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First published online April 10, 2009
doi: 10.1242/10.1242/dev.032425

Development 136, 1539-1548 (2009)
Published by The Company of Biologists 2009
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The mechanism and pattern of yolk consumption provide insight into embryonic nutrition in Xenopus

Paul Jorgensen1, Judith A. J. Steen2,3, Hanno Steen3,4 and Marc W. Kirschner1,*

1 Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
2 F. M. Kirby Neurobiology Center, Children's Hospital Boston, and Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
3 Proteomics Center at Children's Hospital Boston, Boston, MA 02115, USA.
4 Department of Pathology, Harvard Medical School and Children's Hospital Boston, Boston, MA 02115, USA.


Figure 1
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  		Fig. 1. Seryp is a novel component of the YP superficial layer. (A,B) YPs were purified from eggs [low and high density (p) YPs] and tailbud stage embryos (stage 26) and the contents subfractionated into YP crystal and YP supernatant (sup) fractions. Purified YPs and the two derived fractions were analyzed by western blotting (A), Coomassie staining (A) and phosphoprotein staining (B). Seryp species were greatly enriched in the YP supernatant fraction, whereas LV1 and LV2 were nearly exclusively in YP crystals. The phosphoprotein stain identified PHO, PVT1 and PVT2, based on approximate size similarity to the major yolk phosphoproteins described by Wiley and Wallace (Wiley and Wallace, 1981Go). PVT1 also stained with Coomassie and appeared to migrate anomalously when greatly enriched in the YP supernatant. (C,D) Anti-Vtg-Alexa488 ({alpha}-Vtg) and anti-Seryp-Alexa568 ({alpha}-Seryp) antibodies colocalize to the rim of YPs in unfertilized egg sections (C). Anti-Vtg-Alexa488 but not anti-Seryp-Alexa568 bound to the interior of YP crystals that cracked during sectioning (D). Scale bars: 10 µm in C; 5 µm in D. (E) When subjected to isopycnic centrifugation, gently lysed single eggs exhibited lower density, low Seryp YPs and higher density, high Seryp YPs, as well as Seryp that did not sediment. Fractions that contained {rho}=1.13 and {rho}=1.15 density markers are indicated. Smearing on the western blots was likely to be due to Percoll in the gel wells.

 

Figure 2
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  		Fig. 2. Seryp is taken up by oocytes from plasma, probably through interaction with Vitellogenin. (A) Seryp species have different electrophoretic mobilities. Female X. laevis plasma contains a 54 kDa Seryp species, the precursor of the egg forms p50 and p48. Early oocytes contain a ~60 kDa protein that cross-reacts strongly with anti-Seryp antibody. Asterisk indicates cross-reaction with highly abundant serum Vitellogenin. Gel loading was approximately normalized by oocyte volume. (B) Reciprocal co-immunoprecipitation of Vitellogenin and Seryp from female X. laevis plasma. Antibody cross-linked beads were prepared from IgG purified from pre-immune and immunized anti-Vtg rabbit serum, as well as from pre-immune and immunized anti-Seryp rabbit serum. Western blots were sequentially probed with anti-Vtg and anti-Seryp antibodies.

 

Figure 3
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  		Fig. 3. Seryp is proteolyzed prior to Vitellogenin. (A) By bulk measurement (western blotting), Vitellogenin derivatives (LV1, LV2) and Seryp species (p50, p48, p30) decline at later stages of development. Extracts are ordered by time post-fertilization (pf) and Nieuwkoop-Faber stage (NF). Load: 1/500 (top blot) or 1/50 (bottom blot) of single embryos. (B) Quantitative dot blotting of extracts prepared from single embryos confirms that Seryp is consumed prior to Vitellogenin during development. Absolute levels of LV1 and Seryp p50 were obtained from standard curves relating antibody binding (fluorescence intensity) to absolute amounts of each protein. Each dot is the average for a single embryo ±1 s.d. (C) Seryp could not be detected in many YPs in tailbud embryos. Notochord (1) and prosencephalon (2) (yellow boxes) from a sagittal section of a tailbud embryo (top, stage 26, scale bar: 100 µm) were immunostained with anti-Vtg and anti-Seryp and imaged by confocal microscopy (bottom, scale bars: 10 µm). Blue arrows highlight YPs without detectable Seryp.

