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Using Xenopus as a model system for an undergraduate laboratory course in vertebrate development at the University of Bordeaux, France.
Olive M
,
Thiebaud P
,
Landry M
,
Duvert M
,
Verna A
,
Barillot W
,
Theze N
.
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The goal of this laboratory course is to introduce vertebrate developmental biology to undergraduate students, emphasizing both classical and contemporary aspects of this field. During the course, the students combine the use of living Xenopus laevis material with active tutorial participation, with the aim of illustrating how the fertilized egg can generate the diversity of cell types and complexity of pattern seen only a few days later in the embryo. Special emphasis is given to the observation and manipulation of living material. The laboratory course includes a comprehensive analysis of both oogenesis and early development and is divided into two overlapping parts that combine tutorial and practical approaches. The first part is devoted to oogenesis; oocytes are sorted out, allowed to mature in vitro and observed in histological section. In the second part, students perform an in vitro fertilization of Xenopus eggs and a mesoderm and neural induction assay of animal cap explants. Successful induction of the explants is confirmed by morphological, histological and molecular analyses. Finally, the students observe and comment on selected slides to illustrate the organization of the body plan of the amphibian embryo at an early stage of organogenesis.
Fig. 2. Early embryos and animal cap explants obtained by students. (A) 2-cell and 4-cell embryos viewed from the animal pole. (B) 16-cell embryo. (C) Neurula-stage embryo viewed from the dorsal side. (D) Animal cap (ac) explants dissected out from blastula embryos (bl). (E) Animal caps treated with 1 ng (1), 10 ng (10) or 100 ng (100) of activin. (F) bFGF-treated (f) and control (c) animal caps. (G) Histological section of an animal cap control. (H) Histological section of an activin-treated animal cap. (I) Histological section of a bFGF-treated animal cap. epi, epidermis; mus, muscle; mes, mesothelium. Bar (D-F): 500 μm
Fig. 3. Embryo morphology, muscle gene expression and animal cap induction analysis. Scanning microscopy photographs (A-F). (A) Transverse section through the blastula showing blastocoel cavity (bc) and ectodermic animal cap (ac). (B) Blastocoel roof of (A) at higher magnification. (C) Control animal cap. (D) Late gastrula showing yolk plug (yp). (E) Transverse section through the gastrula showing archenteron (ar), ectoderm (ect), mesoderm (mes) and endoderm (end) cells. (F) Activin-treated animal cap. (G) Whole-mount in situ hybridization of tadpoleembryo made with an MLC1f/3f mRNA antisense digoxygenin probe. (H) In situ hybridization, transverse section of tadpoleembryo, with an MLC1f/3f mRNA S35 antisense probe. Somites (s) are strongly labeled but not the heart (h). (I) RT-PCR analysis of animal cap explants treated with activin (a), bFGF (f) or caffeine (ca), or untreated (c). The amplified fragments correspond to N-CAM (N), MLC1f/3f (M) or ODC (O) mRNAs.
Fig. 4. Observation of Xenopus laevis ovaries and oocytes. (A) View of an ovary. (B) Group of early stage (I-III) oocytes. (C) Stage-I to stage-VI oocytes sorted by students. (D) Progesterone-treated oocytes showing a typical white spot (arrow) at the animal pole, indicative of maturation. Inset shows animal (ap) and vegetal (vp) pole views of stage-VI oocytes, and an equatorial view. (E) Germinal vesicle (gv) extracted from a stage-VI oocyte, 1.2 mm in diameter.
Fig. 5. Histological, cytological and histochemical observations of the principal features of Xenopus laevis oogenesis. Stained, paraffin-embedded Xenopus laevis ovary sections. (A) Trichrome staining showing oocytes at various stages. (B,C) Unna blue staining showing previtellogenic stage-II oocyte. (D) P.A.S. staining of a vitellogenic oocyte. (E,F) Unna blue staining of vitellogenic oocyte. Bars: 100 μm in A-D and F; 25 μm in E.
ig. 6. Histological sections of embryos illustrating body plan organization. Trichrome stainings of paraffin-embedded Xenopus laevis tailbud sections. (A) Sagittal section of the anterior third of an embryo. (B-D, F) Transverse sections. (B) is a section through the pharynx and gills and (C) is through the trunk. (D) is a detail of intestine and peritoneum. (E) Sagittal section illustrating metamerization. (F) is a detail of various viscera. ch.pl., choroid plexus; mes, mesentery; oes, esophagus; p.e., pericardial epithelium; rhom, rhombencephalon. Bars: 200 μm.
