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Keywords:

  • gene regulatory network;
  • aboral ectoderm;
  • sea urchin

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

The temporal and spatial expression patterns of regulatory genes are required for building a gene regulatory network (GRN). The current ectoderm GRN model for the sea urchin embryo includes pregastrular specification functions in the oral (OE) and aboral ectoderm (AE). Unlike the OE, which is resolved into several subdomains, the AE is considered a simpler territory due to the lack of detailed gene expression studies in this territory. Here, we perform temporal and spatial gene expression studies on the eight transcription factor genes constituting the AE GRN. Based on the differential gene expression patterns, we conclude that the AE contains at least three subdomains at the mesenchyme blastula stage. We also performed immunostaining for pSmad1/5/8 to monitor the activation of the BMP signaling pathway. The dynamic changes in the expression patterns of these transcription factor genes and the nuclearization of pSmad1/5/8 may provide a foundation for resolving the AE GRN. Developmental Dynamics, 2011. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Developmental gene regulatory networks (GRNs) provide the causal linkages between transcription factors and genomic regulatory sequences. Furthermore, GRNs explain the developmental mechanisms of an embryo. The most powerful demonstration to date of the ability of a complete GRN to explain every aspect of a developmental process involves the specification of skeletogenic cells in the sea urchin embryo (Oliveri et al.,2008). A GRN model for oral (OE) and aboral ectoderm (AE) specification in the same embryo has recently been established based on a large-scale perturbation analysis (Su et al.,2009). During sea urchin development, prospective oral and aboral ectodermal cells are morphologically indistinguishable until the gastrula stage. After gastrulation, the archenteron bends to the oral side, and the OE begins to flatten. The OE later invaginates to form a stomodeum, which then fuses with the tip of the archenteron to create a mouth. The AE differentiates into a squamous epithelium that covers the majority of the larval body. Although the morphology of the oral and aboral ectoderm cells is similar at the blastula stage, many regulatory genes have started to be expressed differentially in these two territories during the process of primary mesenchyme cell (PMC) ingression (Su,2009). Based on the gene expression patterns and perturbation analysis, the OE is resolved into several subdomains in the blastula embryo, which includes the ciliary band, the border with the endoderm, the central face region, and, within the central face region, the stomodeum (Su et al.,2009). Unlike the multi-subdomains in the OE, no subdomains have been identified in the prospective AE. Therefore, the AE has been thought to be a simpler territory than the OE, which contradicts the polyclonal origin of the AE as revealed by lineage tracing experiments (Davidson et al.,1998).

Using fluorescent lineage tracer injected into individual blastomeres of eight-cell sea urchin embryos, Cameron et al. showed that the animal blastomere quartet (Na), the vegetal blastomere quartet (VA), and the four lateral blastomeres (left and right NL and VL) all contribute to the AE (Cameron et al.,1987). At the pluteus stage, Na progenies become the abanal AE, which covers the midregion of the AE opposite of the anus opening. The great granddaughter cells of VA, left and right VL, contribute to the AE at the apex and the left and right anal plate AE, respectively, at the pluteus stage (Cameron et al.,1987,1990,1991). In addition, the granddaughter cells of the left and right NL develop into the left and right lateral AE (Cameron et al.,1990). Although the AE is derived from six different progenitor cells, only one cell type arises from it, which is a squamous epithelium that forms the wall of the pluteus larva. The multi-origin AE, which becomes a simple territory, is supported by the expression patterns of differentiation genes. The AE-specific markers cytoskeletal actin CyIIIa, Spec1, Spec2A, and arylsulfatase genes are expressed in the AE of the blastula embryo with no distinguishable subdomains (Davidson et al.,1998). Therefore, the specification of the AE occurs presumably by mechanisms that do not depend on the cell lineages.

The eight transcription factor genes hmx, klf7, tbx2/3, nk2.2, irxa, msx, dlx, and hox7 are expressed in the AE at the blastula stage. Perturbation analysis revealed that there are extensive feedback relationships among these AE regulators (Su et al.,2009). Because of the lack of detailed temporal and spatial expression profiles for these genes and their complex feedback relationships, it is difficult to resolve the epistatic relationships within the AE GRN. Additionally, it is known that BMP signaling is required for the specification of the AE in sea urchin embryos (Duboc et al.,2004). Despite being expressed in the OE, knockdown of BMP2/4 results in the down-regulation of several transcription factor genes that are normally expressed in the AE (Duboc et al.,2004; Su et al.,2009). Therefore, it is believed that BMP proteins diffuse from the oral to the aboral ectoderm and their effect in the OE is antagonized by the BMP antagonist Chordin, which is also expressed in the OE (Bradham et al.,2009; Lapraz et al.,2009; Su et al.,2009). Recently, Lapraz et al. confirmed in the sea urchin Paracentrotus lividus that phosphorylation and nuclearization of Smad1/5/8 (pSmad1/5/8), the BMP signaling transcription factor, occurs in the AE of the blastula embryos and that Chordin restricts effective BMP signaling in the AE (Lapraz et al.,2009).

