Isolation of sea star transcription factors
orthologs of regulatory genes that have known or suspected roles in patterning the axial neuroectoderm of many protostome and deuterostome embryos were isolated using a candidate gene approach. Recombinants for the following seven genes that encode homeodomain proteins were obtained: retinal homeobox
), optix-like homeobox 3
), gastrulation and brain-specific homeobox
), lim domain homeobox 2
) and paired box homeobox 6
), as well as members of the Nkx gene family, nk2.1
. We also identified partial sequences of eyes absent
), the ets family gene pea3
, and two C2H2 zinc-finger genes, zic
and krupple-like factor 13
). The following four winged-helix forkhead box genes were isolated: foxq2
. A complete list of orthologs, sequence lengths, and orthology of gene sequences is provided in Additional file 1
Animal-vegetal patterning of the sea star blastula ectoderm
Sea star late blastulae have a morphologically distinct animal-vegetal (AV) axis that is first readily observed when elongation of cells at the vegetal pole results in a noticeable thickening of epithelium termed the vegetal plate
]. During gastrulation, the vegetal plate invaginates to produce the mesoderm and endoderm of the larva, leaving the remaining animal epithelium as ciliated ectoderm [18
]. At this stage, no obvious morphological differences in ectodermal cell type have been observed, but we nonetheless reveal here a remarkable complexity of regulatory states within the ectoderm (summarized in Figure ).
Figure 1 Comparison of orthologous neuroectodermal gene expression domains among the deuterostomes. (A-E) Indirectly developing echinoderms. (F) Directly developing hemichordate. (G) Generalized vertebrate. Sea stars (Figures 1A-1C) and sea urchins (Figures 1D (more ...)
Transcripts of sea star regulatory genes are localized throughout the animal ectoderm in overlapping concentric domains along the AV axis. Some of these transcripts, such as those of zic, foxq2, rx and nk2.1, are found only in the animal-most ectoderm (Figures ). Of these genes, zic appears to be most closely localized to the animal pole, while expression of foxq2 and rx overlaps with zic, yet extends further. Transcripts of six3 and klf13 (Figures and ) also are detected in the animal-most ectoderm of blastulae; however, they show a still broader distribution. Although there is no clear cell morphology that demarcates the boundary between the animal ectoderm and the vegetal plate endomesoderm, we observe a ring of nk1 expression above the vegetal plate (Figure ) that partially overlaps with endodermally localized gatae transcripts (Figure ). The nk1 expression domain therefore likely marks the vegetal-most ectoderm of the blastula. foxj1 and pea3 (Figures and ) are expressed throughout the ectoderm.
Figure 2 Nested concentric expression domains pattern the axial ectoderm of sea star, P. miniata, blastulae. Embryos are oriented with the animal pole up. (A-G) Whole mount in situ hybridization (WMISH). (A) zic, (B) foxq2, (C) rx, and (D) nk2.1 expression is (more ...)
Taken together, the spatial expression of these regulatory genes demonstrates that the ectoderm of the sea star blastula is patterned along the AV axis in at least five nested concentric domains (summarized in Figure ).
Regulatory gene expression within the ectoderm of the ciliary bands and animal pole domain
During gastrulation, the ectoderm appears to undergo very little morphological change other than the coalescence of cilia within two bands: a preoral ciliary band that loops above the opening of the mouth and a postoral ciliary band that loops below it and around the aboral surface at the "back" of the embryo (Figure ) [7
]. Transcripts of several genes that were distributed broadly throughout the ectoderm prior to gastrulation are later expressed within the ectoderm of the ciliary bands of the larva following gastrulation (for example, foxj1
in Figures , pea3
as summarized in Figure and as previously reported for otx
]). At present, it is unclear if these patterns of expression reflect a migration of ectodermal cells to the sites of the future ciliary bands or if there is another patterning mechanism that restricts the earlier broad expression. Other transcripts are first detected at this stage within the ectoderm on the oral side of the gastrula and then later within the ciliary bands (for example, transcripts of foxg
; Figures and and , and and , respectively). A two-probe whole mount in situ
) of foxg
, a gene localized to the aboral ectoderm, further highlights the oral side restriction of foxg
transcripts in the gastrula (Figure ).
Figure 3 Heterogeneous regulatory patterning of the larval ciliary bands as visualized by WMISH. (A) Schematic describes the position of the two larval ciliary bands (red) from oral (left) and lateral (right) views. A, anus; CB, ciliary band; M, mouth. (B-F) (more ...)
