QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 26 January 2005
doi: 10.1242/dev.01657

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ng, E. S. Articles by Elefanty, A. G. Search for Related Content PubMed PubMed Citation Articles by Ng, E. S. Articles by Elefanty, A. G. Social Bookmarking                         What's this?

The primitive streak gene Mixl1 is required for efficient haematopoiesis and BMP4-induced ventral mesoderm patterning in differentiating ES cells

Elizabeth S. Ng^1,*, Lisa Azzola¹, Koula Sourris¹, Lorraine Robb², Edouard G. Stanley¹ and Andrew G. Elefanty^1,

¹ Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, VIC, 3800, Australia
² The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3050, Australia

View larger version (107K): [in a new window]   Fig. 1. Analysis of gene expression in differentiating ES cells showing the progressive loss of stem cell markers and the sequential acquisition of transcripts indicative of specific stages of embryonic development. RT-PCR on RNA isolated from undifferentiated ES cells (day 0) and EBs harvested at daily intervals (days 1-6) was performed using primers specific for the genes indicated. -ve, no template.

View larger version (122K): [in a new window]   Fig. 2. GFP fluorescence of day 4 Mixl1GFP/w EBs. Low-magnification bright-field (A) and epifluorescence (B) images showing that most EBs contain GFP-positive cells. (C) Confocal image showing that GFP-positive cells are present both on the surface and distributed throughout the EB. (D) High magnification of the EB in C, showing GFP-positive cells located on the surface of the EB have a flattened morphology reminiscent of endoderm (arrowhead).

View larger version (16K): [in a new window]   Fig. 3. Time course of GFP expression of in differentiating Mixl1GFP/w heterozygous and MixlGFP/GFP null ES cells as determined by flow cytometry. Both lines initiated GFP expression at day 2.5 and peak expression was seen at day 4. The frequency of GFP-positive cells diminished more quickly in Mixl1 heterozygous cells than in those deficient in Mixl1. The percentage of GFP-positive cells is shown in the lower right of each plot.

View larger version (36K): [in a new window]   Fig. 4. Expression of E-cadherin and GFP in differentiating Mixl1 wild-type (Mixl1w/w), heterozygote (Mixl1GFP/w) and null (Mixl1GFP/GFP) ES cells. (A) Flow cytometric analysis of day 3 EBs showed that more than 98% of cells were E-cadherin positive and more than a third of these also expressed GFP from the Mixl1 locus. (B) Time course of E-cadherin and GFP expression in differentiating Mixl1GFP/W ES cells. Initially, E-cadherin-positive (E-cad+) stem cells differentiate into a transient double-positive population (E-cad+ GFP+) corresponding to cells at the primitive streak stage of development. After day 3, Mixl1-expressing (GFP+) nascent mesoderm downregulated E-cadherin.

View larger version (40K): [in a new window]   Fig. 5. Expression of FLK1 and GFP in differentiating Mixl1 wild-type (w/w), heterozygote (GFP/w) and null (GFP/GFP) ES cells. (A) Time course of FLK1 and GFP expression showing that the onset of FLK1 expression is delayed and that the frequency of FLK1-positive cells was reduced in Mixl1-deficient ES cells. (B,C) Summary of data (n=3-6) demonstrating a lower percentage of FLK1+ cells (B) and a reduction in the ratio of double-positive GFP+FLK1+ to GFP+ cells (C) in day 4 EBs derived from two independent Mixl1-deficient ES cell lines compared with their heterozygote counterparts. The Mixl1-null lines M916 and M3C5 were derived from Mixl1-heterozygous ES cell lines M147 and M114, respectively. Mixl1w/w is the parental ES cell line. The error bars represent 1 s.d. and the P values indicated were derived using a two-tailed t-test.

