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.

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