Establishment of lateral pre-antennal hemispheres, the stomodeum and the labrum (S16-20)

During stages 16, 17 and 18, the pre-antennal, laterally projected hemispheres undergo significant cell proliferation. The cells become smaller, more densely packed and have point-shaped nuclei. Arched ridges reaching from the medial area to the lateral hemispheres show the highest density of nuclei, indicating multiple layers of cells (Figure 5 A1, A2, A3, B4, red arrows). In contrast, the periphery of an individual pre-antennal hemisphere appears to be composed of fewer cells (Figure 5 A1, A2, B4, areas adjacent to red arrows). The An1 and An2 appendages elongate and produce additional articles (Figure 5 A2, A4, B2, B4, C2, C4).

Their development is advanced in comparison to the thoracic appendage anlagen (Figure 5 B2, see also Figures 74 and 75). At the onset of stage 18, two small opposing medial outgrowths, situated between the An1 and An2 segments, mark the onset of stomodeum formation. (Figure 5 C1, blue arrows; compare to A4, blue arrow). During stage 18, the stomodeum begins to invaginate. At the beginning of stage 19, the two opposing outgrowths are clearly visible on lateral positions within the deepening stomodeal opening. Subsequently, they will be referred to as “lateral stomodeal projections” (Figure 5 D1, stomodeal field is highlighted red, lateral stomodeal projections are highlighted white; D2-D4, red arrows indicate the anterior and posterior boundaries of the stomodeal field). The anterior ridge of the stomodeum becomes first visible as a short horizontal stripe with a high number of densely clustered nuclei (Figure 5 E1, red arrowhead), similar to the outlines of the developing An1, An2 and Mn appendages. As the oesophagus invaginates during the stages 19 and 20, the stomodeal field itself becomes smaller (Figure 6). Anterior of the stomodeum, the labrum becomes visible at stage 19 (Figure 5 E2, F3, red asterisks). It begins to protrude distally, tending towards the posterior (Figure 6). At the end of stage 20, the labrum is visibly composed of two lobes that begin to grow over the distinct stomodeal opening (Figure 6 A3, A4, C3, C4). At stage 20, just posterior to the labrum, the brink of the stomodeum is visible as a raised medial ridge at the level of the An2 segment (Figure 6 C4; Browne et al., 2005).

During stage 19, the lateral portions of the pre-antennal hemispheres expand. As a consequence, they appear wedge-shaped (Figure 5 E1, E2, F2, F4) and extend in part to the dorsal face of the egg (Figure 5 F1). As a consequence, the anterior borderline appears now more V-shaped, as the expanding preantennal hemispheres conclude an angle of about 100-120° (Figure 5 E1, F2, white arrows; F1, black arrows). The delineation between embryonic

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tissue and the extra-embryonic dorsal region of the egg is clearly cut (Figure 5 E1, F1).

During stage 19 the paragnaths become visible just medial to the mandibles (Figure 6 C1-C3;

Browne et al., 2005).

Figure 5 (next page): Parhyale head development from stage 16 to the onset of stage 20. Shown are grey-scale images of nuclear labelled embryos (SYTOX®) and one grey-scale DIC image (F1). In all images, anterior is up. A Early stage 17 Parhyale embryo in dorsal (A1), tilted anterior (A2), lateral (A3) and anterior (A4) view. Red arrows indicate the arched ridges that show the highest density of nuclei within the pre-antennal hemispheres. Yellow asterisks mark the An1 appendage anlagen. The blue arrow (A4) points towards a cluster of cell that will subsequently form one of the lateral oesophageal projections (see also C1). The embryo had been subjected to Ph kni1 WMISH (3.4.7). B Stage 17 (B1, B2) and late stage 17 (B3, B4) Parhyale embryos. Yellow and orange asterisks mark the elongating An1 and An2 appendage anlagen, respectively. B1 Tilted anterior and B2 tilted ventral view of a stage 17 embryo. In B2, the thoracic appendage anlagen of T2, T3 and T4 are labelled (see also al1S17). The embryo had been subjected to Ph al1 WMISH (see R). B3 Anterior view, focal plane on the medial region at the level of the An1 segment, and B4 Anterior view, focal plane on the pre-antennal ridges of a late stage 17 embryo. A red arrow (B4) indicates the left pre-antennal ridge (see also A1-A3).