Here, we performed quantitative PCR (QPCR) and in situ hybridization on embryos from early blastula to mesenchyme blastula stages at 2-hr intervals to reveal the detailed temporal and spatial expression profiles of the eight AE regulators. Knowing the timing and domain of expression of these genes will help us to decipher the causal linkages between them. Furthermore, the staining pattern of pSmad1/5/8 was compared to the expression domains of the transcription factor genes expressed in the AE. With future comprehensive perturbation analysis, the AE GRN could be resolved into subdomains, and the relationships between BMP signaling and the regulator genes could be deciphered. Finally, understanding the dynamic expression patterns of these aboral regulators may elucidate the molecular mechanism that homogenizes the multi-origin AE as it develops.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Temporal Expression Profiles

To determine the onset of zygotic gene expression of the eight transcription factors constituting the sea urchin AE GRN, we performed quantitative PCR (QPCR) on samples collected from the early blastula stage (12 hr post-fertilization (hpf)) to the mesenchyme blastula stage (24 hpf) at 2-hr intervals. The expression profiles were grouped into four categories based on the timing of their expression. First, Sp-Hmx and Sp-Klf7 were both maternally transcribed and turned on zygotically at 14 and 16 hpf, respectively (Fig. 1A). Second, Sp-Tbx2/3 and Sp-Nk2.2 were zygotically expressed at 16 hpf (Fig. 1B). Third, Sp-IrxA and Sp-Msx were transcribed at 18 hpf (Fig. 1C), and, fourth, the last group, composed of Sp-Hox7 and Sp-Dlx, was transcribed at 20 hpf (Fig. 1D). These expression profiles mostly support the results of a recent high-resolution measurement of the regulatory genes in early sea urchin development using an RNA counting device termed the NanoString nCounter (Materna et al.,2010). The absolute transcript numbers between these two approaches, however, are different in many cases, which may be due to the fundamental differences between these two methods. The QPCR method we used measures the relative abundance of transcripts, while the NanoString nCounter directly counts the transcript number using a fluorescent barcode attached to a sequence-specific hybridization probe (Geiss et al.,2008). Nevertheless, both sets of data demonstrate that both Sp-Hmx and Sp-Klf7 are expressed maternally and turn on zygotically within 12 to 16 hpf. Of the other remaining genes, Sp-Nk2.2 was expressed first (15–16 hpf) followed by Sp-Tbx2/3. Sp-IrxA and Sp-Msx were expressed next and at the same time. Sp-Dlx was then expressed followed by Sp-Hox7, which was expressed last. To determine the spatial expression patterns of each of these genes and their relationships to each other, we performed in situ hybridization on embryos collected from 12 to 24 hpf at 2-hr intervals.

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Figure 1. Eight transcription factor genes constituting the AE GRN turn on at different time points during the blastula stage. Transcript levels of each of the eight genes were measured by QPCR at 2-hr intervals from 12 to 24 hpf. The Y-axis is the transcript level normalized to the ubiquitin expression level at the same stage. All four graphs are in the same scale. The expression profiles are grouped into four categories based on the timing of their expression. A: Sp-Hmx and Sp-Klf7 are expressed maternally. The inset is an enlarged graph on a smaller scale showing the zygotic gene activation of Sp-Hmx and Sp-Klf7 between 12 to 16 hpf. B: Sp-Tbx2/3 and Sp-Nk2.2 begin to be expressed at 16 hpf. C: Sp-IrxA and Sp-Msx are first detected at 18 hpf. D: Sp-Dlx and Sp-Hox7 are transcriptionally activated at 20 hpf.

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Co-Localization of Sp-Hmx and Sp-Tbx2/3 Expression in the Animal Aboral Ectoderm

Sp-Hmx is a member of the H6 homeobox gene family and was first discovered in a library screening for homeobox genes (Martinez and Davidson,1997). It was later confirmed to be an AE-specific gene expressed in the blastula-stage embryo (Howard-Ashby et al.,2006; Su et al.,2009). Sp-Hmx was expressed ubiquitously in the unfertilized egg (Fig. 2A). Specific expression in the AE was first detected at 14 hpf (Fig. 2B), and the expression became stronger after 18 hpf (Fig. 2C). The belt expression domain of Sp-Hmx was clearly observed from the aboral side at the blastula stage (Fig. 2D). At the mesenchyme blastula stage (24 hpf), the width of the Sp-Hmx expression domain along the oral-aboral axis covered almost three-fourths of the circumference of the embryo when it was observed from the vegetal pole (Fig. 2E).

