Winning RS and Sargent TD (1994), Pagliaccio, a member of the Eph family of recep...

XB-ART-21213

Mech Dev 1994 Jun 01;463:219-29.

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Pagliaccio, a member of the Eph family of receptor tyrosine kinase genes, has localized expression in a subset of neural crest and neural tissues in Xenopus laevis embryos.

Winning RS , Sargent TD .

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Cranial neural crest cells arise from neural folds in the embryonic head and differentiate to produce most of the cartilages and bones of the skull and the somatosensory ganglia of several cranial nerves, among other tissues. Since the molecular basis of the determination of these cells is poorly understood, we have begun a search for molecules involved in signal transduction in cranial neural crest. From a Xenopus laevis cranial neural crest cDNA bank, we have cloned a cDNA encoding a putative receptor tyrosine kinase, which we call Pagliaccio (Pag). Pag RNA is present transiently in visceral arch 3, probably representing neural crest cells in this tissue. Pag is also expressed in the forebrain, rhombomeres r3 and r5 of the hindbrain and in the pronephros. Based on this localized expression, we propose that Pag may play a role in the differentiation of cranial neural crest and other tissues.

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Species referenced: Xenopus laevis
Genes referenced: epha4 tbx2

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	Fig. I. Schematic representation of construction of the cranial neural crest eDNA bank. Poly (A+) RNA prepared from cranial neural crest was reverse transcribed, using a random hexamer (N6) tagged with an adaptor sequence (adaptor l) as a primer. Second strand eDNA synthesis was primed with a random hexamer tagged with a different adaptor sequence (adaptor 2) and extended with the Klenow fragment of DNA polymerase I. Adaptor sequences are listed in the Materials and Methods.
	Fig. 2. A. Schematic representation of the Pag eDNA clone. The 5' and 3' untranslated regions are represented by horizontal lines; the open reading frame is shown as a variably shaded box. Vertical lines in the extracellular domain indicate the positions of 19 cysteine residues conserved in all Eph..class kinases. The open circle above the kinase domain represents a tyrosine residue that is conserved in Eph-class kinases and is a putative autophosphorylation site (Sajjadi et al., 1991). Asterisks indicate stop codons. The DNA sequence around the start codon is shown, to indicate the in-frame upstream stop codon and translation initiation consensus sequence (underlined; Kozak, 1992). B. The deduced amino acid sequence of Pag. The putative signal peptide and transmembrahe domain are underlined, and the catalytic domain is delineated by arrows. The 19 conserved cysteine residues are indicated by filled circles, and the putative autophosphorylated tyrosine is indicated by an open circle, The Pag eDNA sequence is available through GenBank under accession number L26099.
	Fig. 3. Developmental expression of Pag, determined by Northern blotting. A. Northern blot of total RNA from oocyte, unfertilized egg and various embryonic stages, probed with Pag cDNA. The positions and sizes of the three bands are indicated, as are the positions of 28S and 18S ribosomal RNA. B. Northern blots of poly (A+) RNA from oocytes and stage 23 embryos, probed with Pag cDNA. The two lanes are from the same gel, but the oocyte lane is from an exposure ten times longer than that of the embryonic lane. The sizes of the three RNA bands shown in panel A are indicated by arrows.
	Fig. 4. The spatial expression pattern of Pag in Xenopus embryos, as determined by whole mount in situ hybridization. Except for panels A and D, in each case the anterior end of the embryo is to the left. The bar in each panel represents I mm. A. Gastrula-stage embryos (stage I I ), lateral view (left) and vegetal view (right). yp, yolk plug. B. Dorsal view of a neural plate-stage embryo (stage 14). The two paired stripes of expression in the neural plate are indicated by arrows. C. Lateral view of a neural fold-stage embryo (stage 16). Expression in rhombomeres (r) and forebrain (fb) is indicated by arrows, d, dorsal side; v, ventral side. D. The same stage as in C, anterior view. A tract of cells expressing Pag is observed adjacent to r5 (large arrow). A narrower, more ventral, tract of cells that also expresses Pag is indicated by a small arrow. E. Lateral view of a stage 22 (post-neurula) embryo; orientation is as in panel C. The two tracts of cells shown in panel D are indicated by large and small arrows, respectively. F. Lateral view ofa tailbud-stage embryo (stage 26). Pag expression in otic vesicle (small arrow) and pronephros (large arrow) is shown. G. Lateral view of a late tailbud-stage embryo (stage 37). Pag expression in rl (solid arrow) and above and below the stomodeum (open arrows) are indicated.
	Fig. 5. Transverse sections of embryos at stages 14 (A), 16 (B), 22 (C), 24 (D) and 26 (E) from whole mount in situ hybridization with Pag probe. The ventral limit of visceral arch Pag expression is indicated by a large arrow in each panel. Circumpharyngeal Pag expression is indicated by a small arrow in panel E. Due to the plane of section, Pag expression in r5 is not visible in some panels, ep, epidermis; np, neural plate; nf, neural folds; nt, neural tube; pc, pharyngeal cavity. The bar represents 1 um.
	Fig. 6. Comparison of the expression patterns of Pag (panel A) and Krox 20 (panel B) in stage 26 embryos, by whole mount in situ hybridization. The position of the otic vesicle (ov), adjacent to r4, is shown for reference. The same pair of rhombomeres, r3 and r5, express both Pag and Krox 20. The bar represents 1 mm.
	Fig. 7. Sections of embryos from whole mount in situ hybridization with Pag cRNA probe. Asymmetry of staining in some sections is due to slight obliquity in the plane of section. A. Transverse section through a stage 26 embryo at the level of rhombomere rS. Pag expression in r5 (r) and visceral arch 3 (v3) are shown. B. Transverse section of the same embryo at the level of the otic vesicle (ov), showing Pag expression in the dorsal epithelium of the vesicle. C. Transverse section farther caudad in the same embryo. Pag expression in the pronephric intermediate mesoderm (p) is indicated. D. Transverse section through a stage 37 embryo, at the level of r5 (r) and the heart (h). Staining in the innermost layer of the rhombomere (ventricular layer) is shown. E. Frontal section through a stage 37 embryo, showing staining in the ventricular layer of the forebrain (fb). e, eye; ov, otic vesicle. The bars in panels A and D represent l ram; panels B and C were at the same magnification as A, and panel E was at the same magnification as D.