Quantitative assessment of Hox complex expression in the indirect development of the polychaete annelid Chaetopterus sp

Abstract

A prediction from the set-aside theory of bilaterian origins is that pattern formation processes such as those controlled by the Hox cluster genes are required specifically for adult body plan formation. This prediction can be tested in animals that use maximal indirect development, in which the embryonic formation of the larva and the postembryonic formation of the adult body plan are temporally and spatially distinct. To this end, we quantitatively measured the amount of transcripts for five Hox genes in embryos of a lophotrochozoan, the polychaete annelid Chaetopterus sp. The polychaete Hox complex is shown not to be expressed during embryogenesis, but transcripts of all measured Hox complex genes are detected at significant levels during the initial stages of adult body plan formation. Temporal colinearity in the sequence of their activation is observed, so that activation follows the 3'-5' arrangement of the genes. Moreover, Hox gene expression is spatially localized to the region of teloblastic set-aside cells of the later-stage embryos. This study shows that an indirectly developing lophotrochozoan shares with an indirectly developing deuterostome, the sea urchin, a common mode of Hox complex utilization: construction of the larva, whether a trochophore or dipleurula, does not involve Hox cluster expression, but in both forms the complex is expressed in the set-aside cells from which the adult body plan derives.

Figure 1

Single-strand probe excess titrations for transcripts of two Hox genes and the Brachyury gene in the polychaete worm Chaetopterus: CH-Hox1 (A), CH-Hox2 (B), and CH-Bra (C). These examples are representative of the data sets that provided the measurements reported in this paper. Total RNA was extracted from unfertilized eggs and embryos at the indicated stages and in increasing amounts reacted with ³²P-labeled antisense RNA probes (see ref. and Material and Methods for probes and procedures). Mean background levels (averages of two measurements on samples containing yeast RNA alone) for each probe are indicated with an arrow: for CH-Hox1, 38 and 41 cpm; for CH-Hox2, 2,792 and 2,889 cpm; for CH-Bra, 236 and 239 cpm.

Figure 2

Transcripts of Hox cluster genes and the Brachyury gene, per embryo or larva. Histograms indicate transcript numbers for the indicated stages of embryogenesis: 0 h, unfertilized eggs; 6 h, early gastrula; 12 h, swimming prototrochophore; 24 h, L1 larva; and 48 h, L2 larva. The dots indicate the number of cells per organism (same ordinates). Each panel represents a single data set. Transcript numbers were calculated from the slopes as described (ref. ; see Fig. 1), taking into account the amount of total RNA per embryo (6.5 ng; see Materials and Methods) and the lengths and specific activities of the probes. Hox probes are from Irvine and Martindale (21); the Brachyury probe is described in Materials and Methods.

Figure 3

Hox gene expression in larvae of Chaetopterus: CH-Hox2 (A) and CH-Hox4 (B). Plastic thin sections were made from WMISH prepared by Irvine and Martindale (21). (A) CH-Hox2 expression pattern in an L1-stage larva sectioned horizontally with the anterior end upward. The cells expressing the gene are in the posterior region occupied by the teloblasts, which generate much of the adult endomesodermal derivatives that constitute the body wall of the segmented trunk. (B) CH-Hox4 expression in an L2-stage larva. The plane of section is slightly oblique to the longitudinal axis, and the gut appears almost in cross section. As for CH-Hox2, the cells expressing the CH-Hox4 gene product appear to be the progeny of the endomesodermal teloblastic set-aside cells.