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doi: 10.1242/10.1242/dev.00099

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Expression of pair-rule gene homologues in a chelicerate: early patterning of the two-spotted spider mite Tetranychus urticae

Peter K. Dearden^1,2,*, Cameron Donly² and Miodrag Grbi^1,

¹ Department of Biology, University of Western Ontario, London, Ontario N6A 5B7, Canada
² Southern Crop Protection and Food Research Center, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario, N5V 4T3, Canada
^* Current address: Biochemistry Department, University of Otago, PO Box 56, Dunedin, New Zealand

View larger version (164K): [in a new window]   Fig. 1. Embryogenesis in the two-spotted spider mite. (A-D) Early cleavages. (A) Uncleaved egg. The nucleus is just starting to become visible in the centre of the egg. (B) First division, the egg nucleus (dark area) has divided. A clear membrane between the two blastomeres is visible (arrow). (C) Second division. (D) Blastoderm stage embryo. Nine divisions have taken place forming a cellular blastoderm. (E-F) Formation of the `germ disc', and the germ band. (E) At 20-22 hours AEL a thickened portion of the germ band is visible (arrow), probably in ventral regions, which we interpret to be the germ disc. The germ disc starts as an ovoid swelling (E,F), and then flattens (G). Flattening of the germ disc is quickly followed by the appearance of leg primordia on both sides of the ventral midline (viewed from the anterior in H). (I-L) Formation of the germ band, limb primordia and limb outgrowth (lateral view). All embryos are viewed with anterior to the left and dorsal up. (I) The initial limb primordia form by 23 hours AEL. P, pedipalps; 1-4, walking legs. Primordia for the chelicerabearing segment and the opisthosoma germ band become visible soon after. (J) C, chelicerae. (K,L) Limb buds grow and become jointed. (M-P) Formation of the germ band, limb primordia and limb growth (ventral view). All embryos are viewed from the ventral side, with anterior to the left. Embryos are of the same stage as those in I-L. Scale bars: 50 µm.

View larger version (101K): [in a new window]   Fig. 2. Dextran injections. (A-C) A two-cell stage embryo 60 minutes after injection with tetramethylrhodamine dextran into a single blastomere. Dye has diffused into both blastomeres. (D-F) A four-cell stage embryo 60 minutes after injection with tetramethylrhodamine dextran. Dye remains localised in the injected blastomere. (G-H) A 16-cell stage embryo 60 minutes after injection with tetramethylrhodamine dextran. Dye remains localised in the injected blastomere. Scale bars: 20 µm.

View larger version (65K): [in a new window]   Fig. 3. Sequence analysis. (A) Multiple sequence alignment of the most conserved domain of runt-like proteins including Tu-run. Dr_Runt1, Danio rerio runt; Aml1, human AML1 protein; Sp_runt, Strongylocentrotus purpuratus; Cs_runt 1 and 2, Cupiennius salei runt1 and 2; Lozenge, Drosophila melanogaster loz; Dm_runt Drosophila melanogaster runt. (B) Multiple alignment of the C-terminal VWRPY motif of Runt-like proteins. He_Runt1, Heliocidaris erythrogramma; Gg_runtb, chick runtB; Xl-aml1, Xenopus laevis AML-like protein 1; Mm_runt1 and 3, mouse runt 1 and 3; Hs-aml1 and 2a, human AML-like proteins 1 and 2a; Dr_runtb, Danio rerio runt b; Dm_loz, Drosophila melanogaster lozenge; Ds_runt, Drosophila simulans runt; Ce_runt1, Caenorhabditis elegans runt1. (C) Unrooted maximum likelihood cladogram of the multiple alignment shown in A. (D) Amino acid sequence alignment of the cloned region of Tu-pax3/7 with other PgIII genes. Boxed region shows the amino acids used for the phylogenetic analysis shown in F. Sa_PB1 and 2, Schistocerca americana Pairberry 1 and 2; Dm_prd, Dm_gbp-D, Dm_gbp-P, Drosophila melanogaster paired, gooseberry paired distal and gooseberry paired proximal respectively; pax 7 and 3, mouse pax7 and 3. (E) Unrooted maximum likelihood cladogram of the region of the alignment shown in D including representatives of all pax group genes. Pax1-9, mouse Pax proteins; pox m, pox messo; spark, sparkling; ey, eyeless proteins from Drosophila melanogaster; paxD and paxb from Acropora millepora. (F) Unrooted maximum likelihood cladogram of the boxed region of the alignment in D. Tu-run GenBank accession number, AY148194; Tu-pax3/7 GenBank accession number: AY148194.

View larger version (90K): [in a new window]   Fig. 4. Expression of Tu-run and Dll in spider mite embryos. (A-C) Detection of Tu-run using in situ hybridisation. Embryos are oriented with dorsal up and anterior to the left. Arrowhead indicates the first walking leg segment in all panels. (A) Tu-run RNA is first detected in five pairs of rings in ventral regions of the embryo (blue). (B) The posterior side of each ring of cells loses detectable Tu-run expression, forming stripes across the ventral midline. (C) Expression of Tu-run becomes undetectable in stripes in the epidermis, and segmental stripes appear in the nervous system. (D-F) Lateral view of embryos hybridised with an RNA probe for Tu-run RNA (blue), and stained with an antibody that detects Dll protein (brown). Embryos are oriented with dorsal up and anterior to the left. Dll protein marks the developing limb buds. (D) The rings of cells expressing Tu-run RNA entirely surround cells expressing Dll protein. (E) As Tu-run comes to be expressed in stripes of cells, these stripes lie directly anterior to Dll-expressing cells. (F) As epidermal expression of Tu-run RNA becomes undetectable, both Dll protein (asterisk) and Tu-run RNA are expressed in cells in the nervous system. (G-I) Ventral view of embryos hybridised with an RNA probe for Tu-run RNA (blue), and stained with an antibody that detects Dll protein (brown). Embryos are oriented looking down on the ventral surface with anterior to the left. (G) Ventral view of Tu-run RNA surrounding Dll protein-expressing cells. (H) Stripes of Tu-run RNA-expressing cells lie directly anterior to cells expressing Dll protein. (I) Ventral view of the central nervous system showing Tu-run RNA-expressing cells. Arrow marks the fourth walking leg primordium. (J-L) Spider mite embryos stained for Dll protein (black). Embryos are oriented with dorsal up and anterior to the left except L where the embryo is oriented looking down on the ventral surface. Distal-less is initially expressed only in limb buds. (J) Expression of Dll protein in the chelicerae, pedipalp limb buds and the four walking legs. Dll expression in the fourth walking leg is out of focus. (K) Expression of Dll in the limbs as they grow. (L) At the late germband stage, Dll is expressed in a ring of cells in proximal regions of the limb, and a broad domain of cells at the distal tip. Scale bars: 50 µm.

