Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

Iwasaki-Yokozawa, Sawa; Nanjo, Ryota; Akiyama-Oda, Yasuko; Oda, Hiroki

doi:10.1186/s12915-022-01421-0

Research article
Open access
Published: 06 October 2022

Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

Sawa Iwasaki-Yokozawa¹,
Ryota Nanjo^1,2,
Yasuko Akiyama-Oda^1,3,4 &
…
Hiroki Oda ORCID: orcid.org/0000-0003-4424-988X^1,2

BMC Biology volume 20, Article number: 223 (2022) Cite this article

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A Publisher Correction to this article was published on 17 December 2022

This article has been updated

Abstract

Background

The process of early development varies across the species-rich phylum Arthropoda. Owing to the limited research strategies for dissecting lineage-specific processes of development in arthropods, little is known about the variations in early arthropod development at molecular resolution. The Theridiidae spider, Parasteatoda tepidariorum, has its genome sequenced and could potentially contribute to dissecting early embryonic processes.

Results

We present genome-wide identification of candidate genes that exhibit locally restricted expression in germ disc forming stage embryos of P. tepidariorum, based on comparative transcriptomes of isolated cells from different regions of the embryo. A subsequent pilot screen by parental RNA interference identifies three genes required for body axis formation. One of them is a GATA-like gene that has been fast evolving after duplication and divergence from a canonical GATA family gene. This gene is designated fuchi nashi (fuchi) after its knockdown phenotypes, where the cell movement toward the formation of a germ disc was reversed. fuchi expression occurs in cells outside a forming germ disc and persists in the endoderm. Transcriptome and chromatin accessibility analyses of fuchi pRNAi embryos suggest that early fuchi activity regulates chromatin state and zygotic gene activation to promote endoderm specification and pattern formation. We also show that there are many uncharacterized genes regulated by fuchi.

Conclusions

Our genome-based research using an arthropod phylogenetically distant from Drosophila identifies a lineage-specific, fast-evolving gene with key developmental roles in one of the earliest, genome-wide regulatory events, and allows for molecular exploration of the developmental variations in early arthropod embryos.

Background

The early processes of development and reproductive strategies of animals vary among species. In certain metazoan phyla, including Chordata and Arthropoda, a high degree of early developmental variations among species is observed, despite the identification of similar morphological traits and gene expression patterns during mid-embryogenesis in each phylum [1,2,3,4,5,6,7]. Mechanisms of animal evolution that account for varied developmental trajectories preceding the phylotypic period are poorly understood.

The species-rich phylum Arthropoda, comprising three major groups (Chelicerata, Myriapoda, and Pancrustacea [Crustacea plus Hexapoda]), conspicuously shows the diversification of early developmental processes without the disruption of stable embryonic traits, such as the elongating germ band and repetitive body units. Genetic mutation screens and a range of molecular and genetic techniques are feasible for the model organism Drosophila melanogaster, which has contributed to the identification of numerous essential genes and their interactions in body axis formation, germ layer formation, and segmentation. The Drosophila paradigm of genetic programs for early embryogenesis has allowed for candidate-gene approaches toward elucidating the evolution of developmental mechanisms in other insects or arthropod species. Such candidate-gene approaches, however, are biased with respect to the detection of key developmental genes; having relatively low consideration for lineage-specific, fast evolving, or orphan genes [8, 9]. Conversely, comparative genomics in Arthropoda and other metazoan phyla has revealed lineage-specific genomic events, including gain/loss, expansion, or divergence of a gene or a gene family [10,11,12,13]. bicoid, spätzle, and gurken in Drosophila, which are key regulators in the organization of the major embryonic axes, are examples of lineage-specific or fast-evolving genes [14,15,16]. Considering these Drosophila cases and similar ones in different phyla [17,18,19], an unbiased way of identifying key developmental genes is required. Therefore, several studies that used non-Drosophila insect species have performed genetic mutation or gene knockdown screens by assessing larval cuticle phenotypes [20,21,22,23]. However, the identification and characterization of novel genes with key functions in specific processes of early embryogenesis are hardly feasible in any non-Drosophila arthropods, particularly in non-insect arthropods.