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Figure 2. Sp-Hmx expression co-localizes with Sp-Tbx2/3 expression in the animal aboral ectoderm. Whole mount in situ hybridization of Sp-Hmx (A–E) and Sp-Tbx2/3 (F–J) was performed at different embryonic stages. The earliest specific expression of Sp-Hmx and Sp-Tbx2/3 in the AE is indicated by the arrow and arrowhead, respectively. K–M′: Double FISH of Sp-Hmx and Sp-Tbx2/3. Every individual column from K to H′ is the same embryo observed under DIC or different fluorescent filters. S–V, E′–H′, and I′–M′ are the full projections of the confocal Z stacks from the double FISH. The developmental stage is indicated at the bottom left corner, and the observed view is indicated at the bottom right corner of each panel. abv, aboral view; lv, lateral view (left-side view); vv, vegetal view.

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It is known that the T-box family transcription factor Tbx2/3 is involved in the formation of the oral-aboral axis of sea urchin embryos (Croce et al.,2003; Gross et al.,2003). A recently published ectoderm GRN placed Sp-Tbx2/3 as one of the first regulators in the AE (Su et al.,2009). Sp-Tbx2/3 expression was detected as early as 16 hpf, and its expression became prominent by 18 hpf (Fig. 2F). The Sp-Tbx2/3 expression domain extended to the aboral endomesoderm by 20 hpf (Fig. 2G). As PMC ingression proceeded, Sp-Tbx2/3 staining became very strong in the vegetal AE and endoderm (Fig. 2H). Although the Sp-Tbx2/3 expression domain extended further toward the vegetal pole than the Sp-Hmx domain at 24 hpf (Fig. 2I), the widths of their expression domains at the equator of the embryo were similar (Fig. 2E, J). To further compare the expression domains of Sp-Hmx and Sp-Tbx2/3, we performed double fluorescent in situ hybridization (FISH) and examined the fluorescent signals from lateral, vegetal, and aboral views of the embryos. The FISH results were similar to the results from the in situ hybridization followed by AP/NBT-BCIP staining. Sp-Hmx was expressed in a belt domain in the animal half of the AE, and this domain covered to approximately three-fourths of the embryo (Fig. 2K–R). The expression domain of Sp-Tbx2/3 was as wide as the Sp-Hmx domain and had a similar animal border. The vegetal border of the Sp-Tbx2/3 expression domain, however, extended toward the vegetal plate (Fig. 2S–V). At the mesenchyme blastula stage, the expression of Sp-Hmx remained at the animal AE, whereas Sp-Tbx2/3 was expressed in the whole AE and the aboral side of the vegetal plate (Fig. 2W–H′). The confocal Z stacks of the embryo viewed from the left side at different developmental stages further confirmed the overlapping area of Sp-Hmx and Sp-Tbx2/3 expression in the animal AE, and Sp-Tbx2/3 extended more vegetally as development proceeded (Fig. 2I′–M′). The expression of Sp-Tbx2/3 is very dynamic during the blastula stages, and we will discuss this subject in the Nuclearization of pSmad1/5/8 in the Central Aboral Ectoderm section.

Sp-Nk2.2 and Sp-Klf7 Are Expressed in Both the Oral and Aboral Ectoderm

Sp-Nk2.2 was first identified in a genome-wide search for homeobox transcription factor genes and is expressed in the AE (Howard-Ashby et al.,2006). Later studies also confirmed its expression in the AE at the blastula stage (Poustka et al.,2007; Su et al.,2009). Close examination of the expression patterns of Sp-Nk2.2 revealed that initially, at 18 hpf, it was expressed at one side of the ectoderm near the vegetal plate (Fig. 3A,B). Two hours later, Sp-Nk2.2 was expressed in both sides of the ectoderm (Fig. 3C). At the mesenchyme blastula stage, the two expression domains were clearly separated, and one domain was narrower than the other (Fig. 3D). Double FISH confirmed that the early Sp-Nk2.2 expression domain was located at the oral side, because it was opposite to the Sp-Tbx2/3 expression domain in the AE (Fig. 3E–H). The late Sp-Nk2.2 expression domain was broader than the early domain and resided in the vegetal part of the Sp-Tbx2/3 expression domain in the AE (Fig. 3I–P). Initially, the vegetal border of the AE expression domain of Sp-Nk2.2 was similar to that of the Sp-Tbx2/3 expression domain (Fig. 3I–P). In the 24-hpf embryos, unlike the expression of Sp-Tbx2/3, which extended to the endoderm, the expression of Sp-Nk2.2 stayed in the AE (Fig. 3Q–T). Although the weak expression of Sp-Nk2.2 in the OE had not been mentioned in previously published reports, its expression is visible in the published data (Poustka et al.,2007; Su et al.,2009).