View larger version (39K): [in a new window]   Fig. 6. Comparison of haematopoietic development from differentiating Mixl1 (w/w), heterozygote (GFP/w) and null (GFP/GFP) ES cells. (A) Flow cytometric analysis of day 6 EBs showing Mixl1-deficient cells failed to efficiently generate TER119-positive erythroid precursors despite their ability to form significant numbers of CD34-positive cells. (B) Summary of methylcellulose culture experiments (n=3) indicating that Mixl1-deficient day 4 EBs contained significantly fewer blast colony-forming cells (BL-CFCs) and primitive erythroid precursors (EryP). Data were derived from experiments performed with two independent Mixl1-deficient ES cell lines and their wild-type and heterozygote counterparts. The Mixl1-null lines M916 and M3C5 were derived from Mixl1-heterozygous ES cell lines M147 and M114 respectively. Mixl1w/w is the parental ES cell line. The error bars represent 1 s.d. and the P values indicated were derived using a two-tailed t-test. GF, growth factors (VEGF, SCF, IL3, EPO). No EryP colonies were seen in the absence of growth factors (-GF).

View larger version (51K): [in a new window]   Fig. 7. (A-D) Blast colonies develop into haematopoietic and endothelial cells. Blast colonies (A), typically composed of 50-200 round cells emanating from a dense core, proliferated in liquid culture (B) supplemented by VEGF, SCF, IL3 and EPO to give haematopoietic cells of myeloid and erythroid lineages (C, May-Grunwald-Giemsa stained cytocentrifuge preparation) and adherent endothelial cells (D) visualized by antibodies to CD31. (E,F) Frequency of blast colony-forming cells (Blast-CFCs) in GFP+FLK1+, GFP+FLK1-, GFP-FLK1+, GFP-FLK1- populations of cells isolated from differentiating Mixl1GFP/w ES cells. (E) In day 3 EBs, Blast-CFCs were distributed throughout the four populations, reflecting the dynamic nature of the differentiation process at this time (see Discussion). (F) By day 4, the GFP+FLK1+ fraction contained the highest frequency and the majority of Blast-CFCs. UN, unsorted. Error bars represent 1 s.d. (n=3 for day 3; n=4 for day 4).

View larger version (59K): [in a new window]   Fig. 8. Experiments examining the developmental relationship between GFP+FLK1+, GFP+FLK1-, GFP-FLK1+ and GFP-FLK1- populations isolated from differentiating Mixl1GFP/w ES cells at day 2.8 (A), day 3 (B) and day 4 (C). At time points shown, a sample was removed from each culture and the EBs disaggregated. Cells were sorted into populations based on expression of GFP and FLK1 and recultured overnight. The following day, the sorted and unsorted populations, as well as EBs, from the continuing differentiation cultures were analysed for expression of GFP and FLK1.

View larger version (27K): [in a new window]   Fig. 9. Differentiation of Mixl1 heterozygote (GFP/w) and null (GFP/GFP) ES cells in serum-free (SF) media supplemented with BMP4 or activin A. In SF media, activin A (A) was a weaker inducer of GFP expression in Mixl1GFP/w cells than BMP4 (B). In comparison with serum-induced differentiation (C), the magnitude of the response to SF+BMP4 media was less and GFP induction was delayed. There was no GFP expression in SF media alone. (D) BMP4 increased the percentage of viable cells isolated from day 4 and day 5 cultures of ES cells differentiated in SF media (P<0.01). Error bars represent 1 s.d. and P values were calculated using a two-tailed t-test (n=7). (E) The previously observed defect (see Fig. 5A) in the ability of Mixl1-null ES cells to generate FLK1+ cells at day 4 was exacerbated in SF+BMP4 differentiation cultures. At d7, differences in frequency of FLK1-positive cells between the two cell lines had diminished and both cell lines were able to generate CD34-positive cells. (F) Summary of methylcellulose culture experiments showing that Mixl1-deficient ES cells generated fewer haematopoietic CFCs in SF+BMP4 differentiation cultures than their heterozygous counterparts.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter