FIGURE SUMMARY
Title

Two sox9 genes on duplicated zebrafish chromosomes: expression of similar transcription activators in distinct sites

Authors
Chiang, E., Pai, C.-I., Wyatt, M., Yan, Y-L., Postlethwait, J., and Chung, B.-C.
Source
Full text @ Dev. Biol.

Expression of zebrafish sox9a and sox9b in different adult tissues by RT-PCR. Total RNA from different tissues was used for RT-PCR analysis followed by hybridization to the sox9a or sox9b cDNA probe. Plasmids bearing sox9a and sox9b were used as control templates to monitor PCR fidelity and hybridization specificity. Actin primers were used to determine whether equal amounts of RNA were used for each PCR.

In situ hybridization shows transcripts of sox9a in zebrafish testis and sox9b in the ovary. (A, C) Whole-mount (B, D) sections of testis and ovary. Transcripts of sox9a (A, B) were detected in the zebrafish testis. Scattered cells across the mature testis express sox9a (arrows in A and B). Cryosection revealed that these cells are Sertoli cells (arrows B), which are irregularly shaped and lie close to the edge of the large membrane-bound cyst-like structures in the testis. Spermatogenesis proceeds synchronously within each cyst. Different stages of spermatogenesis can be seen in different cysts. Large and round shaped germ cells are early stage spermatocytes (cy). These spermatocytes decrease in size as spermatogenesis progresses and develop into spermatids (t). Arrowheads in B indicate the edge of one of the cysts. Transcripts of sox9b (C, D) were evident in the previtellogenic oocytes of the ovary. I, follicles at the primary growth stage (stage I); II, follicles at the cortical alveolus stage (stage II); III, stage III vitellogenic follicles.

Expression patterns of sox9a and sox9b in the head during the pharyngula stage. (A, B) Lateral view and (C, D) dorsal view of the 24 h head. In (A) and (B), both sox9a and sox9b transcripts were detected in the epiphysis (ep), forebrain, and the ventral midbrain. Expression of sox9a in the diencephalon (d) of the forebrain extends dorsally, whereas sox9b expression is mainly at the ventral diencephalon. t, telencephalon. Only sox9a transcripts are detected in the midbrain-hindbrain boundary (MHB), pharyngeal pouches (arrowheads in C), and the head mesenchyme (ms) in the caudal hindbrain. Transcripts of sox9b are detected in the eye (e) close to the midline and in six stripes of the hindbrain, with the highest expression in the four posterior rhombomeres (D). Otic vesicle (ov) marks the position of the caudal hindbrain. (E–G). Double staining with sox9a (red) and sox9b (blue) at 34 h. Both whole mount (E) and cryosections (F, G) show that both genes are expressed in cells around the ventricle (arrow in F) and in two areas (arrow in G) in the dorsal hindbrain. In the head mesenchyme (ms) and segmented pharyngeal arch primordia, which are shown in the whole mount (as arrowheads in E) or in sections (labeled as 1st, 2nd, 3rd), only sox9a transcripts were detected. As in panel D, the retina (r), but not the lens (l) of the eye (e), retains sox9b expression. (H–J) Double staining with sox9a (red) and col2a1 (blue) of the head. The precondensed mesenchyme in the segmented pharyngeal arches express only sox9a, not col2a1 (arrowheads in H and 2nd in I). Transverse sections through the rostral hindbrain (I) and at the level of the ear (J) confirm that the transcripts of the two genes overlap in the head mesenchyme (ms) which lie beneath or to the side of the neural tube. Expression of col2a1 in the epithelium (ep) of otic vesicle is much stronger than that of sox9a. Only a few epithelial cells are positive for both sox9a and col2a1 staining (arrow in J). Expression of col2a1 was also detected in the midline structures, including the floor plate (fp) and notochord (nd), but not in CNS.

Expression of sox9a and sox9b in the developing pharyngeal arches. (A, B) Lateral view of the 45 h embryo. Both sox9a and sox9b were detected strongly in the pectoral fin buds (pf), most segments of pharyngeal arch primordia, such as mandibular (m), hyoid (h), and ceratobranchial (cb) arches, CNS, and otic vesicles (ov). Expression patterns of the two genes in the brain and otic vesicle are not identical. (C, D) Ventral view of head at 72 h. Differentiating cartilage elements of pharyngeal arches express both sox9a and sox9b; the level of sox9b expression is lower and concentrated in the ceratobranchial. Other abbreviations: cb, ceratobranchials; ch, ceratohyal; mc, Meckel’s cartilage; pq, palatoquadrate; tb, trabeculae. (E, F) Double staining of 76 h head with col2a1 in blue and sox9a in red (E), or col2a1 in red and sox9a in blue (F). Expressions of the two genes overlap in most cartilaginous tissues in the cranio-facial and pectoral fin skeletons. The distribution of col2a1 in these elements is less restricted than that of sox9a.

Expression of sox9a and sox9b in the developing pectoral fins. (A, B) Double staining of sox9a (red) and sox9b (blue) at the early fin bud stage. (A) Dorsal view of rostral trunk shows that both genes are expressed in the developing fin bud with broader sox9a expression. (B) Lateral view. Both transcripts are restricted to the mesoderm of the fin bud. (C, D) Dorsal view of rostral trunk at 60h. Both sox9a and sox9b transcripts are expressed in precartilaginous mesenchyme of the elongated pectoral fins but not in ectodermal fin fold (ff).

Binding of Sox9a and Sox9b to specific DNA sequences. (A) The sequences of oligo used in the protein–DNA-binding experiment. The sequence of the known binding site for SOX9 is underlined. Electrophoretic mobility-shift assay was performed by incubating 32P-labeled col2c2 (B) or HMG (C) probe with in vitro translation products derived from plasmids encoding no protein (pSG5), mouse SOX9, or zebrafish Sox9a or Sox9b followed by electrophoresis. A 100-fold excess of unlabeled oligo was added as competitor. “-” refers to samples with no competitors. Zebrafish Sox9a and Sox9b recognize the col2c2 motif and the HMG consensus sequence as effectively as does mouse SOX9. The protein–DNA complex can be competed away by unlabeled col2c2, but not by the unrelated kuc1. The unbound probe has run off the gel in this set of experiments.

Transactivation domain analysis of Sox9a and Sox9b. (A) Structure of expression plasmids. The Gal4 DNA-binding domain (DBD) was linked to different lengths of the C-terminal domain or full-length Sox9 protein. The numbers above each box represent residue numbers. The reporter contains the chloramphenicol acetyltransferase (CAT) gene driven by a minimal TATA box and 5 copies of the Gal4 recognition sequence. (B) CAT activity of the reporter pG5E1bCAT cotransfected with different effector plasmids after normalization. (C) Western blot of 20 μg protein from each cell lysate after transfection probed with anti-Gal4 antibody. Effector proteins could be detected in all transfections.

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Reprinted from Developmental Biology, 229, Chiang, E., Pai, C.-I., Wyatt, M., Yan, Y-L., Postlethwait, J., and Chung, B.-C., Two sox9 genes on duplicated zebrafish chromosomes: expression of similar transcription activators in distinct sites, 149-163, Copyright (2001) with permission from Elsevier. Full text @ Dev. Biol.