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Figure 3. Sp-Nk2.2 and Sp-Klf7 are expressed in both the oral and aboral ectoderm. A–D: The expression patterns of Sp-Nk2.2 at different blastula stages. Double FISH of Sp-Nk2.2 with Sp-Tbx2/3 (E–T) and Sp-Nk2.2 with Sp-Klf7 (U–J′) reveal the expression domains of Sp-Nk2.2 and Sp-Klf7 in both the oral and aboral ectoderm. The initial expression of Sp-Nk2.2 in the OE is indicated by arrows. The initial expression of Sp-Klf7 in the AE is denoted by the arrowhead.

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Sp-Klf7, also known as Sp-Z86, belongs to the krüppel-like zinc finger family (Materna et al.,2006). Previous studies concluded that Sp-Klf7 is expressed in the AE at the mesenchyme blastula and early gastrula stages (Materna et al.,2006; Su et al.,2009). A detailed analysis of the Sp-Klf7 expression pattern revealed that even though the maternal transcript distributed ubiquitously during the cleavage stages, the earliest specific expression in the ectoderm was observed in the 18-hpf embryo (Fig. 3W). More importantly, like Sp-Nk2.2, Sp-Klf7 was also expressed in both the oral and aboral ectoderm (Fig. 3A′). However, unlike the initial expression of Sp-Nk2.2 in the OE followed by its expression in the AE, Sp-Klf7 was first detected in the AE and then later in the OE (Fig. 3U–B′). The signal intensity of the Sp-Klf7 in situ hybridization was generally very weak, which may be due to its low expression level (Fig. 1A). Furthermore, we observed that once both Sp-Nk2.2 and Sp-Klf7 were expressed in the oral and aboral ectoderm, their expression domains completely overlapped at the mesenchyme blastula stage (Fig. 3C′–J′).

Sp-IrxA and Sp-Msx Are Co-Expressed in the Vegetal Aboral Ectoderm

The homeobox transcription factor gene Sp-IrxA was discovered in a genome-wide study (Howard-Ashby et al.,2006). It is expressed in the AE (Howard-Ashby et al.,2006; Poustka et al.,2007) and is one of the transcription factor genes in the AE GRN that has extensive positive feedback relationships with Sp-Tbx2/3, Sp-Dlx, and itself (Su et al.,2009). Sp-Msx is also a homeobox transcription factor that was first isolated by Dobias et al. (1997). It was later shown to be expressed in the AE of the mesenchyme blastula embryo (Su et al.,2009). Our detailed study on gene expressions revealed that Sp-IrxA was first detected by in situ hybridization at 18 hpf in the AE near the vegetal plate (Fig. 4A). Its expression became stronger and expanded toward the animal pole as the embryo develops (Fig. 4A–E). Sp-Msx was also initially detected in a region near the vegetal plate at 20 hpf (Fig. 4G). Unlike the expression of Sp-IrxA, which spread animally, the expression of Sp-Msx remained in the vegetal AE of the mesenchyme blastula embryo (Fig. 4G–K). At 24 hpf, the expression domain of Sp-IrxA was broader than the Sp-Msx domain along the animal-vegetal and oral-aboral axes (Fig. 4E,F,K,L). The onset and initial expression pattern of Sp-IrxA and Sp-Msx appeared similar; therefore, we performed double FISH to determine if there are any differences in their expression patterns. Similar to the AP/NBT-BCIP staining, Sp-IrxA was first detected in the AE at 18 hpf, whereas Sp-Msx expression could not be detected at this time (Fig. 4M–T). The double FISH results also confirmed that as the embryo developed, Sp-IrxA expression expanded toward the animal pole and the oral ectoderm, whereas the expression of Sp-Msx remained strong in the vegetal AE (Fig. 4U–F′). Double FISH of Sp-Nk2.2 and Sp-IrxA showed that, except for the small Sp-Nk2.2 OE expression domain, the expression domains of Sp-Nk2.2 and Sp-IrxA completely overlapped in the central AE (Fig. 4G′–J′). Therefore, we concluded that at the mesenchyme blastula stage, the expression domains of Sp-Nk2.2, Sp-Klf7, and Sp-IrxA completely overlapped in the AE.

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Figure 4. The expression domain of Sp-Msx in the vegetal aboral ectoderm resides in the Sp-IrxA expression domain. Whole mount in situ hybridization for Sp-IrxA (A–F) and Sp-Msx (G–L) was performed at different blastula stages. The brackets denote the area of Sp-IrxA expression expanding from the vegetal AE toward the animal pole and the expression of Sp-Msx remaining in the vegetal AE during the blastula stages. Double FISH of Sp-IrxA and Sp-Msx (M–F′) show that the expression domain of Sp-Msx resides in the vegetal part of the Sp-IrxA expression domain. Double FISH of Sp-Nk2.2 and Sp-IrxA (G′–J′) reveal that the expression domains of Sp-Nk2.2 and Sp-IrxA overlap in the AE. The initial expression of Sp-Nk2.2 in the OE is indicated by arrows.

