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The cell proliferative activity of the Myc family of basic helix-loop-helix/leucine zipper (bHLHZip) transcription factors is dependent upon binding to the ubiquitous Max protein. In the absence of heterodimerization with Max, Myc protein is unable to efficiently bind to DNA and activate transcription. Members of the Mad family of transcription factors are thought to modulate the cell proliferative effects of the c-myc proto-oncogene by binding to Max, directly competing with the Myc protein for both heterodimerization and DNA binding. Consistent with a role in down-regulating cell division, the murine mad genes are expressed in embryonic tissues undergoing differentiation, often during or shortly after the down-regulation of myc gene expression. Here, we report the isolation and characterization of the first Xenopus mad family member, Xmad4. Maternal Xmad4 transcripts are present at high levels in the oocyte and in the cleavage stage embryo, but almost disappear by the neurula stage. Zygotic expression of the Xmad4 gene is initiated in the epidermis of the late neurula stage, and shortly thereafter, Xmad4 is transiently detectable in the cement and hatching glands. At later stages, expression is also observed in the developing pronephros and liver. Unlike the murine mad4 gene, we find that multiple Xmad4 splice variants exist in Xenopus and that these variants are differentially expressed in both the embryo and the adult. Despite the demonstrated antagonistic role of Mad proteins in the regulation of Myc activity, we show that the over-expression of Xmad4 in the cleavage-stage embryo has no detectable phenotypic effect, suggesting that Myc function is dispensable during early embryonic development.
Fig. 3. Differental expression of Xmad4 splice variants analyzed by RT-PCR. A: Developmental profile of Xmad4 from the oocyte to tadpole stages. The position of each of the three splice variants is indicated. Input cDNA levels were estimated using the Xmax sequence (lower panel), which is expressed at constant levels throughout early Xenopus development (Tonissen and Krieg, 1994). B: Diagramatic representation of the dissections of early and mid tailbud stage embryos. C: Distribution of XMad4 splice variants in dissected embryonic tissues. The ubiquitousXmax sequence is used to indicate relative amounts of RNA in the different dissected fractions (lower panel).
Fig. 4. Xmad4 expression in adult Xenopus tissues assayed by RT-PCR analysis. The source of RNA is indicated at the top of each lane. The presence of equivalent amounts of cDNA template in the different tissue samples was confirmed using the Xmax sequence (data not shown).
Fig. 5. Xmad4 expression profile by whole-mount in situ hybridization. In all panels, anterior is to the left. A: Dorsal view of a late neurulaembryo showing strong Xmad4 expression in the ectoderm immediately adjacent to the neural tube. B: Anteriorlateral view of an early tailbud stage embryo. Note expression in the Y-shaped hatching gland (black arrow) and the more ventral cement gland (white arrow). C: Lateral view of a mid-tailbud stage embryo showing Xmad4 expression in the pronephros (black arrow). The darkness of the head is background, due to the extended period of staining required to visualize the relatively low level of transcription in the kidney. D: Close-up view of a mid tailbud stage embryo. Xmad4 expression is detected in the embryonic liver (arrow).
Fig. 6. Developmental expression pattern of the c-myc gene. A: Anterior view of an early neurula stage embryo showing c-myc expression in the cranial neural crest (arrowheads) and future cement gland (arrow). B: Posterior view of the same embryo shown in (A). Note strong expression in the cells immediately surrounding the newly closed blasto- pore. C: Lateral view of an early tailbud stage embryo showing c-myc staining in the neural crest entering the posterior pharyngeal arches (arrows), as well as in the eye (arrowhead) and the brain (white arrowhead). D: Lateral view of a mid tailbud stage embryo. c-myc expression is retained in the pharyngeal arches, most strongly in arch 2 (short arrow). Other domains of expression include the eye (long arrow), floor of the pharynx (small arrowhead), and cells of the first pharyngeal arch (large arrowhead). E: Lateral view of a tadpole stage embryo. Note expression in the pancreatic anlage (arrow), as well as in cells adjacent to the liver (arrowhead). F: Sagital section through a gastrula stage embryo stained for c-myc. Expression is strongest in the animal pole (arrowhead). G: Cross-section through an early neurula stage embryo. c-myc expres- sion is strongest in the intermediate mesoderm (large arrowhead) and the embryonic ectoderm (arrow). Paraxial mesoderm is also weakly positive (small arrowhead). H: Cross-section of a mid tailbudembryo. The future liver is positive for the c-myc gene. I: Cross-section of a mid tailbud stage embryo. c-myc expression is strong in the peripheral optic cup (arrow- head) and dorsal brain (arrow). J: Cross-section of a mid tailbud stage embryo. Note expression in dorsal somite (arrowhead), as well as in the dorsal gut (arrow).
mxd4 (MAX dimerization protein 4) gene expression in a Xenopus laevis embryo as assayed by in situ hybridization, NF stage 24. Antero-lateral view: Dorsal up, anteriorleft
myc-c () gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage x, anterior view, dorsal up.