View larger version (57K): [in a new window]   Fig. 5. Expression of Tu-pax3/7 in blastoderm stage embryos. (A,C,E,G,I) Surface view of blastoderm stage embryos hybridised for Tu-pax3/7 RNA. (B,D,F,H,J) Deeper focal plane of blastoderm stage embryos hybridised for Tu-pax3/7 RNA. (A,B) Tu-pax3/7 RNA is first expressed in three stripes of cells across the ventral surface, corresponding to the pedipalp segment, the second walking leg and the fourth walking leg. (C,D) Expression of Tu-pax3/7 RNA becomes apparent in a stripe in the first walking leg segment (asterisks). (E,F) Embryo with four detectable stripes of Tu-pax3/7-expressing cells, corresponding to the pedipalps, the first and second walking legs and the fourth walking leg. (G,H) Expression of Tu-pax3/7 RNA becomes apparent in a stripe in the third walking leg segment (asterisks). (I,J) Embryo with five detectable stripes of Tu-pax3/7 expression, corresponding to all the segments of the prosoma except the chelicera segment. A small stripe is also forming posterior to the fourth walking leg segment (arrows). Tu-pax3/7 expression is starting to disappear from cells in the centre of each stripe in the prosoma. Scale bars: 50 µm. P, pedipalp; 1-4, walking legs. Anterior to the left, dorsal up.

View larger version (113K): [in a new window]   Fig. 6. Expression of Tu-pax3/7 and Distalless. (A-D) Lateral view of embryos hybridised for Tu-pax3/7 RNA (blue). Anterior to the left, dorsal is up. (A) Surface view of an embryo showing the full pattern of Tu-pax3/7 RNA stripes in the prosoma. (B) Deeper focal plane of an embryo showing one stripe of cells expressing Tu-pax3/7 RNA in the opisthosoma, as well as the prosoma pattern. (C) Later epidermal expression of Tu-pax3/7. Cells expressing Tu-pax3/7 RNA are visible in all the prosoma segments and in three segments in the opisthosoma. (D) Late expression of Tu-pax3/7 in the nervous system. (E-H) Ventral view of embryos hybridised for Tu-pax3/7 RNA (blue) and stained for Dll protein (brown). Anterior to the left. (E) Cells within the anterior region of the Tu-pax3/7 RNA-expressing stripes in the prosoma begin to express Dll protein. In cells expressing Dll protein, expression of Tu-pax3/7 RNA rapidly becomes undetectable. (F) Dll expression spreads across the entire Tu-pax3/7 expression domain. Tu-pax3/7 becomes restricted to a square group of cells just ventral to the Dll-expressing limb bud. (G) Dll expression retreats from the proximal regions of the limb bud. (H) Expression of Tu-pax3/7 in the nervous system. The arrow indicates the primordia of the fourth walking leg. (I-L) Lateral view of embryos hybridised for Tu-pax3/7 RNA (blue) and stained for Dll protein (brown). Anterior to the left, dorsal up. (I) Expression of Dll protein appears in Tu-pax3/7-expressing cells in each stripe. These cells rapidly lose expression of Tu-pax3/7. (J) Expression of Dll and loss of Tu-pax3/7 expression spreads across the stripe. (K) Dll expression retreats from proximal regions of the limb. (L) Expression of Tu-pax3/7 in the nervous system. Dark staining in the anterior of the embryo is caused by damage during preparation. (M-P) Embryos oriented to examine expression of Tu-pax3/7 RNA (blue) and Dll protein (brown) in the opisthosoma. Anterior parts of the germ band curl away from the focal plane to the left; embryos are viewed from the ventral surface. (M) One broad stripe of Tu-pax3/7-expressing cells is visible posterior to the fourth walking leg stripe (marked by expression of Dll). This is joined by a second stripe (N), and a third (O). Later, expression of Tu-pax3/7 RNA is visible in three stripes in the central nervous system (P). Arrowheads indicate the first walking leg segment. Scale bars: 50 µm.

View larger version (26K): [in a new window]   Fig. 7. Schematic diagram of PgIII expression and segment identity in the anterior segmented germ band in Drosophila, Schistocerca and Tetranychus. Dotted horizontal lines define the boundaries of homologous segments in insects and chelicerates as described by Damen et al. (Damen et al., 1998) and Telford and Thomas (Telford and Thomas, 1998). Solid horizontal lines represent parasegment boundaries in Drosophila, and possible parasegment boundaries in Schistocerca and Tetranychus. Solid green bars represent engrailed expression in Drosophila; solid blue bars represent primary stripes of PgIII genes; hatched blue bars represent secondary stripes. Pair-rule modulated stripes of PgIII genes in insects and Tetranychus form with a different register.