Results
- Cell lineage analysis of the mandibular segment of the amphipod Orchestia cavimana reveals that the crustacean paragnaths are sternal outgrowths and not limbs

The early cell lineage of the mandibular region (E(0))

After gastrulation, the blastopore is closed. The germ disc consists of about 250 cells (Fig. 2A). The descendants of the macromeres B (left) and D (right) form the anterior parts of the germ disc by migration towards the middle (for details see Wolff and Scholtz [39]). At this time there is no regular cellular pattern in the ventral ectoderm. The first recognizable transverse ectoderm row occurs in a germ disc with about 400 cells (Fig. 2B). This row marks the border between the naupliar (head lobes, the segments of the first and second antennae and of the mandibles) and the post-naupliar (the segment of the first maxillae up to the telson anlage) regions of the developing embryo. This first recognizable cell row forms the genealogical unit E(1) which provides most of the material for the segment of the first maxilla (Fig. 2C). The cells anterior to E(1) show a typical spatial configuration. According to Scholtz [36] we named this region E(0), which forms part of the prospective mandibular segment (Fig. 2C). The cells of E(0) are somewhat smaller and rounder than the more posterior cells. They are arranged in two rows of at least 6 cells on either body half (Fig. 2C, 3A–B). The anterior row is designated as E(0)a and the posterior row as E(0)p (Fig. 3A,B). It is not clear whether these two rows originate from one transverse ectoderm row by longitudinally oriented cell divisions. A distinct unpaired column of midline cells as is typical for the post-naupliar region (Fig. 2E) does not exist in the naupliar part of the germ band. In the median area of the posterior naupliar region E(0) we found about 10–15 smaller cells in a V-shaped arrangement (Fig. 3A,B).

At about this stage, cells of the anterior row E(0)a divide more or less synchronously (mitotic wave) in a longitudinal direction. The products are the anterior row E(0)aa and the posterior row E(0)ap (Fig. 3A,B). The following division pattern of cells in row E(0)aa is not clear in detail but the cells divide symmetrically in each body half (see supplemental material). In row E(0)ap a detailed pattern could not completely be described but some features of the more medially lying cells of this row could be traced. The cells E(0)ap1 und E(0)ap2 are the first to divide in a longitudinal direction whereas E(0)ap3 divides a bit later in a horizontal direction (Fig. 4E). The cell E(0)ap4 shows a delayed division. In relation to the division cycles in row E(0)a, the cells of E(0)p are delayed in their division.

During the second mitotic wave in row E(0)aa, E(0)p2 is the first cell that divides in row E(0)p. It divides in a longitudinal direction. More or less at the same time the midline cell E(0)p0 divides also in a longitudinal direction. Subsequently the cells E(0)p3, E(0)p5, and E(0)p6 divide likewise in a longitudinal direction E(0)p2 (Fig. 4B,C). At the same time the relatively large cell E(0)p1 divides in a horizontal direction (Fig. 4C). At last, the cell E(0)p4 undergoes a division, again in a horizontal direction (Fig. 4D). In this developmental stage small buds of the naupliar appendages (first and second antennae, mandible) become visible (Fig. 4F). This invariant cleavage pattern in the prospective mandibular region eventually produces a reproducible arrangement of cells in the posterior region of E(0)(Fig. 4E).

This is an important pre-condition for our detailed lineage study of the mandibular segment. Unfortunately, we could not reconstruct the complete lineage pattern of the more anterior lying region E(0)a.

The clonal composition of the mandibular segment and its appendages

Identified cells of the transverse ectoderm rows around the boundary between the naupliar and post-naupliar regions were labeled in-vivo during the early germ band stage. The identification of cells is the prerequisite for the analysis of further cell fates and the clonal composition of morphological structures. Since a single cell approach was not feasible throughout, we divided the region E(0) from median to lateral into three sections. The median area, area I, begins lateral to the midline and ends before E(0)p2. The area II starts from E(0)p2 and comprises cell E(0)p3. The most lateral area III reaches from E(0)p4 to the end of the visible row formation (Fig. 4E). The successful in-vivo markings could be assigned to the corresponding areas.

