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First published online 19 July 2006
doi: 10.1242/dev.02485

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Lhx5 promotes forebrain development and activates transcription of secreted Wnt antagonists

Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA.

View larger version (77K): [in a new window]   Fig. 1. Lhx5 promotes forebrain development. In this and subsequent figures, the probes used for whole-mount in situ hybridization are listed in the upper right corner of each panel. Genotypes or experimental manipulations are indicated in the lower left corners. Developmental stages are indicated in the lower right corners. Unless otherwise noted, gastrula stage embryos are orientated in animal pole view, rostral to the top; post-gastrulation stage embryos in lateral view, rostral to the top and dorsal to the right. ctl, control embryos. (A-D) lhx5 is expressed in rostral regions during embryonic development. (A,B) Dorsal to the right, lateral (A) and animal pole (B) views. (D) A gap can be seen between the lhx5 and pax2a expression domains. (E-N) lhx5 gain of function causes expansion of the forebrain. (E,F) Forebrain boundaries are marked by broken lines. (G,H) Dorsal view, bud stage, pax6a expression. (I-N) Dorsal view, rostral to the top. Embryos were dissected and flat mounted in glycerol after in situ hybridization. Partial overlays of panels were made with PhotoShop (Adobe) and brightness in the overlapped regions was adjusted so that the backgrounds match. (O-V) Inhibition of Lhx5 function compromises forebrain development. (U,V) Red arrowheads mark the forebrain-midbrain boundary. (W-Z) lhx5 morpholino knockdown alters pax6a and pax2a expression. Embryos were dissected and flat mounted in glycerol after in situ hybridization. Dorsal view, rostral to the top. Scale bar in Z: 250 µm for A-H,Q-V; 200 µm for I-K; 167 µm for L-N; 150 µm for O,P; 100 µm for W-Z.

View larger version (57K): [in a new window]   Fig. 2. Lhx5 partially rescues forebrain deficiencies induced by ectopic Wnt signaling. (A) lhx5 gain of function partially rescues the eyeless phenotype of wnt8a mRNA-injected embryos. Injected embryos were scored after the Prim-5 stage (24 hours post-fertilization). The percentages of embryos eyeless (blue) or with two eyes (green), or malformed as a result of dorsalization or injection (yellow), were measured. (B-D) lhx5 gain of function partially rescues eye development in mbl (axin1) mutants. Ventral views, rostral to the left. (E-G) lhx5 gain of function partially restores emx3 expression in mbl (axin1) mutants. Genotypes of mutant and partially rescued embryos were verified by RFLP. (H-J) lhx5 gain of function partially restores six3b expression in mbl (axin1) mutants. WT, wild-type embryos. Scale bar in J: 150 µm for B-D; 250 µm for E-J.

View larger version (17K): [in a new window]   Fig. 3. Zebrafish Sfrps are closely related to Sfrps from other vertebrates. Phylogenic tree of the Sfrps from zebrafish (Dre), frog (Xla), mouse (Mmu) and human (Hsa). The Sfrp from sea urchin (Spu) is used as the outgroup to root the tree. The probability values from the MrBayes output are given at the nodes of the branches. The five Sfrps described in this study are labeled in blue and three previously described zebrafish Sfrps are labeled in green. Protein sequences used in the alignments are available upon request.

View larger version (53K): [in a new window]   Fig. 4. Sfrps antagonize Wnt signaling and partially restore forebrain marker expression in mbl mutant embryos. (A) Sfrp1a and Sfrp5 rescue eye development in wnt8a mRNA-injected embryos. Injected embryos were scored after 24 hours post-fertilization. (B-D) sfrp1a or sfrp5 gain of function expands the six3b expression domain. (E-G) sfrp1a or sfrp5 gain of function expands the emx3 expression domain. Embryos were dissected and flat mounted in glycerol after in situ hybridization. Animal pole views, rostral to the top. (H-M) Overexpression of Sfrps partially restores emx3 (J) and six3b (M) expression in mbl embryos. Dosage injected was 150 pg of sfrp1a mRNA and 100 pg of sfrp5 mRNA per embryo. Genotypes of mutant and partially rescued embryos were verified by RFLP. WT, wild-type embryos. Scale bar in M: 250 µM for B-M.

