The Fox/Forkhead transcription factor family of the hemichordate Saccoglossus kowalevskii

Abstract

Background: The Fox gene family is a large family of transcription factors that arose early in organismal evolution dating back to at least the common ancestor of metazoans and fungi. They are key components of many gene regulatory networks essential for embryonic development. Although much is known about the role of Fox genes during vertebrate development, comprehensive comparative studies outside vertebrates are sparse. We have characterized the Fox transcription factor gene family from the genome of the enteropneust hemichordate Saccoglossus kowalevskii, including phylogenetic analysis, genomic organization, and expression analysis during early development. Hemichordates are a sister group to echinoderms, closely related to chordates and are a key group for tracing the evolution of gene regulatory mechanisms likely to have been important in the diversification of the deuterostome phyla.

Results: Of the 22 Fox gene families that were likely present in the last common ancestor of all deuterostomes, S. kowalevskii has a single ortholog of each group except FoxH, which we were unable to detect, and FoxQ2, which has three paralogs. A phylogenetic analysis of the FoxQ2 family identified an ancestral duplication in the FoxQ2 lineage at the base of the bilaterians. The expression analyses of all 23 Fox genes of S. kowalevskii provide insights into the evolution of components of the regulatory networks for the development of pharyngeal gill slits (foxC, foxL1, and foxI), mesoderm patterning (foxD, foxF, foxG), hindgut development (foxD, foxI), cilia formation (foxJ1), and patterning of the embryonic apical territory (foxQ2).

Conclusions: Comparisons of our results with data from echinoderms, chordates, and other bilaterians help to develop hypotheses about the developmental roles of Fox genes that likely characterized ancestral deuterostomes and bilaterians, and more recent clade-specific innovations.

Keywords: Deuterostome evolution; EH-I-like motif; Fork head; Forkhead; Fox cluster; Fox genes; FoxQ2; Gene regulatory networks; Gill slits; Gut patterning; Hemichordate; Saccoglossus kowalevskii.

Figure 1

Phylogenetic analysis. (A) Phylogenetic analysis of S. kowalevskii Fox genes: The S. kowalevskii Fox proteins group into their predicted families with high support values. Displayed is the Bayesian tree (standard deviation = 0.0109) with Bayesian posterior probabilities values on top of each branch and maximum likelihood values underneath each branch. Stars indicate a different tree topology result from the maximum likelihood analysis which lead to no support value at that position. Branches with posterior probabilities below 50% are collapsed. For gene accession numbers, gene predictions, and alignment see Additional file 1: Table S1, Additional file 2: Table S2, Additional file 3: Table S3 and Additional file 4: Table S4. (B) Phylogenetic analysis of the FoxQ2 family. Phylogenetic analysis of FoxQ2 proteins containing an EH-I-like motif (see Additional file 5: Table S5) result in a tree topology supporting a duplication of the FoxQ2 family at the base of the bilaterians. Displayed is the Bayesian tree (standard deviation = 0.023) with Bayesian posterior probabilities values on top of each branch and maximum likelihood values underneath each branch. Stars indicate different tree topologies which lead to no support value at that position. Branches with posterior probabilities below 50% are condensed. Proteins with a C-terminal EH-I-like motif are highlighted in blue. Proteins with a N-terminal EH-I-like motif are highlighted in yellow. Proteins with a N-terminal and a C-terminal EH-I-like motif are highlighted in yellow and blue. For gene accession numbers, identification of the EH-like motif, and alignment see Additional file 1: Table S1, Additional file 2: Table S2, Additional file 3: Table S3, Additional file 4: Table S4, Additional file 5: Table S5 and Additional file 6: Table S6.

Figure 2

Expression patterns of S. kowalevskii foxA-E. Spatial expression pattern of S. kowalevskii foxA - foxE. Animals are oriented as indicated in cartoons for the corresponding stage if not otherwise specified. For a detailed description of the expression patterns see text. Panels 1 to 5; ectoderm = light gray, mesoderm = light blue, endoderm = dark gray, black arrows in panel 4 point at the forming furrows at the boundary between the proboscis and collar and collar and trunk respectively. Panels 6, 11, 16, 19, 21, and 31 show surface views. Panels 17/18 are lateral views. (10) Panel 10 shows a dorsal view, white arrows points at the forming gill pores, the black arrow points at the gap of ectodermal foxA expression at the dorsal collar, inlay shows lateral view. (15) Inlay in panel 15 shows ventral view on the mouth opening, white arrow points at the mouth opening. (20) White arrow in panel 20 points at the endodermal expression domain of foxB. (30) White star in panel 30 indicates the ectodermal expression domain of foxD at the base of the proboscis. The inlay shows a closeup of the posterior gut region. An: animal pole; Veg: vegetal pole; L: left; R: right; A: anterior; P: posterior; D: dorsal; V: ventral; ao: apical organ; cb: ciliated band; gp: gill pore. Brightness and contrast of pictures were adjusted when appropriate to match overall appearance of the figure.