The embryo had been subjected to Ph kni1 WMISH (3.4.7). C Parhyale embryo at the onset of stage 18. Yellow and orange asterisks mark the An1 and An2 appendages, respectively. C1 Anterior view. Blue arrows point to two small opposing medial outgrowths that will subsequently be recognised as lateral oesophageal projections (see also E2). C2 Tilted ventral view. C3 Tilted anterior view. C4 Lateral view. The embryo had not been subjected to WMISH. D Parhyale embryo at the onset of stage 19. The stomodeum forms. The embryo had been subjected to Ph al1 WMISH (3.2.2.10). D1 Anterior view. The stomodeal field is highlighted red. The lateral oesophageal projections are highlighted white. Yellow and orange asterisks mark the An1 and An2 appendages, respectively. D2 Ventral view. D3 Ventral view, alternative focal plane. D4 Lateral view, sagittal focal plane. Red arrows (D2-D4) indicate the upper and lower boundaries of the invaginating stomodeal field. E Parhyale embryo at stage 19. The embryo had been subjected to Ph gbx WMISH (3.3.2.4). E1 Anterior view. As a consequence of the lateral expansion of the pre-antennal hemispheres, the anterior borderline appears V-shaped with an angle of about 100-120° (white arrows). The anterior ridge of the stomodeum is indicated by a red arrowhead. E2 Anterior view, focal plane on the lateral oesophageal projections (white arrowhead). Anterior of the stomodeum, the outgrowing labrum is recognisable as a dense cluster of nuclei (red asterisk). E3 Ventral view. White arrowheads mark the lateral oesophageal projections. E4 Lateral view, sagittal focal plane. The position of the labrum is indicated by a red asterisk. Red arrows mark the anterior and posterior brinks of the invaginating stomodeum. A white arrowhead points at the lateral oesophageal projections. F Parhyale embryo at the onset of stage 20. The embryo had been subjected to Ph kni1 WMISH (3.4.7). F1 DIC image of the embryo in dorsal view. The anterior end of the embryonic head delineates sharply from the dorsal, extra-embryonic region. The pre-antennal hemispheres conclude an angle of about 100-120° (black arrows). F2 Anterior view, focal plane on the anterior end of the embryonic head. The pre-antennal hemispheres conclude an angle of about 100-120°

(white arrows). F3 Anterior view, focal plane on the labrum (indicated by a red asterisk). The white arrowheads point towards the lateral oesophageal projections. F4 Lateral view, sagittal focal plane. The pre-antennal hemispheres have expanded during stages 18 and 19 and now appear wedge-shaped (white arrows). The position of the labrum is indicated by a red asterisk. Red arrows mark the anterior and posterior brinks of the invaginating stomodeum. A white arrowhead points at the lateral oesophageal projections.

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Figure 5: Parhyale head development from stage 16 to the onset of stage 20.

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Figure 6: Development of stomodeum and labrum in Parhyale during stage 20. Shown are grey-scale images of nuclear labelled embryos (SYTOX®). In all images, anterior is up. Shown is the gradual invagination of the stomodeum along with the elongation of the labrum from the onset of stage 20 (#1) through early stage 20 (#2) and the mid of stage 20 (#3) until the end of stage 20 (#4).

A red arrow indicates the posterior stomodeal ridge. The embryos had been subjected to Ph six3 WMISH (A1, B1, C1; 3.3.1.7), to Ph hbn WMISH (A2, B2; 3.2.2.9), to Ph kni1 WMISH (C2; 3.4.7), to Ph six4 WMISH (A3, B3, C3; 3.3.1.8) and to Ph awh WMISH (A4, B4, C4; 3.2.2.4). A Tilted anterior (A1) and anterior (A2-A4) views. Yellow and orange asterisks mark the distal articles of the An1 and An2 appendages, respectively. Red asterisks mark the left lobe of the bi-lobed labrum in A3 and A4. B Lateral views, sagittal focal plane. Red asterisks mark the distal tip of the labrum. C Ventral views, focal plane on the Mn segments. White arrows indicate the emerging paragnaths (C1, C2, C3).

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