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BACKGROUND: Specific molecules involved in early inductive signaling from anterior neural tissue to the placodal ectoderm to establish a lens-forming bias, as well as their regulatory factors, remain largely unknown. In this study, we sought to identify and characterize these molecules.
RESULTS: Using an expression cloning strategy to isolate genes with lens-inducing activity, we identified the transcriptional cofactor ldb1. This, together with evidence for its nuclear dependence, suggests its role as a regulatory factor, not a direct signaling molecule. We propose that ldb1 mediates induction of early lens genes in our functional assay by transcriptional activation of lens-inducing signals. Gain-of-function assays demonstrate that the inductive activity of the anterior neural plate on head ectodermal structures can be augmented by ldb1. Loss-of-function assays show that knockdown of ldb1 leads to decreased expression of early lens and retinal markers and subsequently to defects in eye development.
CONCLUSIONS: The functional cloning, expression pattern, overexpression, and knockdown data show that an ldb1-regulated mechanism acts as an early signal for Xenopus lens induction.
Figure 1. A: Comparisons of sizes of δldb1 fragment and full-length ldb1; nuclear localization signals (NLS) and MO binding site indicated. The functional LIM-binding domain of ldb1/ldb2 (LIM interaction domain, LID) is shaded. B: Northern analysis of endogenous and synthetic transcripts for ldb1 expression (δldb1 probe). Lanes 1–5: Five embryo equivalents total RNA each from St. VI oocyte, St. 8, St. 10, St. 20, and St. 35 embryos. Lane 6: 1.5 µg St. 14 poly(A)+ RNA. Lane 7: 1 µg pooled synthetic RNA from 10,000 member library fraction. Library pool contains bands corresponding to δldb1 (700–750 bp) and ldb1 (1.5 kb). Oocyte/embryos contain endogenous 3 kb and 1.5–1.8 kb ldb1 transcripts; not δldb1.Download figure to PowerPoint
Figure 2. δldb1 RNA is highly enriched in neural tissue at neural tube stages, and is translated efficiently in oocytes with or without a nucleus. A,B: 35S- labeled protein products from metabolic labeling of oocytes expressing ldb1 or δldb1; δldb1 is abundant. Uninjected oocytes (UN), oocytes injected with 20 ng δldb1 RNA (δ), and oocytes injected with 40 ng ldb1 (ldb1) were cultured in 35S-Met and visualized by SDS-PAGE (A); arrowhead indicates δldb1. δldb1 RNA (20 ng) was injected into normal and enucleated oocytes for translation (B); arrowhead indicates δldb1. C–E: Whole-mount in situ hybridization of ldb1 at Stage 15 (C), anterior neural plate (black arrowhead), lateral expression (arrow), and posterior neural expression (gray arrowhead); Stage 17 (D); anterior neural plate (black arrowhead), lateral expression (presumptive ganglia; arrow), and posterior domain (gray arrowhead); and Stage 18 (E), PLE region (yellow arrowhead) flanked on dorsal side by anterior neural expression (black arrowhead) and posterior side by presumptive cranial ganglion expression (arrow) of ldb1.Download figure to PowerPoint
Figure 3. δldb1-induced activation of early lens, olfactory, and cement gland markers in ectodermal recombinants. A: Schematic of recombinant procedure; Stage 10–11 δldb1 RNA-injected ectoderm combined with Stage 11–11.5 lineage-labeled ectoderm, cultured to the equivalent of Stages 18–26, processed for in situ hybridization with foxe3, nrl-maf, dlx5, and ag1, and sectioned for analysis. B,C: Expression of foxe3 and lineage labeling of ectoderm, Stage 24. D,E: Expression of nrl-maf and lineage labeling of ectoderm, Stage 26. F,G: Expression of dlx5 and lineage labeling of ectoderm, Stage 18. H,I: Expression of ag1 and lineage labeling of ectoderm, Stage 18. Arrowheads indicate identical locations on each pair of images. Table: Expression of genes in recombinants; markers of the lens, nose, and cement gland are detected in δldb1-injected recombinants, markers of neural and neural crest tissue are not.Download figure to PowerPoint
Figure 4. The ability of anterior neural tissue to induce foxe3 expression is enhanced by ldb1. A: Schematic diagram of recombinant procedure; Stage 11–11.5 lineage-labeled animal cap ectoderm combined with control anterior neural plates or anterior neural plates from embryos injected with δldb1 RNA into the animal pole at the 1-cell stage, cultured to Stage 23, processed for in situ hybridization, and sectioned for analysis. B,C: Expression of foxe3 (B) and lineage labeling (C) of responding ectoderm in recombinant with control neural plate (39% expressing foxe3). D,E: Expression of foxe3 (D) and lineage labeling (E) of responding ectoderm in recombinant with δldb1-injected neural plate (64% expressing foxe3).Download figure to PowerPoint
Figure 5. Expression of foxe3 and rax decreased in ldb1 MO-injected embryos. A,B: Control in situ hybridization expression pattern of foxe3 (A) and rax (B) at Stage 26. C,D: ldb MO-injected embryos, expression of foxe3 (C) and rax (D) at Stage 25–26. E,F: ldb1 MO- and δldb1 RNA-coinjected embryos, expression of foxe3 (E) and rax (F) at Stage 25–26.Download figure to PowerPoint
Figure 6. Eye morphology and retinal pigmentation are disrupted, and lens γ-crystallin expression is reduced in ldb1 MO-injected embryos. A–C: Sections of control (A), ldb1 MO-injected (B), and ldb1 MO + δldb1 RNA-injected (C) embryos at Stage 34. D–F: In situ hybridization for γ-crystallin in control (D), ldb1 MO-injected (E), and ldb1 MO + δldb1 RNA-injected (F) embryos at Stage 34.Download figure to PowerPoint
Figure 7. Expression of rax and dll1 reduced in embryos injected unilaterally with ldb1 MO. A–D: Embryos injected into one of two dorsal blastomeres at the 4-cell stage with 35 ng ldb1 MO and 45 ng FLDX, then cultured to Stage 21 (A,B) or Stage 15 (C,D) and processed for in situ hybridization for rax (A,C); lineage label in B, D. E,F: Embryo injected into one of two dorsal blastomeres at the 4-cell stage with 35 ng ldb1 MO and 45 ng FLDX, then cultured to stage 21 and processed for in situ hybridization for dll1(E); lineage label in F. Arrows indicate reduction in expression on the injected side compared to the uninjected side.Download figure to PowerPoint
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