Figure 3

Expression patterns of S. kowalevskii foxF-L1. Spatial expression pattern of S. kowalevskii foxF - foxL1. Animals are oriented as indicated in cartoons of Figure 2 (1-5) for the corresponding stage if not otherwise specified. For a detailed description of the expression patterns see text. Panels 4, 8, 9, and 17-19 show surface views. (5) White arrow in panel 5 points at endodermal expression domain of foxF at the tip of the proboscis, black arrow points at the heart-kidney complex. Inlay shows dorsal view of the pharynx, black arrow points at the heart-kidney complex. White asterisk indicate expression in the pharyngeal mesoderm. (9) Panel 9 shows dorsal surface. Inlay shows ventral surface. (10) Arrow in panel 10 points at dorsal mesoderm. Inlay shows dorsal view of the pharynx, black arrow points at dorsal mesoderm. (15) Black arrow in panel 15 points at posterior endoderm expression of foxI. Inlay shows dorsal view of the forming gill pores. (20) Inlay shows dorsal view of the forming gill pores. Black arrow heads point to gill pouch endoderm. (30) Inlay shows dorsal view of the forming gill pores. An: animal pole; Veg: vegetal pole; L: left; R: right; A: anterior; P: posterior; D: dorsal; V: ventral. Brightness and contrast of pictures were adjusted when appropriate to match overall appearance of the figure.

Figure 4

Expression patterns of S. kowalevskii foxN1/4-Q2-3. Spatial expression pattern of S. kowalevskii foxN1/4 - foxQ2-3. Animals are oriented as indicated in cartoons of Figure 2 (1-5) for the corresponding stage if not differently specified. For a detailed description of the expression patterns see text. Panels 4, 6, 29, and 30 show surface views. Panels 14/15 and 23-25 show light stained embryos. For longer stained embryos see Additional file 11: Figure S3. (5) Black arrow points at expression domain of foxN1/4 in the ventral ectoderm at the posterior tip of the trunk, white arrow points at the ciliated band. Inlay shows ventral view of the posterior tip of the trunk, black arrow points at expression domain of foxN1/4. (7-9) White arrows point to cells with high levels of foxP expression in the proboscis ectoderm. An: animal pole; Veg: vegetal pole; L: left; R: right; A: anterior; P: posterior; D: dorsal; V: ventral. Brightness and contrast of pictures were adjusted when appropriate to match overall appearance of the figure.

Figure 5

Expression summary. (A-I) Expression summary of all S. kowalevskii Fox genes with clear localized expression patterns. (A-E) Blastula -, Gastrula -, 36 h embryo -, 48 h embryo -, 72 h embryo - surface view. (F-H) Gastrula -, 36 h embryo -, 48 h embryo - cross section. (I) Cross section of the gill pore area of a 72-h-old embryo. For details see text. *Potential co-expression is inferred from single gene expression analysis. No double in situ hybridization was performed. **The expression of foxF is very dynamic and only a more detailed analysis will be able to show all expression domains at any given developmental time point. An: animal, Veg: vegetal, A: anterior, P: posterior, D: dorsal, V: ventral.

Figure 6

Examples of conserved Fox gene expression domains. (A-E) FoxB expression (blue) in multiple species. The expression of FoxB seems to be correlated to the expression of chordin, a BMP inhibitor. The endodermal gut expression domain seems to be unique to echinoderms and hemichordates. (F-I) FoxD expression (blue) in the hindgut of different species. The conservation of the expression in the hindgut across deuterostomes indicates that it was likely already present in the hindgut of the deuterostome ancestor. (J-O) Ectodermal expression domains of FoxQ2 (blue) in multiple species across phyla. Expression across bilateria suggest a conserved role in apical (apical neural) patterning. Expression at the aboral side in cnidarians suggests a deep evolutionary origin for this expression in patterning terminal neural structures. For literature summary see Table 1 and for additional discussion see Additional file 12. A: anterior, P: posterior, D: dorsal, V: ventral, oa: aboral, o: oral.