The midline-region of E(0)

The median region of the mandibular segment does not consist of an unpaired column as is known for the post-naupliar ectoderm (the so-called midline, see [40,41]). From the onset, about 10 smaller cells are more sunken into the yolk than are the surrounding symmetrically arranged cells. Thereby, the median cells form a typical V-shaped region (Fig. 3A). During ongoing development a clear separation (in comparison with surrounding lateral cells) is recognizable (Figs. 3C, 4F). The surface of this median region loses its cellular character and has a smooth appearance.

The fate of area I

In relation to the more lateral adjoining cells (area II and III) the cells of area I of E(0) are retarded in cell division during segment formation. They form only a small portion (10–15 cells) of the early mandibular segment, and have their origin in the first column of region E(0) – E(0)a1 and E(0)p1.

In vivo labeling of cell E(0)a1 shows that its descendants form the medio-anterior part of the developing humps of the paragnaths. In addition, a median part of the mandibular sternite is formed by E(0)a1-descendants.

The descendants of E(0)p1 form postero-median parts of the paragnaths and adjacent areas of the sternite (Fig. 5A–C, 1). A few clones of E(0)p1 are found within the neuro-ectoderm in the posterior region of the mandibular segment and form nervous structures. Finally, after further development the ectodermal descendants are involved in the formation of postero-median parts of the paragnaths.

In general, area I forms the median mandibular sternite and median parts of the paragnaths. The clonal composition of a developed paragnath and developed parts of the sternite reflects the early a/p-arrangement of the rows.

The fate of area II

The lateral adjoining area of area I is area II, which comprises the descendants of column 2 and 3. It forms lateral parts of the paragnaths and median parts of the mandible. Descendants of column 2 are found in more lateral parts of the paragnath and in antero-median parts of the mandible (Fig. 6A–C, 2). Interestingly, these median parts of the early mandibular bud are later differentiated into the molar process.

The cells of column 3 give rise to median parts of the mandible (Fig. 6D–E, 2). As in area I, the descendants of the anterior row E(0)a form more anterior parts and according to that the descendants of row E(0)p proliferate more posterior parts. As well, some cells of the sternite have their origin in cells of area II.

In general, area II is responsible for the formation of lateral parts of the paragnaths and more median parts of the mandibles.

The fate of area III

Area III lies laterally adjacent to area II and comprises columns 4 and 5 of region E(0). They form lateral parts of the mandible and neighboring parts of the tergite. That is why the cell E(0)p4 produces cell material for the outer (postero-lateral) part of the mandible and lateral bordering tergite (Fig. 7A–C, 3). In contrast, descendants of row E(0)a in area III form more anterior parts of the outer (antero-lateral) mandible and parts of the adjacent tergite. Remarkably, the lateral portion of the mandibles differentiates by cell proliferation into the prospective pars incisiva.

Summarizing for area III, column 4 forms lateral parts of the mandibles and adjacent tergites and column 5 is not involved in the development of mandibles or paragnaths.

Surrounding areas of E(0)

The fate of the cells anterior to the region of E(0) has not yet been resolved in detail. Furthermore, it is not clear if there is a stereotypic cell division pattern at all forming the anterior embryonic head. Some labeling reveals that median cells of E(0) form part of the stomodaeum and more lateral cells form part of the second antennae.

posterior

Beginning with row E(1) the typical grid-like pattern of the post-naupliar germ band of Orchestia is established. The cell division pattern of E(1) differs somewhat from that of the more posterior rows (compare Figs. 2D and 4E) (for details see [36]). The 4-D analysis in row E(1) shows that its descendant cells are not involved in the formation of the mandibular segment (Fig. 4E) Hence, in contrast to the post-naupliar segments, in the mandibular segment the posterior genealogical boundary corresponds with the segment border.