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First published online 11 March 2009
doi: 10.1242/dev.030619

Development 136, 1295-1303 (2009)
Published by The Company of Biologists 2009
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Steel factor controls primordial germ cell survival and motility from the time of their specification in the allantois, and provides a continuous niche throughout their migration

Ying Gu1,2, Chris Runyan1, Amanda Shoemaker1, Azim Surani3 and Chris Wylie1,*

1 Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
2 Molecular and Developmental Biology Graduate Program, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
3 Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.


Figure 1
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  		Fig. 1. Expression of Steel factor. (A) Whole-mount Steel factor (red) staining of E7.5 Stella-GFP embryos; PGCs in the allantois (boxed) are green. Residual Stella expression in other regions of the embryo at this early stage is also green. (B) Higher magnification views of whole-mount Steel factor staining (red) of E7.5 embryos. PGCs express the Stella-GFP transgene, as well as Steel factor at this stage (arrows in B). Steel factor-expressing cells surround the PGCs. (C) Same image as in B, without the green channel, to allow visualization of Steel factor staining in the PGCs (arrows). (D) Whole-mount staining of an E8.5 Stella-GFP embryo. PGCs are clustered in the hindgut diverticulum, immediately adjacent to Steel factor-expressing cells of the visceral endoderm. (E) Transverse section through an E8.5 hindgut diverticulum (shown by the dashed line in D). The ventral aspect of the hindgut is strongly stained for Steel factor, and PGCs are confined to this ventral region of the gut. (F) Differential interference contrast image of the region labelled `Dorsal' and `G' in E, to show outline of the whole hindgut. (G) Transverse section through an E9.0 hindgut. Both the ventral and dorsal hindgut is stained for Steel factor; PGCs have moved to the dorsal region of the gut. (H) Transverse section through an E10.0 embryo. Steel staining is lost from the hindgut, but is enriched in the dorsal mesentery and body wall. (I) Immunostaining of a section from an E10.5 Steel-/- embryo demonstrating the antibody specificity (lack of red signal). (J) Whole-mount immunostaining of Steel factor in an E7.5 embryo fixed in 2% TCA, which increases the antibody reaction with Steel factor. (K) RT-PCR analysis of membrane-bound and soluble Steel factor. cDNA was prepared from dissected E7.5 allantoides, whole embryo and E10.5 genital ridges. AL, allantois; Ao, aorta; G, hindgut; VE, visceral endoderm. Scale bars: 100 µm in A,D,E,G-I; 50 µm in B,C,J.

 

Figure 2
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  		Fig. 2. Time-lapse frames of E7.25 and E7.5 Stella-GFP embryos. (A) Three frames at t=0, t=78 minutes and t=156 minutes of a movie started at E7.25. At t=0, PGCs, detected by the expression of Stella (green), are visible in the allantois (AL). Residual expression of Stella is also seen elsewhere in the embryo. PGCs start to spread downwards towards the epiblast. Arrow indicates approximate boundary between the allantois and the proximal epiblast. (B) Three frames at t=0, t=280 minutes and t=560 minutes of a movie started at E7.5. PGCs move from the allantois into the posterior epiblast of the embryo during the movie time period. Arrow indicates approximate boundary between the allantois and the proximal epiblast. The PGC population expands into proximal epiblast. Scale bars: 100 µm.

 

Figure 3
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  		Fig. 3. Effects of Steel factor on PGC numbers at E7.5. (A) PGC numbers at E7.5 were significantly reduced in Steel-/- embryos compared with in Steel+/+ and Steel+/- littermates (red bars). PGC numbers were significantly rescued in Steel-/- embryos by the loss of a single allele of Bax (blue bars). n indicates the number of embryos used for quantitation. * and **, P<0.05. Bars represent the mean±s.e.m. (B) Examples of embryos of each genotype used to generate the PGC counts. Scale bars: 100 µm.