In arthropods, many model species have continued to emerge, with the genome sequenced [10, 24]. There have been numerous technical merits with respect to the study of early embryogenesis in one of them, the common house spider—Parasteatoda tepidariorum, and distinct differences from Drosophila have been observed in the early embryogenesis mechanism [25]. These differences include early completion of cellularization [26, 27] and involvement of Hedgehog signaling in establishing the polarity of the first embryonic axis and regulating symmetry-breaking movement of the Dpp signaling center during the orthogonalization of the first and second embryonic axes [28,29,30]. In the P. tepidariorum experimental system, genetic regulations during early embryonic processes can be investigated using a simple gene knockdown technique, that is, parental RNA interference (pRNAi) [29]. Research using P. tepidariorum has been empowered by genomic and bioinformatic resources [25, 31,32,33,34,35,36], thus providing the foundation for identifying key genes involved in P. tepidariorum development.

The egg of P. tepidariorum is spherical in shape (Fig. 1). As the development starts, the egg nuclei synchronously divide, approaching the surface of the egg (stage 1). Cellular organization of the embryo is established by the time the nuclei increase to 16 [26]. The initial blastoderm comprises approximately 64 cells (stage 2; 11 h after egg laying [AEL]), showing spherical symmetry at the morphological level. This symmetry is broken at approximately 15 h AEL by the emergence of uneven cell densities toward the formation of a germ disc within a hemisphere of the egg (stage 3) (Fig. 1; Additional file 1: Movie S1), which side is designated the embryonic side. At the pole on this side, a small number of cells are apically constricted, thus forming a blastopore [25, 28]. Zygotic gene activities are required for the process of germ-disc formation [37]. Although most blastoderm cells participate in the formation of the germ disc, cells derived from around the abembryonic pole do not, and remain in the non-germ-disc region (Fig. 1; see also Additional file 1: Movie S1). The formed germ disc is sharply demarcated by the difference in cell density and comprises a single layer of more than 1000 cuboidal epithelial cells [38]. Cells at the center of the germ disc, corresponding to the blastopore, internalize to become the central endoderm (cEND) cells and the cumulus mesenchymal (CM) cells [28, 32]. The latter cell populations are clustered and act as a dynamic source of Dpp signaling to promote the development of the dorsal side of the embryo [28, 29] (Fig. 1). Cells at and near the rim of the germ disc internalize to become the peripheral endoderm (pEND) and mesoderm (pMES) cells [32]. A line of cells along the rim of the germ disc express specific genes, including a P. tepidariorum hedgehog homolog (Pt-hh), that contributes to the anterior–posterior patterning of the embryonic field [30, 39, 40]. The germ disc is transformed into a bilaterally symmetric germ band through orchestrated cell rearrangements, while being progressively patterned [29, 38] (Fig. 1).

The formation of the sharply demarcated germ disc is an early morphogenetic event in the embryogenesis of Theridiidae and some other Araneoidea spiders [27, 28, 37, 41,42,43,44], but it is not a general feature of spider (Araneae) embryonic development [45,46,47]. There are many other variations in early developmental processes among spider species, including the presence/absence of a visible cumulus and various modes of gastrulation [44, 48]. The spider lineage provides diverse developmental trajectories prior to reaching the embryonic traits typical of arthropods.

To explore the variation in the process of early embryonic development across the phylum Arthropoda at molecular resolution, we conducted comparative transcriptomes of cells isolated from different regions of the embryo at the germ-disc forming stage in P. tepidariorum. Through a pRNAi-based functional screen of differentially expressed gene candidates, we identified a lineage-specific GATA-like gene, fuchi nashi (fuchi), whose knockdown hindered the completion of germ-disc formation. We obtained genome-wide datasets that represented gene expressions and chromatin accessibilities affected by fuchi knockdown in early embryos. Our findings showed that fuchi regulates zygotic gene activation to promote endoderm specification and pattern formation in the early stage of the Theridiidae spider embryo. This study provides new molecular clues for exploring the mechanisms for diversifying early developmental trajectories in the phylum Arthropoda.

