Hourdry J et al. (1988), Localization of c-myc expression during oogenes...

XB-ART-27157

Development 1988 Dec 01;1044:631-41.

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Localization of c-myc expression during oogenesis and embryonic development in Xenopus laevis.

Hourdry J , Brulfert A , Gusse M , Schoevaert D , Taylor MV , Mechali M .

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The expression of the proto-oncogene c-myc during oogenesis and embryonic development was followed by in situ hybridization using a cytological protocol adapted to amphibian embryos. The c-myc RNA was highly expressed in the cytoplasm of young oocytes and was further diluted during oocyte growth without specific localization. From the neurula stage on, new myc transcripts were detected and the whole embryo appeared positive with antisense myc RNA probes relative to control sense RNA probes. In addition, a spatial localization of high levels of the transcript was also observed in specific areas of the developing embryo, including the epidermis, gill buds, optic vesicles and lens placodes. These observations might indicate a specific role of the c-myc gene during the differentiation of these tissues. Alternatively, this high level of myc expression might prevent such tissues from entering into terminal differentiation during the growth of the embryo.

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Species referenced: Xenopus laevis
Genes referenced: myc tbx2

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	Fig. 1. Histological sections of Xenopus oocytes and embryos embedded by the ester-wax procedure. Xenopus oocytes and embryos were fixed and embedded as described in Materials and methods and 7-5 pm sections were stained with toluidine blue. (A) Stage-I and -IV oocytes. Stage-I oocytes are previtellogenic, the nucleus is clearly visible, has a regular shape and nucleoli can be seen at its periphery (thin arrow). Stage-IV oocytes have accumulated yolk platelets (y), the outline of the nucleus is irregular and the number of peripheral nucleoli decreases (thick arrow); a rim of toluidinic cytoplasm separates the nucleus from the surrounding yolk platelets (asterisk), xlOO. e, oocyte envelopes (ovarian and follicular epithelia). (B) Late morula stage-5 embryo (Nieuwkoop & Faber, 1967). Large blastomeres containing larger yolk platelets are located at the vegetal hemisphere (thick arrow) and smaller blastomeres at the animal hemisphere (thin arrow); b, blastocelic cavity, x50. (C) is a detail of B; a blastomere is dividing (asterisk), xlOO. (D) Stage-10 early gastrula embryo. Gastrulation is indicated by the appearance of a slightly bent furrow (arrow); the fold on the left of this furrow is the dorsal lip of the blastopore (star); at this level, the future chordomesoderm invaginates (cm), ec, ectoderm; en, endoderm, x70. (E) Detail of D, X140. (F and G) Stage-22 neurula embryo: parasagittal sections. (F) General view, x50; (G) detail of the dorsal region, xllO. c, notochord; de, dorsal endoderm; ep, epidermis; g, gut; n, neural tube; s, mesodermal cords beginning to divide into metamerized somites; y, yolk endoderm.
	Fig. 2. Localization of c-myc RNA during oogenesis. In situ hybridization of myc 35S-DNA probes (A,B) and myc antisense 35S-RNA probes (C-F) to Xenopus oocytes sections. A high level of labelling was detected throughout the cytoplasm of young oocytes both with DNA (A,B) or RNA probes (C,D). In later stage-Ill and -IV oocytes (E,F), the density of silver grains is lower and still uniformly distributed in the cytoplasm. The white ring around the oocyte (arrow) is due to pigment granules in the cortex of wild-type oocyte. (G) Hybridization with a pUC18 DNA probe and (H) with a sense myc RNA probe: no signal above background can be observed. Labels as Fig. 1. (A,C,E-H) Darkfield illumination; (B,D) brightfield illumination. (E) X120; (A,F,G) X130; (B,C,D,H) X180.
	Fig. 3. Quantification of in situ hybridization signals during oogenesis. Quantification of silver grain density was done as described in Materials and methods. (A) Values were corrected for the grain density background as a function of oocyte diameter. Values are the average of several measurements in different sections. The average background values were 9xlO~3 grains per im\2 for RNA probes and 7xlO~3 grains per fim2 for DNA probes. (B) The number of grains per oocyte was calculated from the oocyte diameter and the thickness of the section (7-5 /an) with correction for the background level. It was compared with the number of myc RNA transcripts as determined by both slot blot and Northern blot hybridizations (Taylor et al. 1986). Histograms are displayed as a function of oocyte growth.
	Fig. 4. Localized expression of c-myc in neurula embryos. Stage-22 (neurula) Xenopus embryo sections were hybridized with a 35S-labelled antisense c-myc RNA probe. (A,C,D,F) Dark-field illumination; (B,E,G) bright-field illumination. (A,B) Sagittal sections. The neural tube (n), notochord (c) and dorsal endoderm (en), exhibit a signal above the background of the whole embryo, g, gut; ep, epidermis. (C-G) Frontal sections. In (C) the neurula is oriented so that the anterior region is on the right side. The section becomes partially sagittal on the right side due to the twisting of the embryo by the cytological protocol. The cut plane crosses both the foregut or pharyngeal cavity (ph) and hindgut near the blastopore (bl) which remains as a posterior opening of the gut (future anus). (D) and (E) are an enlarged detail of C (rectangle on the right side). This detail shows the region located anterodorsally toward the pharyngeal cavity and including the pharyngeal endoderm (en), neural tube (n) and head epidermis (ep). The hybridization signal is generally high in these tissues, with some areas more intensely labelled (thick arrows). Thin arrow, neural lumen. (F) and (G) are another detail of C (rectangle on the left side). Signals are detected over yolk-laden endoderm (y). The grain density is higher both over the posterior epidermis (ep), the mesoderm, and immediately underlying endoderm (star), all of which are composed of moderately yolk-laden blastomeres. (C) x80; (D,E) X170; (A,B) X190; (F,G) x200.
	Fig. 5. Localized expression of c-myc in the tailbud embryo. Embryo frontal sections were hybridized with a 35Slabelled antisense c-myc RNA probe. (A,B,E) Dark-field illumination; (C,D,F) bright-field illumination. A is a general view which shows that the probe hybridized intensely to epidermis (ep), optic cups (op) and lateral pharyngeal walls (arrows). Abbreviations: cep, caudal epidermis; en, yolk-laden endoderm; ph, pharyngeal cavity. B and C are a detail of A in the cephalic region. (D) Caudal region. (E) Frontal section of the anterior part of the embryo showing an intense signal in the right optic cup (op), the left lens placode (le) and the cephalic epidermis (thick arrow). Periphery of the right optic cup is preferentially labelled (thin arrow) and might include the wall of the cup; the less preferentially labelled inner region might include part of the cup cavity. The left optic cup (asterisk) is more weakly labelled because the cut plane barely crosses the cup. g, gut. (A,D) x50; (B,E,F) x90; (C) x!40.