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Sp-Dlx and Sp-Hox7 Are Gradually Expressed in the Central Aboral Ectoderm

Sp-Dlx was first identified in a genome-wide search identifying homeobox transcription factor genes, and it is known to be expressed in the ectoderm (Howard-Ashby et al.,2006). Further studies showed that Sp-Dlx is expressed in the AE and has a positive feedback relationship with Sp-IrxA (Su et al.,2009). A Sp-Hox7 fragment was originally isolated from a genomic DNA library (Angerer et al.,1989) using the orthologous gene Hbox1, isolated from the sea urchin Tripneustes gratilla, as a probe (Dolecki et al.,1986). Later, the complete sequence of Sp-Hox7 was determined, and it was confirmed to be a paralog of the Hox7 gene group (Martinez et al.,1997,1999). Sp-Hox7 is expressed in the AE of the blastula stage embryo (Su et al.,2009), and its mRNA retracts to a small area around the apex of the pluteus larva (Angerer et al.,1989). The detailed in situ hybridization results showed that both Sp-Dlx and Sp-Hox7 were first detected in the AE near the vegetal plate of the 20-hpf embryo (Fig. 5A, F). As development proceeded, the expression domains of both genes expanded animally with the strongest signal in the vegetal AE (Fig. 5A–J). Double FISH of Sp-Dlx and Sp-Hox7 showed that the expression domains of both genes were almost identical at the vegetal and animal boundaries (Fig. 5K–V). As development progressed, however, the expression of Sp-Hox7 expanded toward the OE at the vegetal border, whereas Sp-Dlx maintained a narrower domain in the central AE (Fig. 5W–Y). Therefore, during the mesenchyme blastula stage, Sp-Dlx and Sp-Hox7 were gradually expressed in the central AE.

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Figure 5. Sp-Dlx and Sp-Hox7 are gradually expressed in the central aboral ectoderm. The expression domains of Sp-Dlx (A–E) and Sp-Hox7 (F–J) were detected by in situ hybridization followed by NBT-BCIP staining. Double FISH patterns of Sp-Dlx and Sp-Hox7 were visualized using a fluorescent microscope Zeiss Axio Imager (K–R) or a Leica confocal microscope (S–Y).

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Nuclearization of pSmad1/5/8 in the Central Aboral Ectoderm

The BMP signal is one of the major inputs of the AE GRN (Su et al.,2009); therefore, we performed immunostaining with a commercial pSmad1/5/8 antibody to monitor the activation of Smad-mediated BMP signaling from the early blastula to the mesenchyme blastula stage. Nuclearization of pSmad1/5/8 was first detected at one side of the ectoderm at 18 hpf (Fig. 6A–F). The nuclear pSmad1/5/8 signal was confirmed to be in the AE, which is opposite of Sp-Chordin expression in the OE (Bradham et al.,2009; Lapraz et al.,2009; Su et al.,2009) (Fig. 6D–I). Next, we performed a combination of pSmad1/5/8 staining and Sp-Tbx2/3 FISH to compare the region of pSmad1/5/8 nuclearization relative to the Sp-Tbx2/3 expression domain. The expression of Sp-Tbx2/3 was detected at 16 hpf (Fig. 6J–L). Two hours later, the nuclearization of pSmad1/5/8 was observed in the AE (Fig. 6M–O). At the mesenchyme blastula stage, we observed three different patterns of nuclear pSmad1/5/8 and Sp-Tbx2/3 expression domains (Fig. 6P–X). These different patterns may represent the dynamic and progressive changes that occur in pSmad1/5/8 nuclearization and Sp-Tbx2/3 expression during the mesenchyme blastula stage. To clarify these dynamic changes in Sp-Tbx2/3 expression, we classified its expression patterns into 5 groups: (1) no expression, (2) AE, (3) AE and aboral endomesoderm, (4) diminished in the AE, and (5) aboral endomesoderm expression (Fig. 6Y–C′). The proportions of the expression patterns between 16 to 26 hpf were analyzed (Fig. 6D′). At 22 hpf, 50% of the embryos showed Sp-Tbx2/3 expression in the AE and aboral endomesoderm, and the expression was diminished in the AE in 42% of the embryos. At 24 hpf, we started to see the expression only in the aboral endomesoderm in 42% of the embryos and this expression pattern increased to 84% at 26 hpf. From the changes in the proportions during development, we conclude that the three different expression patterns at 24 hpf reflect the chronological changes that occurred in Sp-Tbx2/3 expression. At the mesenchyme blastula stage, the three expression patterns of Sp-Tbx2/3 seem to negatively correlate with the changes in pSmad1/5/8 nuclearization intensity. At the early mesenchyme blastula stage, the pSmad1/5/8 nuclearization region was smaller than the Sp-Tbx2/3 expression domain, which marked the whole AE and the aboral endomesoderm (Fig. 6P–R). The nuclear signal of pSmad1/5/8 became stronger in the AE by the mid-mesenchyme blastula stage, while the level of Sp-Tbx2/3 transcript decreased in this area (Fig. 6S–U). At the late mesenchyme blastula stage, the strong pSmad1/5/8 signal became broader while the expression of Sp-Tbx2/3 diminished in the AE (Fig. 6V–X). Interestingly, the expression of Sp-Tbx2/3 in the aboral endomesoderm remained unchanged (Fig. 6P–X). This attenuation in Sp-Tbx2/3 expression in the AE at the mid-mesenchyme blastula stage was also observed in Figure 2 (Fig. 2I, G′). Furthermore, we used 3D surface plots to quantify the staining intensity of pSmad1/5/8 and Sp-Tbx2/3. The plots clearly showed an area with a high pSmad1/5/8 peak and a Sp-Tbx2/3 valley in the AE at the mid-mesenchyme blastula stage (Fig. 6E′–H′). The Sp-Tbx2/3 valley then enlarged as the pSmad1/5/8 peak became broader in the AE by the late mesenchyme blastula stage (Fig. 6I′,J′). To compare the spatial relationship between pSmad1/5/8 staining and Sp-Tbx2/3 expression, the aboral half of the embryo was projected to the X-axis from the animal to the vegetal pole. The intensity plot further showed the progressive attenuation of Sp-Tbx2/3 expression in the pSmad1/5/8 peak region (Fig. 6K′–M′). This correlation implies that strong Smad-mediated BMP signaling negatively regulates the expression of Sp-Tbx2/3 in the AE. This idea contradicts the positive role BMP signaling plays in the activation of Tbx2/3 in the sea urchin P. lividus embryo (Duboc et al.,2004; Lapraz et al.,2009). Alternatively, in the previous large-scale ectoderm GRN analysis, morpholino knockdown of Sp-BMP2/4 in S. purpuratus embryos did not significantly alter the transcript level of Sp-Tbx2/3 quantitatively (Su et al.,2009). Therefore, given that Sp-Tbx2/3 expression was observed prior to pSmad1/5/8 nuclearization in the AE and the negative correlation between the Sp-Tbx2/3 expression domain and pSmad1/5/8 nuclear intensity at the mesenchyme blastula stage, we hypothesize that Smad-mediated BMP signaling is not the initial input for Sp-Tbx2/3 expression and that Sp-Tbx2/3 expression is negatively regulated by Smad-mediated BMP signaling at the mesenchyme blastula stage. Further studies on the regulation of Sp-Tbx2/3 expression by BMP signaling will elucidate the molecular mechanism regulating this process.

