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Biochem Biophys Res Commun
2011 Aug 19;4121:170-4. doi: 10.1016/j.bbrc.2011.07.068.
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Xenopus Dbx2 is involved in primary neurogenesis and early neural plate patterning.
Ma P
,
Zhao S
,
Zeng W
,
Yang Q
,
Li C
,
Lv X
,
Zhou Q
,
Mao B
.
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The evolutionarily conserved Dbx homeodomain-containing proteins play important roles in the development of vertebrate central nervous system. In mouse, Dbx and Nkx6 have been suggested to be cross-repressive partners involved in the patterning of ventralneural tube. Here, we have isolated Xenopus Dbx2 and studied its developmental expression and function during neural development. Like XDbx1, from mid-neurula stage on, XDbx2 is expressed in stripes between the primary motoneurons and interneurons. At the tailbud stages, it is detected in the middle region of the neural tube. XDbx2 acts as a transcriptional repressor in vitro and over-expression of XDbx2 inhibits primary neurogenesis in Xenopus embryos. Over-expression of XDbx genes represses the expression of XNkx6.2 and vise versa. Knockdown of either XDbx1, XDbx2 or both by specific morpholinos induces lateral expansion of XNkx6.2 expression domains. These data reveal conserved roles for Dbx in primary neurogenesis and dorsoventral neural patterning in Xenopus.
Fig. 1. Alignment and phylogenetic analysis of X. laevis Dbx proteins. (A) Alignment of the XDbx1 and XDbx2 protein sequences. Identical amino acids are high-lighted by
black background. The conserved eh1 domain and homeodomain are underlined. (B) Phylogenetic analysis of human, rat, mouse, X. tropicals and laevis, zebrafish and
Drosophila Dbx family proteins. ClustalW alignment, Poisson correction model, and Bootstrap test (500 replicates) were used for the neighbor-joining (NJ) tree construction.
The accession number of the proteins used are Mus musculus Dbx1, Ensembl protein ID: ENSMUSP00000032717; Mus musculus Dbx2, Ensembl protein ID:
ENSMUSP00000060424; Rattus norvegicus Dbx1, Ensembl protein ID: ENSRNOP00000019739; Rattus norvegicus Dbx2, Ensembl protein ID: ENSRNOP00000009143; Homo
sapines Dbx1, Ensembl ID: ENSP00000227256; Homo sapines Dbx2, Ensembl protein ID: ENSP00000331470; Danio rerio Dbx1a, NP_571233; Danio rerio Dbx1b, Ensembl
protein ID: ENSDARP00000013350; Danio rerio Dbx2, Ensembl protein ID: ENSDARP00000057729; Xenopus tropocalis Dbx1, XP_002940015; Xenopus tropocalis Dbx2,
XP_002932867; Xenopus leavis Dbx1, NP_001079210.
Fig. 2. The temporal and spatial expression patterns of XDbx. (A) RT-PCR analysis of the developmental expression of XDbx1 and XDbx2. (B–G) The expression patterns of
XDbx1 and XDbx2 revealed by in situ hybridization. (B–E) Stage 15, dorsal view, anterior to the left. The red broken lines indicate the expression boundary of XDbx2. (F–G)
Stage 32, lateral view, anterior to the left. The black broken lines indicate the position of corresponding sections showed in F’ and G’. Br, branchial arches.
Fig. 3. XDbx2 acts as a transcriptional repressor in vitro and is involved in Xenopus primary neurogenesis. (A) The schematic structures of XDbx2 and its truncated constructs.
(B) Schematic diagram of the pGAL4-TK-Luc reporter construct. GAL4 BE, GAL4 binding elements. (C) Luciferase reporter assay showing the transcriptional repressor activities
of XDbx2 and its truncates. HEK293T cells were transiently transfected with 100 ng pGAL4-TK-Luc; 10 ng pRL-TK and 90 ng indicated plasmids per well of 96-well plate. RLU,
relative light units. ⁄p < 0.05; ⁄⁄p < 0.001. (D–I) In situ hybridization with N-tubulin in embryos injected on one side with control LacZ mRNA and the indicated XDbx
constructs. The arrowheads indicate the reduction of N-tubulin expression in the injected areas. (J–L) The expression of N-tubulin in embryos injected on one side with XDbx1
MO (J), XDbx2 MO (K) and XDbx1/2 MOs (L). The black arrowheads indicate the reduction of N-tubulin expression while the white arrowheads in (J) indicate the expansion of
the lateral N-tubulin expression domain. (M–O) The expression of XIslet1 in the medial stripes was reduced on the sides injected with XDbx1 MO (M), XDbx2 MO (N) or XDbx1/
2 MOs (O). LacZ mRNA was co-injected for tracing the injected sides which were stained in red. (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article).
Fig. 4. Cross-repression between XDbx and XNkx6 during neural patterning in Xenopus embryos. (A, B) Exogenous XDbx1 or XDbx2 repressed the expression of XNkx6.2. (C, D)
XDbx1 was down regulated in XNkx6.1 or XNkx6.2 mRNA injected area. (E, F) The expression domains of XNkx6.2 expanded on the sides injected with XDbx1 MO (E), XDbx2 MO
(F) or XDbx1/2 MOs (G). (H) Statistics of the embryos shown in E–G. The percentage of embryos with normal (N), reduced (R) and expanded (E) Nkx6.2 expression are
presented by green, red and blue boxes, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article).
dbx1 (developing brain homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
dbx2 (developing brain homeobox 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
