Transcriptomics of Early Human Development

Ph.DDOCTOR OF PHILOSOPH

BMP signalling regulates the pre-implantation development of extra-embryonic cell lineages in the mouse embryo.

Pre-implantation development requires the specification and organization of embryonic and extra-embryonic lineages. The separation of these lineages takes place when asymmetric divisions generate inside and outside cells that differ in polarity, position and fate. Here we assess the global transcriptional identities of these precursor cells to gain insight into the molecular mechanisms regulating lineage segregation. Unexpectedly, this reveals that complementary components of the bone morphogenetic protein (BMP) signalling pathway are already differentially expressed after the first wave of asymmetric divisions. We investigate the role of BMP signalling by expressing dominant negative forms of Smad4 and Bmpr2, by downregulating the pathway using RNA interference against BMP ligands and by applying three different BMP inhibitors at distinct stages. This reveals that BMP signalling regulates the correct development of both extra-embryonic lineages, primitive endoderm and trophectoderm, but not the embryonic lineage, before implantation. Together, these findings indicate multiple roles of BMP signalling in the early mouse embryo.This work was supported by a Wellcome Trust programme grant to MZG and an Agency for Science, Technology and Research (A*Star) core research budget to PR.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncomms666

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells

Expression of transporter-associated genes found in corneal endothelium, cultured corneal endothelial cells and corneal stroma.

<p>Transporters expressed in CECs that are at least 2 times higher in young CEC-DM than in stroma, and less than 2 fold difference in expression between young and old CEC-DM. Genes are ranked in descending order of RPM values of young CEC-DM. RPM values are rounded off to the nearest integer, and fold change values are rounded off to the nearest decimal place.</p

The human corneal tissue, the isolated endothelium and the cultured corneal endothelial cells.

<p>A. Corneal stroma with an intact Descemet’s membrane (DM), arrowed (left) and a corneal stromal without the DM layer (right). B. Peeled CEC-DM complex in a typical DM-roll, with the endothelium on the outside. C. Confluent culture of human CECs at the second passage. Scale bars: 100 µm.</p

Expression of SMADs in corneal endothelium, cultured corneal endothelial cells and corneal stroma.

<p>Genes are ranked in descending order of RPM values of young CEC-DM. RPM values are rounded off to the nearest integer.</p

Donor information.

<p>A total of 27 donor corneas consisting of 13 single donor corneas and 7 paired donor corneas were used in this study. Donor age ranged from 19–76.</p><p>A: pooled CEC-DM from five young donors;</p><p>B: pooled CEC-DM from five old donors;</p><p>C: Isolated for cell culture;</p><p>D: young CEC-DME control;</p><p>E: old CEC-DME control.</p><p>OS: oculus sinister.</p><p>OD: oculus dexter.</p

QPCR validation of gene expression in CEC-DM andcorneal stroma (keratocytes) from 3 individual donors, corneal stromal fibroblast cultures from 2 individual donors and CEC culture.

<p>CEC: CEC-DM; stroma: corneal stroma (keratocytes); SF: stromal fibroblast. Student’s t-tests (two-tailed assuming non-equal variance): One asterisk indicates p<0.05; two asterisks indicate p<0.01.</p