Results

Comparative transcriptome analyses of cells isolated from different regions of early P. tepidariorum embryos

To search for genes with locally restricted expression in P. tepidariorum embryos at stage 3, we performed RNA sequencing (RNA-seq) of small cell populations (approximately 10–30 cells) isolated from central (c), intermediate (i), and peripheral (p) regions of the nascent germ disc using glass capillary needles (Fig. 2A, B). Using this strategy, we intended to identify genes that are relatively strongly expressed either at the center or the periphery of the germ disc or both. RNA-seq reads were mapped to the P. tepidariorum reference genome Ptep1.0 (GCA_000365465.1), and then the reads were counted against the AUGUSTUS gene models (aug3.1) [31]. The read counts were compared using edgeR [49] by setting the following three combinations: comparison I, c cells versus i/p cells; comparison II, p cells versus c/i cells; and comparison III, i cells versus c/p cells (Fig. 2C–E). Based on these comparisons, we genome-widely identified candidates of differentially expressed genes (DEGs) (Additional file 2: Tables S1-S3). Using the values of false discovery rate (FDR) and fold-change (FC), the candidate DEGs that exhibit strong expression in c cells (from comparison I), p cells (from comparison II), or both (from comparison III) were prioritized (lower FDR with log₂FC < − 10 [for comparisons I and II] or log₂FC > 10 [for comparison III]). The top 10 genes from comparison I were selected as Group C (Fig. 2C), the top 5 genes from comparison II as Group P (Fig. 2D), and the top 5 genes from comparison III as Group CP (Fig. 2E). The top-ranked g4238 in Group C was listed in Group CP, despite its much stronger expression in c cells than in other cells. To avoid redundancy, g4238 was removed from Group CP. Therefore, a total of 19 genes were systematically selected as high-priority candidate genes that might exhibit locally restricted expression in the stage-3 embryo.

To validate our gene-selection strategy, we examined the expression patterns of the 19 selected genes in stage-3 embryos using chromogenic whole-mount in situ hybridization (WISH) (Additional file 3: Fig. S1). Three of the ten Group C genes (g4238, g14287, and g16467) showed specific expression at the embryonic pole. Furthermore, three of the other Group C genes (g15167, g5118, g20850) and one of the four Group CP genes (g26874) showed specific expression at the embryonic pole and in a broad area on the abembryonic side. The other Group CP and Group P genes, however, showed no specific detectable signals.

Additionally, similar comparative transcriptome analyses of isolated cells were applied to stage-4 and early stage-5 embryos. The resulting DEG lists (Additional file 2: Tables S4, S5) included some of the DEGs identified by the analyses of the stage-3 samples (e.g., g4238, and g132), as well as genes that had been known to show region-specific expression at the corresponding and/or later stages (e.g., Pt-lab1 [g7954], Pt-BarH1 [g8250], Pt-prd2 [g18397], and Pt-hh [g4322]).

These data suggest the effectiveness of our gene-selection strategy based on comparative transcriptome analyses of isolated cells from early P. tepidariorum embryos.

Pilot functional screen identifies three genes required for germ-disc formation and/or axis formation

To identify genes with key functions in the early embryonic development of P. tepidariorum, we screened the 19 candidate DEGs (from the stage-3 samples) using parental RNAi (pRNAi). Double-stranded RNA (dsRNA) synthesized for each gene, as well as that for a non-spider control gene green fluorescent protein (gfp), was injected into at least two adult females. Aliquots of eggs deposited by the females were placed in oil to monitor the developmental process from early stages under the stereomicroscope. Through this screening, we identified three genes whose knockdown resulted in uncommon defects associated with germ-disc formation (g26874 and g7720) and/or cumulus movement (g26874, g7720, and g4238) (Additional file 4: Table S6) [50]. To verify the RNAi specificity, we injected two or three dsRNAs prepared from non-overlapping regions of each positive gene and confirmed that the same phenotypes were produced depending on the genes (Fig. 3A; Additional file 3: Fig. S2; Additional file 5: Movie S2) [50]. In addition, we confirmed through WISH that the transcript levels of the target genes were reduced in the corresponding pRNAi embryos (Fig. 3B).

