The clonal composition of biramous and uniramous arthropod limbs

Abstract

We present the first comparative cell lineage analysis of uniramous and biramous limbs of an arthropod, the crustacean Orchestia cavimana. Via single cell labelling of the cells that are involved in limb development, we are able to present the first complete clonal composition of an arthropod limb. We show that the two main branches of crustacean limbs, exopod and endopod, are formed by a secondary subdivision of the growth zone of the main limb axis. Additional limb outgrowths such as exites result from the establishment of new axes. In contrast to general belief, uniramous limbs in Orchestia are not formed by the loss of the exopod but by suppression of the split into exopod and endopod. Our results offer a developmental approach to discriminate between the different kinds of branches of arthropod appendages. This leads to the conclusion that a 'true' biramous limb comprising an endopod and an exopod might have occurred much later in euarthropod evolution than has previously been thought, probably either in the lineage of the Mandibulata or that of the Tetraconata.

Figure 1

Crustacean limb types. Schematic of the three main types of crustacean limb morphology which also represent the extremes of euarthropod limb morphology in general (left: polyramous/phyllopodous limb, middle: two biramous limbs of a different complexity, right: uniramous limb). The biramous limb consists of a proximal protopod (prp), which is mostly subdivided into coxa (cx) and basis (ba), an often segmented inner endopod (endo) and an outer exopod (exo). The uniramous leg of crustaceans is typically seven segments consisting of coxa and basis—together forming the protopod, the ischium (is), merus (me), carpus (ca), propodus (pro) and the dactylus (da). Biramous and uniramous limbs may possess additional branches forming inner endites (en) and outer exites (ex). The latter are often differentiated as gills. For instance, in the amphipod crustacean, Orchestia cavimana, being used for this study, the biramous pleopods (top) possess an undivided protopod and lack any exites and endites, whereas most of the uniramous thoracopods show two exites differentiated as gill and coxal plate. The uniramous limb (right) is commonly understood as the result of an exopod loss (Boxshall 2004). The polyramous/phyllopodous limb is often interpreted as a special case of the biramous limb type with a low degree of segmentation, many endites and one to several exites (Walossek 1993; Olesen et al. 2001).

Figure 2

Cell lineage of the ventral ectoderm in Orchestia. (a) Scheme of the cell division pattern in the post-naupliar ectoderm of malacostracan crustaceans. Top: Each of the consecutively numbered (E(1) to E(17)) ectodermal post-naupliar genealogical units (row abcd) shows the same development. First, it undergoes two mitotic waves with longitudinal spindle directions. Middle: the result is a grid-like pattern of four rows (from anterior to posterior: a, b, c and d). Bottom: with the first differential cleavages (bars mark sister cells), morphogenesis (limb buds, ganglia and intersegmental furrows) begins. The morphological segments do not match the genealogical units. The segmental border (red dotted line) runs between the descendants of row b and does not match the genealogical border (black lines). Accordingly, limb buds, ganglia and other segmental organs are composed structures formed by the descendants of two adjacent ectoderm rows. (b) SEM-picture of left thoracic segments during differential cleavages. The start of segment formation is indicated by intersegmental furrows and limb buds. To visualize clones of each genealogical cell row (a, b, c and d) and columns, one to nine are colour coded (see (a)). The dotted red lines mark the border of two consecutive morphological segments; the black lines indicate genealogical borders. To the left the midline (ml) is visible. (c) Column 1 (DiI labelling of cell abcd1 of the left half of genealogical unit E(9)). (i) Ventral view (double exposure) focused on the sternite. The descendants of cell abcd1 form parts of the sternal plates (ster) of the thoracic segments 6 and 7 (th6–7). (ii) The same section focused on proximal leg segments. Only a small number of cells (arrowhead) are involved in the formation of the proximal limb segments. (ba, basis; is, ischium; me, merus; ca, carpus; pro, propodus). (d) Column 9 (DiI labelling of cell abcd9 of the left half of genealogical unit E(12)) in the pleonic region. Lateral view (double exposure) of the left body side showing the first three pleonic segments with pleopods. The descendants of abcd9 are not involved in limb formation; they form lateral parts of the tergite (ter) of the first and second pleonic segments (pl1–2).