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Figure 6. The pattern of the nuclear pSmad1/5/8 staining and the Sp-Tbx2/3 expression domain changes during the blastula stages. Immunostaining with pSmad1/5/8 antibody was done in combination with FISH using either Sp-Chordin (A–I) or Sp-Tbx2/3 (J–X) as a probe. Five different Sp-Tbx2/3 expression patterns were detected with the pattern labeled at the top of each panel (Y–C′). D′: Proportions of Sp-Tbx2/3 expression patterns at different developmental stages were counted and the Roman numerals correspond to the expression patterns in Y–C′. The number on top of each bar indicates the total number of embryos counted at each stage. The embryos were observed from the lateral side, oral to the left and aboral to the right (A–C′). 3D surface plots (E′–J′) were derived from the Z-projected images P, Q, S, T, V, and W, respectively. The axes of the embryo indicated in E′ are as follows: A, animal; Ab, aboral; O, oral; V, vegetal. The relative fluorescence intensity of pSmad1/5/8 staining and Sp-Tbx2/3 expression were plotted from the animal (A) to vegetal (V) pole (K′–M′) based on the Z-projected images (P–X). The developmental stage of the embryo is indicated at the top of each panel (A–X, E′–M′). E-MB, early-mesenchyme blastula; M-MB, mid-MB; L-MB, late-MB. The white arrowheads denote the attenuation of Sp-Tbx2/3 expression in the AE.

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Summary of the Gene Expression Subdomains in the Aboral Ectoderm

This study on the detailed temporal and spatial expression patterns of eight transcription factor genes provides the first expression map in the aboral ectoderm of the sea urchin embryo. The onset of gene expression was detected by two different methods, QPCR and in situ hybridization, which showed consistent results. The temporal and spatial dynamic gene expression patterns are summarized in Figure 7 (Fig. 7A,B). The expression of Sp-Hmx begins in the animal AE and stays in this region at the mesenchyme blastula stage. Sp-Tbx2/3 marks the whole AE and extends to the aboral endomesoderm. The expression of Sp-IrxA, Sp-Klf7, Sp-Nk2.2, Sp-Msx, Sp-Hox7, and Sp-Dlx all begin in the vegetal AE and later expand toward the animal pole and the oral ectoderm in different manners. The expression of Sp-Msx remains in the vegetal AE; however, the expression domains of the other five genes expand into the animal boundary of the Sp-Tbx2/3 and Sp-Hmx expression domains as the PMCs ingress into the blastocoel. At the mesenchyme blastula stage, the expression domains of these five genes gradually occupy the central AE (Fig. 7B). The Sp-IrxA, Sp-Klf7, and Sp-Nk2.2 domains overlap and form the broadest region in the central AE. The Sp-Hox7 domain resides in this region, and the Sp-Dlx domain is the narrowest in the central AE. The most vegetal region of the AE will develop into the apex in pluteus larva. These gene expression patterns also foreshadow the change of the embryonic oral-aboral (OA) axis into the larval OA (mouth to apex) axis (Fig. 7C).

