XB-ART-10441Mech Dev. September 1, 2000; 96 (2): 183-95.
A direct screen for secreted proteins in Xenopus embryos identifies distinct activities for the Wnt antagonists Crescent and Frzb-1.
To determine the spectrum of secreted proteins that are present in the extracellular space of early Xenopus embryos, a direct secretion screen was performed. Surprisingly, 24% of previously identified bona fide secretory proteins corresponded to four secreted Wnt antagonists of the same family: frzb-1, sizzled, sfrp-2 and crescent. sfrp-2 and crescent are novel components of the growing cocktail of growth factor antagonists secreted by Spemann organizer cells in Xenopus. Crescent is first expressed at blastula, defining a deep endodermal region that may be homologous to the avian hypoblast. Unlike other members of this family of inhibitors, microinjection of crescent mRNA causes the development of cyclopic embryos, even though the amount of anterior neural tissue is normal. In crescent-injected embryos, studies with specific markers indicate that morphogenetic movements of the anterior midline are abnormal, resulting in a more posterior location of prechordal plate and ventral forebrain markers with respect to the developing eye field. The results are discussed in light of recent findings in zebrafish and Xenopus that suggest that Wnt signaling through non-canonical (non-beta-catenin dependent) pathways plays a pivotal role in the regulation of morphogenetic movements.
PubMed ID: 10960783
Article link: Mech Dev.
Grant support: R37 HD021502-14 NICHD NIH HHS , R37 HD21502-14 NICHD NIH HHS
Genes referenced: actl6a chrd.1 egr2 frzb frzb2 gsc ncam1 nog not otx2 sfrp2 shh szl vax1
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|Fig. 3. Expression of crescent, frzb-1, sfrp-2 and sizzled in Xenopus embryos by whole-mount in situ hybridization. (A–D) Early gastrulae (stage 10.5) in vegetal (A,B,D) and lateral (C) view. crescent, frzb-1and sfrp-2 are expressed in the dorsal and sizzled in the ventral blastopore lip. Note an additional expression domain of sfrp-2 in the prospective dorsal neuroectoderm (ne). (E–H) Neurulae (stage 15) in dorsal (E–G) and ventral (H) view. crescent and frzb-1 transcripts are localized to anterior endoderm and prechordal plate at this stage. sfrp-2 expression is strong in the anterior neural plate (forebrain and eye field) and posterior neural plate (hindbrain and spinal cord), and is absent from the floor plate. sizzled mRNA is expressed in the ventral mesoderm (vm), including ventral blood islands, and the ventral part of the closing blastopore lip (vb). (I–L) Tailbud embryos (stage 20) in lateral view. The crescent and frzb-1 expression domains overlap in the prechordal plate and pharyngeal endoderm. Note the additional crescent domain in the pronephros (pn). sfrp-2 has a complex expression pattern in eyes, neural tube, somites and in a belt-like anterior part of the trunk. sizzled transcripts are located in ventral mesoderm and in developing heart (he).|
|Fig. 4. crescent is expressed in dorsal yolky cells of the blastocoele floor at blastula, in anterior mesendoderm at gastrula, and in the pronephros at tailbud stages. In situ hybridization with a crescent antisense probe was performed on sagittally hemisectioned (A–E,G) or on intact embryos (F,H). (A) Internal view of a hemisectioned blastula (stage 9) showing expression in the yolky dorsal blastocoele floor; most of the transcripts are still nuclear indicating the onset of transcription. (B,C) At later blastula (stages 9.5,10−) crescent labeled cells are found more vegetally in the deep layers of the dorsal endoderm. (D) At the onset of gastrulation (stage 10+) the expression in the deep cells intermingle with a new expression domain in the inner layer of the blastopore lip. (E) At stage 10.5 involuting anterior mesendoderm cells have increased transcript levels, whereas the expression in the leading edge of the dorsal endoderm weakens. (F) Dorsal view of a late gastrula (stage 12) showing expression in the prechordal plate and anterior (pharyngeal) endoderm. (G) Internal view of a late neurula (stage 16) showing strong staining in the anterior endoderm (ae) and in the prechordal plate (pp) underlying the developing forebrain. White arrowhead demarcates the anterior limit of the neural plate or head fold. (H) Late tailbud embryo (stage 28) in lateral view displaying expression in the pronephros (pn); note that expression in anterior endomesoderm has disappeared by this stage.|
|frzb2 ( frizzled-related protein 2) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 10.25, dorsal left, animal pole up|
|frzb2 ( frizzled-related protein 2) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 10.5, dorsal left, animal pole up|
|frzb2 ( frizzled-related protein 2) gene expression in Xenopus laevis embryo longitudinal section, assayed via in situ hybridization, NF stage 16, dorsal up, anterior left.|
|frzb2 (frizzled-related protein 2 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.|
