Condensation and fusion of the head lobes (S13-15)

While the general morphology of the embryonic head observed during stage 12 is maintained during stage 13, the number of cells that each head lobe comprises increases. As a consequence, the embryonic head reaches its largest expansion on the egg's surface (Figure 4 A1-A4). Directly following from that point of embryogenesis, the head lobes start to condense and fuse. At the onset of stage 14, each individual head lobe has narrowed (Figure 4 B2, B3).

Densely packed groups of cells flag the appendage anlagen of An1 and An2 (Figure 4 B1-B4). Bordering the Mn and An2 segments laterally, the midgut anlagen have emerged as distinguishable, ovoid structures (Figure 4 B2, B4, pink asterisks; Browne et al., 2005). The medial cleft that separates the head lobes is still visible as a region of low cell density (Figure 4 B1-B3, red arrowheads). It directly abuts the anterior of the future Mn segment and reaches until the anterior end of the embryo. Within the posterior part of the cleft, at the level of the An2 segment, cells undergo mitoses (Figure 4 B3, green arrow). At the same time, a cluster of cells has emerged at the anterior end of the cleft (Figure 4 B3, blue arrow). In the middle of stage 14, cells cover the medial region of the former separated head lobes at the level of the An2 segment (Figure 4 C1, C2, blue arrows). At the onset of stage 15, the fusion of the head lobes is almost completed. Only a small patch located medially to the An1 appendage anlagen still exhibits lower cell density (Figure 4 D1, D2, blue arrows). The An1, An2 and Mn segments have condensed further. The appendages of these segments start to project distally.

In contrast to the condensing head segments, the lateral midgut anlagen have expanded. Their periphery is composed of closely arranged cells (Figure 4 D1, D2, pink asterisks). Therefore, they delineate sharply from the ectodermal segments of the embryo. In embryos that have completed stage 15, the pre-antennal hemispheres of the embryonic head project laterally. The centres of each of these projections show a high number of densely clustered cells that are arranged in multiple layers (Figure 4 D3, D4, white arrows). The medial cleft between the head lobes has been replaced by a single-layered, triangular sheet of cells (Figure 4 D4, red arrow). This sheet of cells is bordered laterally by the emerging appendage anlagen of the An1, An2 and Mn segments, which are clearly visible as proximal-distal outgrowths (Figure 4 D4, yellow, orange and green asterisks indicate the An1, An2 and Mn appendage anlagen, respectively). The anterior end of the embryo has acquired the shape of an almost horizontal border line that separates densely clustered embryonic cells from few scattered extra-embryonic cells, with the lateral anterior-most parts of the pre-antennal hemispheres bulging

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slightly more anterior than the medial region of the pre-antennal head (Figure 4 D3). The presence of Distal-less protein directly anterior to the An1 segment in a confined area medially within the pre-antennal head indicates the onset of the organisation of the labral developmental field (Browne et al., 2005).

Figure 4 (next page): Parhyale head development during stages 13 to 15. Shown are grey-scale images of nuclear labelled embryos (SYTOX®). In all images, anterior is up. A Stage 13 Parhyale embryo. Red arrowheads mark the anterior end of the medial cleft in all images of this embryo. The embryo had been subjected to Ph hbn WMISH (3.2.2.9). A1 Dorsal view. A blue arrow indicates the dorsal organ. The dorsal organ emerges at stage 10 of Parhyale embryogenesis (Browne et al., 2005). A2 Anterior view. The appendage field of the An1 segment is indicated by a yellow asterisk. A3 Anterior view; the embryo is tilted to the left. Directly bordering the appendage field of the An1 segment (yellow asterisk) laterally, the midgut anlagen have increased in size as compared to stage 12 (Browne et al., 2005). At this stage, the embryonic head reaches its largest expansion on the egg's surface. A4 Ventral view; the embryo is tilted to the left. The appendage field of the An1 segment is indicated by a yellow asterisk and the site of the future Mn segment is marked by a green asterisk. B Parhyale embryo at the onset of stage 14. Red arrowheads mark the medial cleft (B1-3). The appendage field of the An1 segment is indicated by a yellow asterisk (B1-4) and that of the An2 segment by an orange asterisk (B2-4). The lateral, ovoid midgut anlagen are marked by a pink asterisk (B2, B4). At the anterior end within the medial cleft, a cluster of cells has emerged (blue arrows, B1, B2). Within the posterior end of the medial cleft, cells undergo mitoses (green arrow, B3). The embryo had been subjected to Ph six3 WMISH (3.3.1.7). B1 Anterior view. B2 Lateral view; the embryo is tilted to the right. B3 Ventral view. B4 Lateral view. C Parhyale embryo in the middle of stage 14. At the level of the An2 segment, cells cover the medial region of the previously separated head lobes (blue arrows, C1, C2). The remaining medial cleft is marked by red arrowheads (C1, C2). The appendage field of the An1 segment is indicated by a yellow asterisk (C1-4) and that of the An2 segment by an orange asterisk (C1-4). The embryo had been subjected to Ph kni1 WMISH (3.4.7). C1 Anterior view. C2 Anterior view, the embryo is tilted slightly to the right. C3 Ventral view. C4 Lateral view, sagittal focal plane. D Parhyale embryo at the onset (D1, D2) and at the end (D3, D4) of stage 15. D1 Tilted ventral and D2 ventral view of a Parhyale embryo at the onset of stage 15.

Except a restricted medial area at the level of the An1 appendage anlagen (blue arrows), the fusion of the former head lobes is complete. The appendages of the An1 (yellow asterisks), An2 (orange asterisks) and Mn (green asterisks) segments start to project distally. In D1, the left appendage anlagen are marked. In D2, the right ones are. The left (D1) and right (D2) lateral midgut anlagen are marked by pink asterisks. The embryo had been subjected to Ph kni1 WMISH (3.4.7). D3 Dorsal and D4 ventral view of a Parhyale embryo that has completed stage 15. The pre-antennal hemispheres of the embryonic head project laterally. In their central parts, they show densely clustered cells that are arranged in multiple layers (white arrows). Medially, a single-layered, triangular sheet of cells has emerged (red arrow). It is bordered laterally by the appendage anlagen of the An1 (yellow asterisks), An2 (orange asterisks) and Mn segments (green asterisk, D4). The embryo had been subjected to Ph six4 WMISH (3.3.1.8).

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Figure 4: Parhyale head development during stages 13 to 15.

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3.1.3 Establishment of lateral pre-antennal hemispheres, the stomodeum and