 

Figure 4
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  		Fig. 4. Effects of Steel factor on PGC migration at E7.5 (A,B) Frames at t=0 (A) and t=413 minutes (B) from movies of E7.5 Stella-GFP embryos with different Steel genotypes. PGCs are green. (C) The tracks of all PGCs that remained in the plane of the confocal image throughout the movies. The boundary between the extraembryonic tissues (EEM; in the posterior region of the allantois) and the posterior end of the embryo (PEM) is marked by a white line. (D) The overall directions of movement of individual PGCs, made by placing the starting point of each PGC track onto the same point. Scale bars in A-C: 100 µm. (E) The maximum velocity, average velocity, and displacement of E7.5 PGCs with different Steel genotypes. Both the velocities and the displacements are significantly decreased in the absence of Steel factor. n, number of PGCs used for quantitation. Units on the y-axis vary based upon the parameter, and are indicated below the bar charts. (F) Windrose diagram shows that the directionality of PGC migration is altered in Steel-/- embryos, but not randomized. The accompanying diagram shows the trajectory (blue arrow) in the vectorial diagram that allows PGCs to leave the allantois and enter the posterior embryo and the hindgut endoderm (yellow). (G) The percentage of PGCs that enter the posterior of the embryo within the time frame of the movies (413 minutes), starting at E7.5, is significantly reduced in Steel-null embryos. (H) The percentage of PGCs that form clusters is markedly higher in the absence of Steel factor. n, the number of embryos used for quantitation for G and H. *P<0.01; bars represent the mean±s.e.m.

 

Figure 5
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  		Fig. 5. PGC clumping in Steel mutant embryos is not due to upregulation of E-cadherin. (Top) E-cadherin (red) and PGCs (green) in Steel+/+ E7.5 embryos, individually and combined. (Bottom) The same staining but for Steel-/- embryos. No difference in expression of E-cadherin was observed in different Steel genotypes. Scale bars: 50 µm.

 

Figure 6
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  		Fig. 6. Loss of an allele of Bax does not rescue PGC motility. Velocities and displacements of PGCs in movies from Steel+/+, Steel+/- and Steel-/- embryos with and without loss of one Bax allele were assayed as in Fig. 4. No statistically significant differences in PGC motility were seen in Steel-/- embryos when one allele of Bax was lost. n, number of PGCs used for quantitation. Units on the y-axis vary based upon parameter, and are indicated below the bar charts.

 

Figure 7
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  		Fig. 7. Effects of Ack2 blocking antibody on PGC motility at E7.5. (A) The maximum velocity, average velocity, and displacement of E7.5 PGCs were significantly decreased by 10 µg/ml Ack2 treatment for 6 hours. n, the number of PGCs used for quantitation. Units on the y-axis vary based upon parameter, and are indicated below the bar charts. (B) The percentage of PGCs leaving the allantois and entering the posterior embryo within the timescale of movies (6 hours) started at E7.5 was significantly lower following treatment with 10 µg/ml Ack2. (C) PGCs clump more in the presence of 10 µg/ml Ack2 at E7.5. n, indicates the number of embryos used for quantitation for B,C. *P<0.01; bars represent the mean±s.e.m.

 

Figure 8
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  		Fig. 8. Effects of Steel factor on PGC migration at E9.0. (A,B) Movie frames of E9.0 Oct4{Delta}PE:GFP embryos with different Steel genotypes are shown at (A) t=0 and (B) t=420 minutes. PGCs are green. Scale bars: 100 µm. (C) The direction of movement of individual PGCs during the movie period. (D) Both the velocities and the displacements of PGCs are significantly decreased in Steel-/- embryos. n, the number of PGCs used for quantitation. Units on y-axis vary based upon parameter, and are indicated below charts. *P<0.01; bars represent the mean±s.e.m. (F) Windrose diagram shows the directionality of PGC migration. As the blue arrow shows in the accompanying diagram, all PGCs migrated in a net dorsal and slightly rostral direction in the hindgut (yellow), regardless of Steel gene dosage.

 

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© The Company of Biologists Ltd 2009