November 1, 2008;
Xenopus BTBD6 and its Drosophila homologue lute are required for neuronal development.
proteins constitute a subclass of CUL3
interacting BTB proteins whose in vivo function remains unknown. Here, we show that the Xenopus BBP
and the single Drosophila homologue of mammalian BBP
genes lute are strongly expressed in the developing nervous system
. In Xenopus, BTBD6
expression responds positively to proneural and negatively to neurogenic gene overexpression. Knockdown of BTBD6
in Xenopus or loss of Drosophila lute result in embryos with strong defects in late neuronal markers and strongly reduced and disorganized axons while early neural development is unaffected. XBTBD6
knockdown in Xenopus also affects muscle
development. Together, these data indicate that BTBD6
/lute is required for proper embryogenesis and plays an essential evolutionary conserved role during neuronal development.
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Figure 1. XBTBD6 and lute expression during embryogenesis. A: In situ analysis of XBTBD6 expression. a,b: Dorsal views. c: Anterior view. d-i: Lateral views. B: Sections of XBTBD6 stained embryos. a: Transversal section. b: Parasagittal section. c: Horizontal section. d,e: Transversal sections at the level of the eye and spinal cord. f: Horizontal section of the head. g-i: Transversal (g) and parasagittal (h,i) sections. j,k: Transversal sections of the eye. l: Parasagittal section of a tadpole brain. Nieuwkoop-Faber stages are indicated. C: In situ analysis of lute expression. a: Lateral view of a stage 14 embryo. b: Dorsal view of a stage 15 embryo. c: Ventral view of a stage 16 embryo. an, anterior neuroectoderm; ba, branchial arches bcs; bl, blastopore; cg, cement gland area; cnr, cephalic neurogenic region; cns, central nervous system; dlp, dorsal lateral plate mesoderm; e, eye; fb, forebrain; g, gut; h, heart; le, lens; n, notochord; nc, neural crest; np, neurogenic placode; nt, neural tube; ov, otic vesicle; pcv, posterior cardinal vein; pg, pineal gland; pns, peripheral nervous system; s, somites; ; tg, trigeminal ganglia; tel, telencephalon; tect, tectum; vvn, vascular vitelline network. Staging according to Nieuwkoop and Faber ().
Figure 2. XBTBD6 is regulated by proneural and neurogenic factors. A: In situ analysis of neurula embryos (dorsal views) injected with the indicated mRNA. LacZ mRNA was used as a tracer in c,d. B: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of animal cap explants isolated from control embryos (CC) or embryos injected with X-ngnr-1. CE, control embryos.
Figure 3. Knockdown of XBTBD6 affects the expression of late but not early neuronal markers and causes axonal outgrowth defects. A-F: Dorsal views of embryos injected with XBTBD6 MO1 and hybridized with the indicated probes. Reduced staining on the injected side in F is indicated (arrowhead). G-I: Control and injected sides of XBTBD6 MO1 (G,H) or XBTBD6 MO1mis (I) injected embryos in the trunk (G) or head region (H,I) immunostained with a HNK-1 antibody. Arrowheads in G and H points to reduced or disorganized axon tracks on the injected sides. Nuc-LacZ was used as a lineage tracer.
Figure 4. XBTBD6 knockdown affects muscle formation and segmentation. Embryos were injected on one side at the two-cell stage with either XBTBD6 MO1 or XBTBD6 MO1mis together with LacZ mRNA. Embryos were analyzed at gastrula or tadpole stage. A,B: Ventral views of embryos stained with Xbra. C,K: Lateral views of embryos stained with MyoD or c. actin. L: Horizontal section of a tail bud embryo stained with c. actin. M: Outline of muscle segmentation pattern observed in L.
btbd6 (BTB (POZ) domain containing 6) gene expression in Xenopus laevis embryos, NF stage 17, as assayed by in situ hybridization. Anterior view: dorsal up.
btbd6(BTB (POZ) domain containing 6) gene expression in Xenopus laevis embryos, NF stage 29, as assayed by in situ hybridization. Lateral view: anterior right, dorsal up.