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Figure 7. Aboral ectoderm subdomains are revealed by the different gene expression patterns. A,B: The gene expression domains during development are demarcated by the colored lines representing the different genes. To avoid confusion, genes expressed in the animal and vegetal AE domains (A) are presented separately from the genes expressed in the central AE (B). The stage of the embryo is indicated at the top of each drawing. The light green region indicates the Sp-Tbx2/3 expression domain. The OE expression domains of Sp-Nk2.2 and Sp-Klf7 are omitted in B. The embryos were viewed from the lateral side, oral to the left and aboral to the right. The gray dotted line marks the boundary between the ectoderm and endoderm. C: The oral-aboral (OA) axis at the mesenchyme blastula stage (black dotted line) and OA axis in the pluteus larva (red dotted line) are indicated.

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Although BMP signaling is important in patterning the AE of sea urchin embryos, our pSmad1/5/8 staining results suggest that the initiation of Sp-Tbx2/3 expression is not controlled by Smad-mediated BMP signaling. Instead, it has been suggested that a redox-sensitive factor is the initial input that activates Sp-Tbx2/3 transcription in the AE (Su et al.,2009). Except for Sp-Tbx2/3 and Sp-Hmx, all of the genes studied are initially expressed in the vegetal AE, which is derived from the vegetal blastomere quartet, VA, of the eight-cell sea urchin embryo (Cameron et al.,1987). The expression of Sp-IrxA, Sp-Klf7, Sp-Nk2.2, Sp-Hox7, and Sp-Dlx then expands toward the Na, NL, and VL progenies. The dynamic changes in the gene expression patterns in the central AE indicate that the expression of these aboral ectoderm regulators occur independently of the cell lineage. These changes, however, do depend on the positional effects that are the result of induction mechanisms involving signaling pathways. The combination of the induction mechanisms initiated in the vegetal AE and the extensive feedback relationships between these genes may expand their expression domains and establish the cell fate of AE cells as simple epithelial cells. Our detailed gene expression patterns provide a foundation for resolving the AE GRN. The casual linkages in the GRN will elucidate the mechanism regulating the development of this polyclonal origin territory into a distinct cell type.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Animals and Embryos

Adult sea urchins (Strongylocentrotus purpuratus) were obtained from So. California Sea Urchin Co. (Patrick Leahy, Corona del Mar, CA). Gametes were released by shaking adult sea urchins. Fertilization and embryo cultures were carried out in filtered seawater (FSW) and maintained at 15°C.

Temporal Expression Profiles

Primers used for QPCR analysis were previously described in Su et al. (2009). mRNA was isolated from several embryonic stages using the RNeasy Micro kit (Qiagen, Chatsworth, CA) and was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). The resulting cDNA was used as a template for QPCR. Levels of ubiquitin mRNA were used for normalizing samples (Ransick,2004). The QPCR analysis was performed on a Roche LightCycler 480 and using the LightCycler 480 SYBR Green I Master (Roche, Nutley, NJ).

In Situ Hybridization

The cDNA clones of Sp-Tbx2/3, Sp-IrxA, Sp-Dlx, and Sp-Chordin were obtained by PCR or cDNA library screening as previously described (Su et al.,2009). These clones were used directly for probe synthesis. For Sp-Hmx and Sp-Nk2.2, 3′RACE was performed using the FirstChoice RLM-RACE Kit (Ambion, Austin, TX). A 3′ Sp-Nk2.2 clone was successfully isolated, and a fragment containing both the partial coding sequence (CDS) and the 3′UTR was PCR amplified for probe synthesis. Because of a cluster of multiple adenosine monophosphates found in the middle of the Sp-Hmx CDS, the 3′RACE resulted in a fragment containing a partial 5′UTR and CDS that was used for probe synthesis. For Sp-Msx and Sp-Hox7, the 5′UTR sequences were extended by 5′RACE. The full-length sequence of the Sp-Klf7 transcript was obtained by 5′ and 3′ RACE, and the sequence was deposited in the GenBank with the accession number HM347349. Primers used to construct the clones for probe synthesis and the positions of the probes are listed in Supp. Table S1, which is available online. Antisense riboprobes were synthesized using digoxigenin (DIG) or fluorescein RNA Labeling Mix (Roche) with T7 or SP6 RNA polymerases (Promega, Madison, WI). For Sp-Klf7, because of its low expression level, a dinitrophenol (DNP)-labeled probe was made using the LabelIT DNP Labeling Kit (Mirus, Madison, WI).