 

Figure 4
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  		Fig. 4. Activated YPs have been reincorporated into the endocytic system. (A,B) YPs containing Seryp were almost mutually exclusive with YPs that accumulated extracellular dextran. Embryonic tissues were dissected from a tailbud embryo (stage 26) that had been previously injected in the blastocoel at the late blastula stage with fluorescently labeled, fixable dextran-Alexa647. The cells in the dissected tissues were dissociated and were immunostained with anti-Vtg and anti-Seryp. A neuron (A) and notochord cells (B) are presented. The soma of the neuron, boxed in yellow in the left panel, is further magnified in the right panels. Scale bars: 10 µm in A (left), B; 2 µm in A (right). (C) The average percentage of Seryp- YPs (left) and the percentage of Seryp- YPs in individual cells (right) was plotted for seven different dissociated tissues. The number of cells quantified is in brackets.

 

Figure 5
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  		Fig. 5. An atlas of YP consumption during early frog embryogenesis. (A) The percentage of Seryp- YPs in each of the tissues and stages depicted, as well as from unfertilized eggs and mid-neurulae (stage 17/18), was quantified. See supplementary material Table S2 for all numbers. For illustrative purposes, tissues (most of which are strictly internal) are approximately located on drawings (Nieuwkoop and Faber, 1994Go). The average ±1 s.d. of at least three embryos descended from different female frogs is presented. (B)Seryp- (%) was plotted, using the most relevant tissues present at each stage. For instance, somites at stage 27 were most related to the somitogenic mesoderm at stage 14, to the dorsal lip at stage 12, and to the marginal zone of stage 9 embryos and eggs. (C) Activated YPs that have no detectable Seryp are apparent in multiple tissues in early neurulae (blue arrows). A transverse section of an early neurula (top, scale bar: 100 µm) locates tissues (yellow boxes) immunostained with anti-Vtg and anti-Seryp and imaged by confocal microscopy (bottom, scale bars: 10 µm).

 

Figure 6
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  		Fig. 6. Declining cell size during development does not induce YP activation. (A) Large cell size was apparent as decreased nuclear density (DAPI, green) in the prosencephalon of a late neurula treated with hydroxyurea/aphidicolin (HU/APH; bottom), as compared with control siblings (top). Scale bar: 100 µm. (B) Overexpression of CycA2 and 6mycCdk2 increased the frequency of mitotic cells in multiple lineages of late neurulae (top, uninjected control in bottom panels). Transcripts encoding the two proteins were injected into the animal pole at the two-cell stage. Cells expressing 6mycCdk2 were identified with anti-myc 9E10 antibody ({alpha}-myc), mitotic nuclei by anti-phospho-Histone H3 antibody ({alpha}-PH3). Right, single channel images; left, merged images. In contrast to a previous report (Richard-Parpaillon et al., 2004Go), CycA2/myc6Cdk2 increased the mitotic index of the somitogenic mesoderm and of the normally postmitotic notochord (notochord, 7.4% versus 0%; somitogenic mesoderm, 5.2% versus 1.0% mitotic index; injected versus uninjected, n>160 cells). Scale bar: 100 µm. (C-E) The effects of HU/APH treatment and CycA2/6mycCdk2 overexpression on mitotic index, cell size and YP activation were quantified. HU/APH and DMSO control data are derived from the prosencephalon of late neurulae (stage 17-20), whereas CycA2/6mycCdk2 overexpression and uninjected control data are derived from the neural tube of late neurulae (stage 18-19). The average ±1 s.e. is presented. For HU/APH and DMSO control, n=9 embryos for cell size and YP activation and n=6 embryos for mitotic index. For CycA2/6mycCdk2 overexpression and uninjected control, n=8 embryos for all parameters. *P<0.01, Student's t-test. pr, prosencephalon; ac, archentron ceiling; ng, neural groove; nt, notochord; s, somitogenic mesoderm.

 

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© The Company of Biologists Ltd 2009