Figure 3

Clonal composition of biramous limbs (pleopods) in Orchestia. (a) Column 3 (DiI labelling of cell abcd3 of the right half of genealogical unit E(14)). Three-dimensional reconstruction (medial view) of the right pleopods 2–6 (pl2–6). The nuclei are counterstained with Hoechst. The descendants of cell abcd3 (red) form inner parts of the endopod of pleopods 3 and 4 (endo) and parts of the corresponding protopods (prp). (b) Column 5 (DiI labelling of cell abcd5 of the left half of genealogical unit E(12)). Double exposure of a ventral view of the pleonic segments 1–3 (pl1–3). The clone of cell abcd5 (green) forms the inner parts of the exopod (exo) of pleopods 1 and 2 and additionally the inner parts of their protopods. The clone crosses the segmental border (arrowhead). The endopod (endo) is not formed by the descendants of column 5. (c) Column 6 (DiI labelling of cell abcd6 of the left half of genealogical unit E (12)). (i) Lateral view of pleopods 1 and 2. The shape of the typical architecture in proximal protopod (prp), and distal an inner endopod (endo) and an outer exopod (exo) are traced by a white line. (ii) The same detail with fluorescence light shows exclusively descending cells from abcd6. (iii) Double exposure to demonstrate that the descendants of column 6 form only the outer part of the exopod and part of the protopod. In addition, an adjacent part of the tergite (ter) is formed by these cells. (d) Column 8 (DiI labelling of cell abcd8 of the right half of genealogical unit E(15)). Lateral view of a three-dimensional reconstruction of the right pleonic appendages 4–6 (pl4–6). The nuclei are counterstained with Hoechst. The descendants of cell abcd8 form the proximal part of the protopods and a part of the tergites (ter) of the fourth and fifth pleonic segments. Neither the exopod (exo) nor the endopod (endo) is formed by these cells. (e) Colour-coded scheme of the clonal composition of pleopods in Orchestia. A combination of two genealogical units (figure 2). Each colour of the bar represents a column of the ventral ectoderm and its contribution to a pleopod. Endo, endopod; exo, exopod; prp, protopod; ster, sternite; ter, tergite.

Figure 4

Clonal composition of uniramous limbs (thoracopods) in Orchestia. (a) Column 3 (DiI labelling of cell abcd 3 of the left half of genealogical unit E(10)). Three-dimensional reconstruction (median view) of left thoracopods 7 and 8 (th7–8). The nuclei are counterstained with Hoechst. The descendants of cell abcd3 (red) form the inner parts of both limbs along the entire proximal–distal axis. (b) Column 4 (DiI labelling of cell abcd 4 of the right half of genealogical unit E(9)). Three-dimensional reconstruction (lateral view) of right thoracopods 6 and 7 (th6–7). The nuclei are counterstained with Hoechst. The descendants of cell abcd4 (red) form the inner part of both limbs along the entire proximal–distal axis. The insert shows thoracopod 7 rotated at approximately 180° to show an ectodermal cell band (red) formed by the descendants of cell d4. (c) Column 6 (DiI labelling of cell abcd6 of the left half of genealogical segment E(6)). (i) Lateral view of thoracopods 4 and 5. The shape of the typical seven-segmented uniramous limb with coxal plate (cxp), gill (gi), coxa (covered by coxal plate), basis (ba), ischium (is), merus (me), carpus (ca), propodus (pro) and dactylus (da) is marked by black lines. (ii) The same detail (double exposure) shows the descendants of column 6 which form an outer (lateral) part of the limb and a part of the gill and coxal plate. (d) Column 8 (DiI labelling of cell abcd8 of the right half of genealogical unit E(7)). Three-dimensional reconstruction (lateral view) of the right thoracopods 3–5 (th3–5). The nuclei are counterstained with Hoechst. The star indicates a part of the gill that was destroyed during dissection. Most of the descendants of cell abcd8 form parts of the tergite (ter) of thoracic segments 4 and 5. In addition, they form a lateral part of the gill (gi) and the coxal plate (cxp). (e) Colour-coded scheme of the combination of all single results (shown in a–d) to show the clonal composition of a thoracopod in Orchestia. A combination of two genealogical units (see figure 2). Each colour of the bar represents a column of the ventral ectoderm and its contribution in the formation of a thoracopod. (ba, basis; ca, carpus; cx, coxal; cxp, coxal plate; da, dactylus; gi, gill; is, ischium; pro, propodus; ster, sternite; ter, tergite).