XB-ART-9862Dev Biol December 15, 2000; 228 (2): 197-210.
Use of large-scale expression cloning screens in the Xenopus laevis tadpole to identify gene function.
We have conducted an expression cloning screen of approximately 50, 000 cDNAs from a tadpole stage Xenopus laevis cDNA library to functionally identify genes affecting a wide range of cellular and developmental processes. Fifty-seven cDNAs were isolated for their ability to alter gross tadpole morphology or the expression patterns of tissue-specific markers. Thirty-seven of the cDNAs have not been previously described for Xenopus, and 15 of these show little or no similarity to sequences in the NCBI database. The screen and the identified genes are presented in this paper to demonstrate the power, ease, speed, and flexibility of expression cloning in the X. laevis embryo. Future screens such as this one can be done on a larger scale and will complement the sequence-based screens and genome-sequencing projects which are producing a large body of novel genes without ascribed functions.
PubMed ID: 11112324
Article link: Dev Biol
Genes referenced: akt1 egr2 hba1 hoxb9 hoxc9-like lrrfip1 myod1 nkx2-5 nrp1 pax8 rac1 sox2 tbx2
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|FIG. 2. The normal expression patterns of the in situ markers are shown at the stages used in this screen (stage 21 (A) and stage 28 (D)). All embryos are shown with anterior facing left. (A) Anterodorsal views and (D) lateral views. (A) Nrp-1 staining of general neural tissue (eyes, brain, and spinal cord). (B) krox-20 and Hoxb9 were stained simultaneously. krox-20 stains the R3 and R5 rhombomeres of the hindbrain (black arrow) as well as a band of migrating neural crest cells (black arrowhead). Hoxb9 stains the spinal cord (red arrowhead). (C) MyoD stains the somites of the paraxial mesoderm which will give rise to skeletal muscle. (D) Pax-8 stains the pronephric tubule and duct of the developing kidney, as well as the otic vesicle of the developing ear (black arrowhead). (E) Nkx-2.5 stains the developing heart. (F) a-globin stains the developing ventral blood islands.|
|FIG. 6. Pools of mRNAs induce changes in a-globin and Pax-8 expression. Individual clones were not isolated (see text). All embryos are shown with anterior to the left. (A) Uninjected control stained for expression of a-globin (ventral blood islands). (B, C, D) Embryos injected with pools 18A1 (B), 28B1 (C), and 28B2 (D) showed disruption of a-globin and gross morphology. (E) Uninjected control stained for expression of Pax-8 (otic vesicle and kidney). (F, G, H) Pools 120A2 (F), 121A2 (G), and 5A2 (H) perturbed the expression of Pax-8. Embryos injected with 5A2 also showed a distinct head kink.|
|hba1 (hemoglobin subunit alpha) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.|
|Fig 3. Secondary axis, anterior defect, and ectopic pigmentation. (A) 77E19 (ﰄ-catenin) induces a complete secondary axis. Ectopic cement gland marked with red arrow, normal cement gland marked with white arrowhead. (B) Clone 31N15 (Wnt-7b) also induces a complete secondary axis. This embryo has been stained with MyoD. (C) Embryo injected with clone 81C8A/81E10C (similar to KIAA0058) displays anterior defects. (D) Injection with 2H23 (Sox-2). This embryo shows ectopic pigmentation continuous with the cement gland (red arrowhead). (E) Uninjected control. (F) Embryo injected with 17J10 (Forkhead-2) shows ectopic pigmentation.|
|Fig. 4. Apoptosis, epithelial disruptions, and edema. (A) Unin- jected control, stage 10. (B) Embryo injected with clone 19N22 (vex-1) begins to show the mottled phenotype characteristic of apoptosis. (C) Embryo injected with 19K13 (similar to caspase-2) shows more severe apoptosis. (D) At later stages (here, st. 23), the dead cells are extruded into the vitelline space. Once the embryo hatches out, these cells will fall off and the embryo will look morphologically normal. This embryo was injected with 10A7b (similar to trip-7). (E) Embryos injected with 118B6A (similar to xepsin) show an epidermal defect. (F, G) Embryos injected with 101J13 (Rac-1) display ventral edema. (F) Stage 20. (G) Stage 28. Note that at later stages the fluid-filled sac has regressed.|
|FIG. 5. Disruptions of mRNA in situ hybridization patterns. (A, B) Embryos injected with 118B6A (similar to xepsin) show disrupted expression of nrp-1, including loss of eye (black arrows, A) and ectopic nrp-1. (A) Anterior view. (B) Dorsal view. (C) Injection of 118N8 (ras) also disrupts nrp-1. (D) 77K7A (Hoxd3)-injected embryos lose krox-20 expression in rhombomere 3 (red arrows). (E) Embryos injected with 82N15A (similar to Hoxc10) show loss of krox-20 in both rhombomeres 3 and 5. (F) 118N8 (ras) injections also disrupt expression of MyoD.|
|The developing ventral blood island, stained for hba1 (hemoglobin subunit alpha 1, also called alpha-globin) via in situ hybridization, in a X. laevis NF stage 28 embryo, lateral view, anterior left, dorsal up.|