In situ hybridization was performed as previously described (Walton et al.,2006; Yu and Holland,2009) with the following modifications. Embryos were fixed in 4% paraformaldehyde in FSW with 10 mM 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS; Sigma E1894, St. Louis, MO), pH 8, at 4°C overnight. Next, they were washed with FSW and dehydrated with cold methanol. Stored embryos were rehydrated with phosphate buffer saline containing 0.1% Tween-20 (PBST) and pre-hybridized at 60°C with hybridization buffer (50% formamide, 5× SSC pH 7, 1 mg/ml of yeast RNA, 100 μg/ml of heparin, 1× Denhardt's, 0.1% Tween-20, and 5 mM EDTA). Embryos were then hybridized with DIG, fluorescein, or DNP-labeled probe overnight at 60°C. After hybridization, embryos were washed with the hybridization buffer once at 60°C for 10 min, then four times with wash solution (50% formamide, 5× SSC, 0.1% Tween-20, and 5 mM EDTA) at 60°C, followed by two MABT (100 mM maleic acid, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) washes at room temperature. Washed embryos were blocked in MAB blocking buffer (1× MABT, 2% blocking reagent [Roche], and 10% sheep serum) for 1 hr at room temperature and then incubated in alkaline phosphatase (AP) conjugated anti-DIG (Roche), anti-fluorescein (Roche), or anti-DNP antibody (Abcam, Cambridge, MA) overnight at 4°C. Embryos were then washed with MABT and AP buffer (100 mM Tris, pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20, and 2 mM levamisole). The AP-conjugated antibodies were detected using 190 μg/ml of 4-Nitro blue tetrazolium chloride (NBT) and 175 μg/ml of BCIP 4-toluidine salt as substrates. For double fluorescent in situ hybridization (FISH), post-hybridization washes with MABT were replaced with wash solution/2× SSCT (1:1), 2× SSCT, 0.2× SSCT, and TBST (100 mM Tris, 150 mM NaCl, pH 7.5, and 0.1% Tween-20) washes at 60°C. TBST blocking buffer (1×X TBST, 0.5% blocking reagent) was used before adding horse-radish peroxidase (POD) conjugated anti-DIG (Roche), anti-Fluorescein (Roche), or anti-DNP antibody (PerkinElmer). The fluorescent signal was amplified by the TSA Plus Cyanine 3 & Fluorescein system (PerkinElmer, Waltham, MA). Two-channel fluorescence and DIC images of the embryo were visualized and photographed using a Zeiss (Thornwood, NY) Axio Imager.A1 microscope or a Leica (Bannockburn, IL) TCS-SP5-AOBS confocal microscope.

Immunofluorescence With pSmad1/5/8 Antibody

Antibody specific for pSmad1/5/8 was purchased from Cell Signaling Technology (Danvers, MA; no. 9511). For double labeling of Sp-Chordin or Sp-Tbx2/3 RNA and pSmad1/5/8 protein, FISH was performed first using the anti-DIG-POD antibody and the Fluorescein substrate in the TSA Plus system to detect the mRNA. Embryos were then washed with PBST and blocked in PBST blocking buffer (PBS with 0.1% Tween-20 and 3% BSA) for 2 hr at room temperature, followed by incubation with anti-pSmad1/5/8 antibody at a 1:200 dilution in PBST blocking buffer overnight at 4°C. Alexa Fluor 594 goat anti-rabbit antibody (Invitrogen, Carlsbad, CA; 1:400 in blocking solution) was used as a secondary antibody.

Image Analysis

Optical sections or Z projection images acquired at 1–2 μm/slice were processed by Leica confocal software (LAS AF). To visualize the 3D surface plot, Z-projected images were analyzed by an ImageJ (http://rsbweb.nih.gov/ij/) plug-in program, Interative 3D Surface Plot (http://rsbweb.nih.gov/ij/plugins/surface-plot-3d.html). To quantify and compare the spatial relationship between pSmad1/5/8 staining and Tbx2/3 expression, the aboral half of the embryo was projected to the X-axis from the animal (A) to vegetal (V) pole using Plot Profile in ImageJ. The relative fluorescence intensity corresponding to the embryo AV axis was then plotted in Microsoft Excel.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

We thank the personnel in the ICOB core facility for excellent technical assistance. We also thank the personnel at the Marine Research Station, ICOB, for maintaining the sea urchin aquariums.

REFERENCES

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  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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DVDY_22514_sm_supptab.doc34KSupporting Table 1

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