View larger version (70K): [in a new window]   Fig. 5. Lhx5 regulates Sfrp1a and Sfrp5 expression. (A-I) Lhx5 regulates Sfrp1a expression. (A-C) Animal pole views, dorsal to the right. (D-F) Dorsal views, bud stage, sfrp1a expression. (G-I) Flat mounts in glycerol; dorsal views, rostral to the top. 2 ng (H) or 4 ng (I) of lhx5 morpholino was injected. (J-M) Lhx5 cell autonomously regulates Sfrp1a expression. Transplanted cells are labeled in red. sfrp1a transcripts are highlighted by white arrowheads and are apparent as blue dots in the narrow cytoplasm around the nuclei of transplanted cells in controls (J, ctl) and lhx5 mRNA-injected cells transplanted into lhx5 morpholino injected hosts (M). sfrp1a transcripts are not detected in lhx5-en injected cells transplanted into control hosts (K). In transplanted lhx5 morpholino-injected cells, sfrp1a expression is reduced (L). (J-L) 90% epiboly stage; (M) one-somite stage. (N) Mapping of sfrp1a upstream enhancers by transient GFP reporter expression in the forebrain. Fragments are numbered using the first nucleotide of the Sfrp1a start codon as the reference. GFP expression in the forebrain was scored at the 3 somite stage and the Prim-5 stage. The number of + signs indicate relative GFP expression levels. (O) Lhx5 binds to the sfrp1a promoter. Input represents 0.2% (2 µl, 2x) or 0.1% (1 µl, 1x) of starting material for each immunoprecipitation reaction. 2 µl (2x) or 1 µl (1x) of purified DNA product from each sample was used in the PCR. ctl., uninjected embryos treated in parallel; inj., lhx5-GFP mRNA-injected embryos. Mock immunoprecipitation with no antibody fails to amplify from either uninjected or injected groups (data not shown). (P-U) Lhx5 regulates Sfrp5 expression. Flat mounts in glycerol, dorsal views, rostral to the top. (R,T,U) 2 ng (R,T) or 4 ng (U) of lhx5 morpholinos was injected. Scale bar in U: 250 µm for A-F; 100 µm for G-I; 8 µm for J-M; 100 µm for P-U.

View larger version (71K): [in a new window]   Fig. 6. Sfrp compensates for inhibition of Lhx5 function.(A) Sfrp1a and Sfrp5 rescue eye development in lhx5-en mRNA-injected embryos. The percentages of embryos malformed, as a result of dorsalization or injection (yellow), eyeless (blue) or two eyed (green) were measured. (B-E) Ectopic Wnt signaling results in ectopic axin2 expression. (B,C) Animal pole views; (D,E) lateral views, rostral to the left, dorsal to the top. Genotypes were determined by RFLP. (F-I) Ectopic axin2 expression in lhx5 morpholino-injected embryos. Flat mounts in glycerol. Brain boundaries are marked by broken lines. Green arrows indicate the borders of axin2 labeling. (F,G) Dorsal views, rostral to the top; (H,I) ateral views, rostral to the left, dorsal to the top. Eyes were removed to expose the brain. (J-M) sfrp1a or sfrp5 gain of function reduces ectopic axin2 expression in lhx5-en-injected embryos. Animal pole views, dorsal to the left. Scale bar in M: 250 µm for B,C; 125 µm for D,E; 100 µm for F-I; 250 µm for J-M.

View larger version (13K): [in a new window]   Fig. 7. Lhx5 regulates Sfrps that antagonize Wnt signaling in zebrafish forebrain. At gastrula stages, Wnt genes are expressed in the posterior paraxial mesoderm and Wnt signaling activates axin2 expression. Rostral ectodermal cells express Lhx5, which activates the expression of Sfrps. Sfrps in turn antagonize Wnt activity in the rostral ectoderm and axin2 expression is prevented. Similar events are likely to also occur in the presumptive forebrain at segmentation stage. Question mark denotes that other unknown Lhx5 downstream factors may also contribute to the antagonism of Wnt signaling.