The expression patterns at this later stage also show that the regulatory state of the early larval ciliary bands is heterogeneous, for example, nk2.1 and foxd are expressed in part of the preoral ciliary band directly above the mouth (Figures and , respectively), while gbx and nk1 are localized to part of the postoral ciliary band below the mouth (Figures and , respectively). Therefore, while the regulatory state of the ciliary band ectoderm can be defined by a suite of transcription factors (that is, klf13, foxj1, pea3, foxg, otx and hnf-6/onecut), they are further subdivided into pre- and postoral regions on the basis of the localization of foxd, nk2.1, nk1 and gbx.
Other transcripts that we detected within the ectoderm of the blastula remained within the animal ectoderm as gastrulation proceeded in what we define here as the animal pole domain. Unlike sea urchins, the sea star, P. miniata, does not appear to have a morphologically distinct animal pole domain at this stage. Transcripts of foxq2, pax6 and pea3 (Figures ) tightly localize to the animal pole ectoderm, although their vegetal boundaries do not exactly coincide. Transcripts of zic, rx and six3 are expressed within the animal pole domain as well, but even more vegetally throughout the animal ectoderm (Figures ). The vegetal boundary of the animal pole domain therefore is not clearly defined by regulatory gene expression. The preoral and postoral ciliary bands run through the sea star animal pole domain as demonstrated by a two-probe fluorescence in situ hybridization (FISH) using the ciliary band marker, foxg, and the animal pole domain gene, pax6 (Figure ). Thus, despite its lack of morphological regionalization, the animal pole has a distinct regulatory state, as defined by foxq2, pax6, pea3, zic, rx and six3 expression, suggesting that it is a unique territory within the sea star. It is not yet clear whether these genes remain expressed in all cells of the sea star animal pole domain during later stages of larval development or if expression becomes refined to only subsets of cells within this domain.
Figure 4 Gene expression molecularly defines the animal pole domain in the sea star. Embryos are shown laterally, with the animal pole up and oral side to the right. (A-F) WMISH. Expression of (A) foxq2, (B) pax6, (C) pea3, (D) zic, (E) rx and (F) six3 within (more ...)
Comparisons of ectodermal patterning between sea urchin and sea star embryos
At first inspection, the expression patterns of many genes appear markedly different in the earlier blastula stages of sea urchin and sea star embryos. The later restrictions within the animal pole or ciliary bands are, with some exceptions, more similar (Figure ). We suggest that the sea urchin embryo may simply undergo a relatively more rapid specification of these territories, with an associated loss of intermediate domains that we observe in the sea star. Indeed, a careful examination of expression patterns in sea urchin has recently shown that the apical plate in sea urchin consists of at least two regulatory domains: an inner animal pole domain flanked by a ring of six3
]. These two domains in the sea urchin hatched blastula may therefore represent a more apically compressed version of the nested, concentric regulatory domains found in the sea star blastula.
Some of the patterning differences between sea urchin and sea star ectoderm also seem to account for the differences observed in the localization of the pan-neuronal marker, synaptotagmin-B [21
]. For example, similar to the patterns of gene expression that we describe here, synaptotagmin-B is detected broadly throughout the ectoderm of the sea star gastrula, but in the larva it is found primarily in neurons associated with the ciliary bands and animal pole [9
]. In the sea urchin, however, synaptotagmin-B is already localized to the animal pole domain and the presumptive ciliary band by the gastrula stage [9
Although expression of many of the genes within the ciliary bands of the sea star appears conserved in the sea urchin, nk2.1
show clear differences in expression that may be associated with the evolutionary transition from a double looping of the ciliary band around the body of the sea star bipinnaria and hemichordate tornaria to a single looping of the ciliary band observed in sea urchins. This single ciliary band in the sea urchin develops at the junction between oral and aboral ectoderm. nk2.1
are expressed in part of the preoral ciliary band of the oral hood of the sea star, while sea urchin orthologs of these are found in the animal plate ectoderm (compare Figures ). Interestingly, both of these genes in sea urchin appear enriched on the oral side of the embryos [22
]. Thus, we speculate that the preoral ciliary band may have been compressed into the oral-side animal plate territory in sea urchins and that this region within the sea urchin may therefore constitute a different territory than the remaining animal plate.
