor (FGF) signalling is repressed by retinoic acid, and this vitamin A derivative is further required for transcription of neural genes. We show here that exposure to retinoic acid or inhibition of FGF signalling promotes precocious decompaction and central nuclear positioning of differentiation gene loci. Using the Raldh2 mutant as a model for retinoid deficiency, we further find that such changes in higher order chromatin organisation are dependent on retinoid signalling. In this retinoid deficient condition, FGF signalling persists ectopically in the elongating body, and importantly, we find that inhibiting FGF receptor (FGFR) signalling in Raldh2−/− embryos does not rescue differentiation gene transcription, but does elicit both chromatin decompaction and nuclear position change. These findings demonstrate that regulation of higher order chromatin organisation during differentiation in the embryo can be uncoupled from the machinery that promotes transcription and, for the first time, identify FGF as an extrinsic signal that can direct chromatin compaction and nuclear organisation of gene loci

Pregnancy-related factors and the risk of breast carcinoma in situ and invasive breast cancer among postmenopausal women in the California Teachers Study cohort

Abstract Introduction Although pregnancy-related factors such as nulliparity and late age at first full-term pregnancy are well-established risk factors for invasive breast cancer, the roles of these factors in the natural history of breast cancer development remain unclear. Methods Among 52,464 postmenopausal women participating in the California Teachers Study (CTS), 624 were diagnosed with breast carcinoma in situ (CIS) and 2,828 with invasive breast cancer between 1995 and 2007. Multivariable Cox proportional hazards regression methods were used to estimate relative risks associated with parity, age at first full-term pregnancy, breastfeeding, nausea or vomiting during pregnancy, and preeclampsia. Results Compared with never-pregnant women, an increasing number of full-term pregnancies was associated with greater risk reduction for both breast CIS and invasive breast cancer (both P trend < 0.01). Women having four or more full-term pregnancies had a 31% lower breast CIS risk (RR = 0.69, 95% CI = 0.51 to 0.93) and 18% lower invasive breast cancer risk (RR = 0.82, 95% CI = 0.72 to 0.94). Parous women whose first full-term pregnancy occurred at age 35 years or later had a 118% greater risk for breast CIS (RR = 2.18, 95% CI = 1.36 to 3.49) and 27% greater risk for invasive breast cancer (RR = 1.27, 95% CI = 0.99 to 1.65) than those whose first full-term pregnancy occurred before age 21 years. Furthermore, parity was negatively associated with the risk of estrogen receptor-positive (ER+) or ER+/progesterone receptor-positive (PR+) while age at first full-term pregnancy was positively associated with the risk of ER+ or ER+/PR+ invasive breast cancer. Neither of these factors was statistically significantly associated with the risk of ER-negative (ER-) or ER-/PR- invasive breast cancer, tests for heterogeneity between subtypes did not reach statistical significance. No clear associations were detected for other pregnancy-related factors. Conclusions These results provide some epidemiologic evidence that parity and age at first full-term pregnancy are involved in the development of breast cancer among postmenopausal women. The role of these factors in risk of in situ versus invasive, and hormone receptor-positive versus -negative breast cancer merits further exploration

Regulation of cell death in the tailbud by retinoid signalling.

<p>(A–E′, C″, E″) Detection of cell death using Tunel (Apoptag) in chick tailbud at key stages (A) HH16, (B, B′) HH22, (C–C′″) HH24, and (D–E′″) HH26/27. (C″) Some apoptotic cells are detected in mesoderm progenitors (yellow dashed line) and the CNH (red dashed line), distal notochord (black dashed line) at HH24. (C′″) section as in (C″) DAPI stained nuclei confirm tissue organisation. (E″) Increase in apoptosis in the terminal structures by HH27, distal notochord (black dashed line). (E′″) section as in E″ DAPI stained nuclei. (F–H″) Cell death detection using NucView 488 caspase-3 substrate (green) and actin cytoskeleton counter-labelling with Phalloidin (red) in HH20 explanted tailbuds cultured for 24 h in (G–G′) control DMSO only conditions or in (H–H″) the presence of RAR/RXR antagonists. White dashed line, neural tube outline; red dashed line, CNH. CNH region is not well defined in RAR/RXR treated tails. This may reflect ectopic/increased Bra in these conditions. Scale bar, 100 µm.</p

Fate mapping the late tailbud reveals continued cell ingression.

<p>Schematic of DiI labelling experiment for distinct cell populations in HH20/21 tailbud (NT, neural tube; CNH, chordo-neural-hinge; MP mesoderm progenitors) (A); DiI labelling of NT before incubation (B), fixed and analysed in sections (<i>n</i> = 5/5 confined to NT) (B′); after incubation, DiI is restricted to Sox2 positive NT (B′″); DiI labelling of CNH before incubation (C), fixed and analysed in sections (<i>n</i> = 8/11 confined to CNH) (C′); after incubation, DiI is found in NT, MPs, and their derivatives, including Sox2 positive cells in the MP domain (C′″); DiI labelling of MPs before incubation (D), fixed and analysed in sections (<i>n</i> = 6/6 confined to MPs) (D′); after incubation, DiI is restricted to MPs, presomitic mesoderm, somites, and lateral mesoderm and rarely labels Sox2 positive cells in the MP domain (D′″). White arrow, Sox2-expressing/DiI labelled cells in MP domain; white dashed line, region of Sox2 positive cells in the position of the MP domain. (E) Summary of cell movements following DiI labelling (orange) in NT, CNH, and MP domain.</p

Summary data for fate map of the late tailbud.

<p>Summary of contributions of cell groups DiI labelled in HH20 neural tube (NT), chordoneural hinge (CNH), or mesoderm progenitors (MP) to tissues in and derived from the tailbud observed at HH24. <i>n</i>, number of embryos labelled at HH20 for each tissue.</p