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Tcf/Lef HMG box transcription factors are nuclear effectors of the canonical Wnt signaling pathway, which function in cell fate specification. Lef1 is required for the development of tissues and organs that depend on epithelial mesenchymal interactions. Here, we report the effects of lef1 loss of function on early development in X. tropicalis. Depletion of lef1 affects gene expression already during gastrulation and results in abnormal differentiation of cells derived from ectoderm and mesoderm. At tail bud stages, the epidermis was devoid of ciliated cells and derivatives of the neural crest, e.g. melanocytes and cephalic ganglia were absent. In the Central Nervous System, nerve fibers were absent or underdeveloped. The development of the paraxial mesoderm was affected; intersomitic boundaries were not distinct and development of the hypaxial musculature was impaired. The development of the pronephros and pronephric ducts was disturbed. Most striking was the absence of blood flow in lef1 depleted embryos. Analysis of blood vessel marker genes demonstrated that lef1 is required for the development of the major blood vessels and the heart.
Fig. 1. Lef1 depletion affects expression of genes in the ventral and dorsal mesoderm. In situ hybridization for X. tropicalis lef1 RNA at stage 10.5 (A) shows high levels of expression in the lateral and ventral marginal zone, low expression in the ectoderm and in the dorsal marginal zone (dorsal side up). Expression of marker genes in gastrula stage embryos, stage 10.5 (B), dorsal side up. Lef1 depletion does not alter the expression of brachyury, wnt8 or myf5. Msr/APJ expression is down- regulated by lef1 depletion whereas nodal related 3 (nr-3) expression is upregulated. Co-injection of human LEF1 RNA rescues the expression of msr/APJ and partially downregulates nr-3 expresssion.
Fig. 2. Lef1 depletion results in impaired development of different tissues. Non-injected control X. tropicalis embryo, stage 38 (A). Lef1 morpholino oligo injected embryos (B) are short and lack a tail with a dorsal and ventralfin. The eyes are small and pigmentation is absent. Co-injection of mRNA encoding human LEF1 rescues the lef1 MO effects (C). Broken lines in (A-C) indicate level of sections shown in (D-F). Transverse section of non-injected control embryo (D). Lef1 MO injected embryos (E) lack the dorsal longitudinal anastomosing vessel (dlav), the posterior cardinal veins (pcv), the pronephric ducts (pd), the hypochord (hc) and the dorsal aorta. The somites and notochord (N) are poorly differentiated. The spinal cord (sc) lacks nerve fibers and the notochord (N) is smaller in diameter. Embryo co-injected with LEF1 RNA (F) shows rescue of the structures affected by lef1 depletion. 3D reconstructions of transverse sections (G-H of embryos shown in (A,B). Noninjected control embryo (G,G and embryo injected with lef1 MO (H,H. Reconstructions were aligned relative to the otic vesicle.
Fig. 3. Ectodermal derivatives are affected by lef1 depletion. Transverse section of the eye of a control X. tropicalis stage 38 embryo (A) shows differentiation of retina and lens. Transverse section of the eye of a lef1 depleted embryo (B) shows that the retina and lens have not developed properly and that the lens has not separated from the epidermal ectoderm. Detail of the epidermis showing a ciliated cell (arrow) of a control embryo (C). Lef1 depleted embryos (D) lack ciliated cells in the epidermis. Rescue of ciliated cells (arrow) in the epidermis of embryos injected with lef1 MO together with human LEF1 RNA (E). Expression of the neural crest marker gene tfap2a in a control embryo (F) and reduced expression in a lef1 depleted embryo (G).
Fig. 4. Mesoderm formation is affected by lef1 depletion. Somitic boundaries of a control X. tropicalis embryo (A). The intersomitic bound- aries (white arrow heads) are affected by lef1 depletion (B). At the ventral side boundaries are lost. Myod expression in a control X. tropicalis stage 38 embryo (C) in the ventral part of somites (black arrow-head) and migrating hypaxial musculature (open arrowheads). Myod expression is downregulated in the ventral part of somites (black arrow- head) and hypaxial musculature is absent in lef1 depleted embryos (D). Control embryos stained for pax2 expression (E) in the pronephros (black arrow), pronephric duct (open arrowhead indicates junction between pronephric duct and rectal diverticulum) and rectal diverticulum. In lef1 depleted embryos (F) the caudal part of the pronephric ducts did not grow out properly and did not join the rectal diverticula, which were rudimentary (open arrowhead indicates the posterior end of an aberrant pronephric duct). Transverse section of the pronephros (G) showing pronephric tubules in a control embryo (arrows). The pronephros of lef1 depleted embryos (H) remains a single tube (arrow), with an S-shape (cf. Fig. 4F).
Fig. 5. The cardiovascular system is affected by lef1 depletion. Sagittal section of a stage 38 control embryo (A) showing the dorsal aorta and the caudal vein. In lef1 depleted embryos (B), the tail lacks the major blood vessels, the caudal vein and the dorsal aorta. Fli1 expression in the endothelium of a control embryo (C). isv, intersomitic vessels. dlav, dorsal longitudinal anastomosing vessel. pcv, posterior cardinal vein. aorta, dorsal aorta. Lef1 depleted embryos show strongly reduced expression of fli1 (D) at the position of the pcv and the aorta and no indication of intersomitic vessels, dlav or caudal vein. Msr/APJ expression in control embryo (E) and in lef1 depleted embryo (F) showing reduced expression. Endogenous expres- sion of lef1 in the heart of a stage 34 control embryo (G). e, endocardium. m, myocardium. Transverse section of the heart of a control embryo (H) and of a lef1 depleted embryo (I) which shows severe retardation of heart development both for the endocardium and myocardium.
