Intact cluster and chordate-like expression of ParaHox genes in a sea star

Abstract

Background: The ParaHox genes are thought to be major players in patterning the gut of several bilaterian taxa. Though this is a fundamental role that these transcription factors play, their activities are not limited to the endoderm and extend to both ectodermal and mesodermal tissues. Three genes compose the ParaHox group: Gsx, Xlox and Cdx. In some taxa (mostly chordates but to some degree also in protostomes) the three genes are arranged into a genomic cluster, in a similar fashion to what has been shown for the better-known Hox genes. Sea urchins possess the full complement of ParaHox genes but they are all dispersed throughout the genome, an arrangement that, perhaps, represented the primitive condition for all echinoderms. In order to understand the evolutionary history of this group of genes we cloned and characterized all ParaHox genes, studied their expression patterns and identified their genomic loci in a member of an earlier branching group of echinoderms, the asteroid Patiria miniata.

Results: We identified the three ParaHox orthologs in the genome of P. miniata. While one of them, PmGsx is provided as maternal message, with no zygotic activation afterwards, the other two, PmLox and PmCdx are expressed during embryogenesis, within restricted domains of both endoderm and ectoderm. Screening of a Patiria bacterial artificial chromosome (BAC) library led to the identification of a clone containing the three genes. The transcriptional directions of PmGsx and PmLox are opposed to that of the PmCdx gene within the cluster.

Conclusions: The identification of P. miniata ParaHox genes has revealed the fact that these genes are clustered in the genome, in contrast to what has been reported for echinoids. Since the presence of an intact cluster, or at least a partial cluster, has been reported in chordates and polychaetes respectively, it becomes clear that within echinoderms, sea urchins have modified the original bilaterian arrangement. Moreover, the sea star ParaHox domains of expression show chordate-like features not found in the sea urchin, confirming that the dynamics of gene expression for the respective genes and their putative regulatory interactions have clearly changed over evolutionary time within the echinoid lineage.

Figure 1

Identification of the sea star Patiria miniata ParaHox orthologs. (A) Alignment of the homeodomains of the three P. miniata ParaHox genes with representative invertebrate and vertebrate orthologs. Broken lines indicate amino acid identities; the position of PmCdx intron within the homeodomain is indicated by a vertical arrow. The names of the species are: Sp (Strongylocentrotus purpuratus); Pf (Ptychodera flava); Ci (Ciona intestinalis); m (Mus musculus); Dfd (Drosophila melanogaster); Xl (Xenopus laevis); Htr (Helobdella triserialis); Bf (Branchiostoma floridae). ind and caudal are the Drosophila melanogastervorthologs of Gsx and Cdx respectively. (B) The genomic organization of P. miniata ParaHox genes is represented in scale showing separate exons for each gene. Asterisks indicate the homeobox in each gene. kb: kilobases.

Figure 2

Relative temporal expression profiles of ParaHox genes during Patiria miniata development. The graph shows the relative transcript abundance normalized against ubiquitin mRNA levels during sea star development. mRNA levels were measured by QPCR from cDNA templates prepared from whole embryos at the indicated developmental times. The results are expressed as percentage of the maximum value corresponding to PmCdx level of expression at 24 hpf. Due to the low levels of PmGsx transcript abundance, a separate graph has been created for PmGsx alone (see inset) expressing the results as percentage of PmGsx maximum level of expression (24 hpf). PmLox and PmGsx curves have been obtained repeating the experiments with two independent sets of primers. For the detailed experimental procedure see the relative section in Methods. Hpf, hours post fertilization.

Figure 3

Spatial expression patterns of ParaHox genes during Patiria miniata development. Panels A, B, C and D show the expression of PmLox gene. Panels E, F, G, H, I correspond to PmCdx expression. Panels J, K, L, M, N provide the expression domains for PmGsx. In panels A, D, F, L, N and J, embryos and larvae are in frontal view; in B, C, G, H, Ivand M, embryos and larvae are in lateral view; E and K are vegetal views of blastula embryos. Developmental stages are indicated in each panel.

Figure 4

PmLox and PmCdx relative expression domains in the sea star developing gut. (A, B, C) Double in situ hybridization of PmLox (green) and PmCdx (magenta) expression domains coupled with nuclei staining (blue, obtained with DAPI) in sea star embryo and larvae. In A, a gastrula embryo is shown in frontal view. In B and C, larvae are shown in lateral view. Developmental stages are provided in each panel. On the right of each developmental stage, a magnification of the gut domain expressing the two genes is provided, showing first both channels (A’, B’, C’), then the green channel only (A”, B”, C”) and then the magenta panel (A”’, B”’, C”’); nuclear staining is shown in blue. All the pictures represent full projection of confocal z-series. d, days; DAPI, 4',6-diamidino-2-phenylindole.

Figure 5

PmLox ectodermal domain of expression. (A, C, E)PmLox in situ hybridization developed with alkaline phosphatase methodology (A, E) and with fluorescent tyramide cy5 methodology coupled with DAPI staining (C, PmLox transcripts in green, nuclei in blue); B, D and F show acetylated tubulin immunostaining (in magenta) coupled with nuclear staining (in blue). A, B, E, Fvembryos and larvae are shown in frontal view; C and D larvae are shown in lateral view. Developmental stages are indicated in each panel. d, days; DAPI, 4',6-diamidino-2-phenylindole.

Figure 6

Evolution of the ParaHox gene cluster and relative expression domains in bilateral animals. Schematic representation of ParaHox genomic organization and expression patterns in several bilateral animals: the mouse Mus musculus [9,17,23,33,63], the bowfin fish Amia calva [43], the hagfish Eptatretus burgeri [64], the lancelet Branchiostoma floridae [5,11], the sea urchin Strongylocentrotus purpuratus [7,19], the sea star Patiria miniata (present study) and the polychaete Platynereis dumerilii [6]. Arrows indicate ParaHox genes and their orientation in the genome; a continuous line below arrows indicates an intact cluster. The cartoons on the right side of the panel schematize the domains of expression of ParaHox genes in representatives of some of the bilaterian groups. Mouse, amphioxus, sea urchin and sea star embryos are in lateral view; the polychaete embryo is in frontal view. Nervous system domains are depicted with dashed lines. Endodermal structures are in gray. Gsx expression is shown in blue, Xlox in green and Cdx in magenta. Amphioxus Gsx expression in the hindbrain is depicted in light blue, as at the stage of development represented here, this domain of expression is fading out [11]. kb, kilobases; Chr, chromosome. Double-parallel lines indicate long genomic distance.