|Fig. 6. Microinjection of crescent mRNA does not inhibit development of anterior neural structures. (A,B) Overexpression of crescent mRNA enlarges the expression of a dorso-anterior marker in whole embryos. Neurulae (stage 16) in anterior view after whole mount in situ hybridization with Otx-2 and krox-20. (A) Uninjected control; Otx-2 expression is seen in the prospective forebrain (fb) and midbrain (mb). The krox-20 stripes mark rhombomeres 3 and 5. (B) Embryo injected with 4 ng crescent mRNA into the animal pole; the Otx-2 fore- and midbrain domain is enlarged; note that the posterior expression is continuous and not interrupted by a gap as in the uninjected sample. The anterior krox-20 stripe in rhombomere 3 is wider and stripe 5 is missing; Otx-2 staining in anterior endoderm is decreased (C) crescent or frzb-1 in combination with chordin promote dorsalization of the ectoderm (neural induction) in the absence of meso- derm. Animal caps were excised from injected embryos at stage 9, cultured until stage 27 and assayed by RT-PCR. N-CAM is a pan-neural marker, a- actin is a marker for mesoderm induction, and EF-1a a loading control.|
|Fig. 7. Overexpression of crescent, but not of frzb-1 mRNA, perturbs the anterior extension of midline markers. Neurula embryos (stage 14) shown in anterior view were uninjected (left), radially injected with crescent RNA (400 pg, middle) or frzb-1 RNA (400 pg, right), and analyzed by whole mount in situ hybridization. (A) The horseshoe-shaped expression domain of rx-2A demarcates the prospective eye ®eld in the anterior neural plate. Note the posterior indentation of the eye ®eld. (B) In crescent-injected embryos the rx-2A expression domain is narrowed towards the midline and lacks the indentation separating the two eye primordia. (C) frzb-1 injection increases the width of the rx-2A domain. (D) vax-1 expression labels the anteriormost medial section of the neural plate fated to become basal forebrain and optic stalk. (E) crescent blocks vax-1 RNA expression. (F) frzb-1 does not signi®cantly affect vax-1 expression. (G) The midline marker and signaling factor shh is expressed in the anterior pharyngeal endoderm, prechordal plate, notochord, ̄oorplate and prospective ventral forebrain; krox-20 demarcates rhombomeres 3 and 5. The expression in the prechordal plate and notochord is obscured by the staining in the overlying ectoderm. (H) crescent reduces the anteroposterior extent of the shh domain and broadens it; note the signi®cantly shorter distance between the anterior tip of the shh domain and the remaining krox-20 stripe in rhombomere 3. shh expression is absent from the normal position of the prechordal plate, ventral forebrain and anterior endoderm. (I) frzb-1 broadens the shh domain at the level of the prechordal plate, ventral brain, ̄oor plate and notochord; krox-20 transcripts in rhombomere 5 are inhibited by frzb-1, as is the case for crescent. (J) noggin expression is evident in the prechordal plate, noto- chord and in a ®ne arc demarcating the anterior margin of the neural plate. (K) crescent shortens and broadens the midline domain of noggin expres- sion. Importantly, the gap between noggin in the anterior midline and in the anterior border of the neural plate is increased, indicating a posteriorization or loss of the prechordal plate. (L) frzb-1 widens noggin expression along the midline, and decreases the distance between the anterior tip and the anterior border of the neural plate, implying an enlargement of the prechor- dal plate. (M) The anterior midline marker gsc demarcates anterior endo- derm and prechordal plate. (N) crescent downregulates gsc RNA; expression is weaker at the prechordal plate and blocked in pharyngeal endoderm. (O) frzb-1 does not alter gsc in the anterior endoderm but widens its domain of expression at the level of the prechordal plate.|
|Fig. 5. Overexpression of crescent, but not of frzb-1, results in cyclopia. (A± D) Tadpole embryos (stage 40) in anterior view. (A) Uninjected control embryo. Note the two lateral eyes (e) and nasal placodes (white arrow- heads), the medial mouth (m) and cement gland (cg). (B) Embryo after four radial injections with crescent RNA at 50 pg/blastomere. Note that the eyes are fused along the midline, as are the nasal placodes (white arrow- head); the cement gland is smaller and the mouth missing. (C) Embryo injected with crescent RNA at higher doses (100 pg/blastomere) showing a single eye vesicle. The nasal placode, mouth and cement gland are not formed. (D) Embryo after injection with frzb-1 RNA (100 pg /blastomere). The distance between the two lateral eyes and between the nasal cavities (white arrowheads) is increased, the mouth is broadened and the cement gland signi®cantly enlarged. (E±G) Lateral view of swimming tadpole embryos at stage 43. (E) Uninjected control. (F) Embryo radially injected with crescent RNA (200 pg total) with cyclopia and reduced pigmentation. (G) Embryo radially injected with frzb-1 RNA (400 pg total). Note the enlargement of the head and of the ventral ®ssure of the eye (coloboma), which is thought to result from an increase of anterior ventral midline structures.|