Conservation of anterior (animal)-most regulatory patterning with other deuterostomes
Comparisons of the regulatory gene expression patterns that we observed in these indirectly developing sea star embryos with those known in directly developing bilaterians illuminate additional surprising patterns of conservation. We observe a general mapping of gene expression patterns along body axes (compare Figures with Figures ). For instance, in the sea star, foxq2
orthologs are apically expressed within the ectoderm. foxq2
expression in the amphioxus, a basal chordate, is restricted to the anterior-most end of the embryo [24
]. Orthologs of rx
are expressed in the anterior-most neuroectoderm in the hemichordate Saccoglossus
], and they also pattern the anteriorly localized eye primordium in vertebrates [25
]. The Drosophila rx
ortholog is required for brain development [27
]. Orthologs of six3
are expressed in anterior neuroectoderm in members of all three deuterostome phyla [13
]. The most vegetal ectoderm in sea stars is characterized by the presence of nk1
transcripts. In vertebrates, a gbx
ortholog establishes the midbrain-hindbrain boundary [30
]. The zebrafish ortholog of nk1
, is expressed within the midbrain-hindbrain boundary as well, although its expression is not exclusive to this territory [31
]. Expression of nk1
in sea stars, and possibly sea urchins, marks the vegetal (posterior)-most ectoderm.
There is some evidence of additional conservation between the DV and oral-aboral axes as well. The mouse ortholog of nk2.1
) is involved in the formation of motor neurons in the ventral telencephalon [32
]. Saccoglossus nk2.1
orthologs also show a ventral bias in expression [13
]. Furthermore, foxg
plays a role in ventral forebrain development, while lhx2
specifies dorsal telencephalic fates [32
]. We similarly show that expression of sea star orthologs of foxg
is restricted to the oral (ventral) ectoderm, while lhx2
orthologs are expressed within the aboral (dorsal) ectoderm (Figure ).
While in these comparisons we do not intend to convey a tight homology in gene expression patterns across deuterostome phyla, we predict that similarities in the overall patterning are an ancestral innovation and perhaps evidence of maintenance of some elements of a developmental gene regulatory network (GRN) inherited from a common ancestor. Conservation, however, is not maintained for orthologs of genes expressed within regions posterior to the midbrain-hindbrain boundary in chordates and hemichordates as nk1
marks the vegetal-most ectoderm. Also, the overlapping expression of hox
gene orthologs needed to pattern the posterior of many embryos are found only later in echinoderm development within the mesoderm of the rudiment [33
Separation of "retinal" from "anterior neural" regulatory patterning
Vertebrate orthologs of transcription factors such as pax6
play known roles in pattering and specifying anterior vertebrate sensory systems, most notably the eyes [25
]. Furthermore, orthologs of pax6
, the six
gene family members and eya
operate in a similar gene network for retinal determination in both vertebrates and Drosophila
(as reviewed in [35
Having established that orthologs of many regulatory genes involved in anterior neural specification are also expressed within the anterior ectoderm of sea star embryos, we sought to determine if orthologs of transcription factors involved within the retinal determination network are also expressed within echinoderm embryos.
We have already shown that the sea star pax6 ortholog is expressed within the animal pole domain (Figure ), although it is not expressed within the ectoderm of the sea urchin embryo (Figure ). Transcripts of both pax6 and eya in both sea urchins and sea stars, however, are detected in the mesoderm of midgastrulae and then more prominently in one mesodermally derived coelom in late gastrulae (Figures and Figures ). While we were unable to obtain a six1.2 ortholog from the sea star, this gene is expressed also within the mesodermal coelom in the sea urchin (Figures and ).
Figure 5 Retinal determination orthologs are expressed within sea urchin, (Strongylocentrotus purpuratus, Sp), and sea star mesoderm. (A-N) WMISH. (A and B) SpEya, (C and D) SpPax6 and (E and F) SpSix1.2 are expressed at the tip of the archenteron in gastrulae (more ...)
In the sea urchin, expression of two members of the light-sensing rhodopsin family of G-coupled protein receptors, opsin1
, has been shown as early as 1 week [36
]. We were unable to obtain sea star opsin sequences; however, we confirmed the expression of opsin1
1-week-old sea urchin larvae (Figures and ). The morphology of the late larval sea urchin embryos makes it difficult to decipher the precise location of these transcripts within the embryo. We therefore sought to determine if opsin
s collocalize with eya
, which we show is expressed likely within one or both coeloms (depending on developmental timing) in 1-week-old larvae (Figure ). Using a two-probe FISH
, we observe that transcripts of opsin1
colocalized with those of eya
in 1-week-old sea urchin larvae (Figures ). Expression of retinal determination orthologs within the mesoderm of gastrulae and larvae allow for the possibility that these genes operate within a common GRN.
The tightly coupled GRNs for anterior neural and visual sensory structures that are found in vertebrates and also in invertebrates, such as Drosophila
, therefore are spatially separated in echinoderms. The presence of gene transcripts of pax6
, but not, for example, eya
, within the animal ectoderm of sea star bipinnaria larva may indicate a partial retention of an ancestral retinal determination network that once operated within this embryonic territory. This might also explain the absence of apically localized rhabdomeric eyespots, which are characteristic of the indirectly developing tornaria larvae of some hemichordates but were likely lost in the echinoderm lineage [16