Time-lapse microscopy and cell tracking revealed that g26874 pRNAi embryos showed normal blastoderm formation (stage 2) and subsequent initiation of cell density shift toward the formation of a germ disc, in a rather normal way, but they failed to stabilize the boundary of the forming germ disc (25 h AEL), with the cell density shift being reversed (Fig. 3A; Additional file 5: Movie S2; Additional file 6: Movie S3). In all of such severely affected g26874 pRNAi embryos, the initial cell thickening at the embryonic pole took place. In 64% of the g26874 pRNAi embryos (n = 101 from 10 egg sacs), however, CM cell migration was faint or failed to occur. The defects in germ-disc formation were not recovered at later stages, showing no clear sign of cell movements toward the formation of a germ band. In the most severe cases of g26874 pRNAi embryos, cells that had initially shifted toward forming a germ disc reversed their movement to spread across the embryo, followed by abrupt breakdown of the surface cell layer and extrusion of the yolk materials (Additional file 5: Movie S2) [50].

g7720 pRNAi embryos showed normal blastoderm formation followed by cell-density shift (stages 2 and 3), but its development was arrested around the end of stage 3, showing a gradually degenerating germ disc (Additional file 5: Movie S2) [50]. Although cumulus-like cell thickening was formed at the embryonic pole, it persisted there, with the overall morphology failing to develop further.

g4238 pRNAi embryos were morphologically indistinguishable from wild-type and gfp pRNAi (control) embryos until early stage 5. The earliest visible difference of g4238 pRNAi embryos from normal embryos was their failure to initiate migration of the CM cells at early stage 5, followed by their disassembly and dispersal (Additional file 5: Movie S2) [50]. In many of the embryos, surface cells around the closed blastopore formed a tail-like protrusion. Despite experiencing cumulus defects, some g4238 pRNAi embryos displayed asymmetric cell movements toward the formation of a germ band [50].

Molecular characterization of the three identified genes

Developmental transcript profiling in whole wild-type embryos, based on public RNA-seq datasets [36], revealed that there are a few transcripts for g26874 and g4238 at stage 1 (9 h AEL) and increasing levels of the zygotic transcripts prior to peaking at stage 4 (Fig. 3C). In contrast, substantial levels of g7720 transcript appeared to be maternally supplied, with the zygotic transcript expressed at rather uniform levels through development.

Reciprocal blastp/tblastn searches using Drosophila and mouse reference sequence (RefSeq) protein databases at National Center for Biotechnology Informataion (NCBI) and the P. tepidariorum aug3.1 transcript sequences [31] revealed that g7720 encoded an orthologue of Drosophila proximal sequence element A (PSEA)-binding protein 49kD (Pbp49) and mouse small nuclear RNA-activating complex subunit 3 (Snapc3) [51], and g4238 an orthologue of Drosophila Ets98B (D-ets4) and mouse SAM pointed domain-containing Ets transcription factor (Fig. 3D) [52]. g4238 was identical to Pt-Ets4, as reported in a previous study [53], and consistent with our observations.

In contrast to the g4238 and g7720 cases, reciprocal blastp/tblastn searches failed to confirm the presence of Drosophila and mouse orthologues for g26874. The best-hit Drosophila and mouse proteins were Pannier (Pnr), a GATA transcription factor, and GATA-4, respectively (Fig. 3D), but these proteins were significantly closer to GATA family members encoded by g8336 and several other genes in the P. tepidariorum genome. Based on these results, along with those obtained from comprehensive sequence analyses described below, we concluded that g26874 encodes a novel GATA-like gene, which we named “fuchi nashi” (abbreviated fuchi) after its knockdown phenotypes. This Japanese word means rimless.

fuchi is a lineage-specific, fast-evolving GATA-like gene

The family of GATA transcription factors are characterized by the possession of a specific DNA-binding domain, which comprises two GATA-type zinc finger (ZF) motifs (ZF1 and ZF2), typically CX₂CX₁₇CX₂C, and two basic regions followed by each ZF motif (BR1 and BR2) (Fig. 4A, B) [54, 55]. Fuchi had this GATA-type DNA-binding domain, although its sequence was highly divergent from those of Drosophila Pnr and mouse GATA-4. To comprehensively understand the diversity of GATA family proteins among metazoans and the phylogenetic origin of fuchi, we exhaustively collected and manually aligned amino acid sequences of GATA family proteins from P. tepidariorum, other spider and non-spider chelicerate species as well as from other selected arthropod and non-arthropod metazoan species using publicly available bioinformatic resources (Additional file 7: Tables S7, S8). As some of the sequences were divergent, common alignment tools could not help to align them without complicated gaps. The resulting sequence alignment revealed that the metazoan GATA family members were classified under canonical and noncanonical types. The canonical type was characterized by a BR1 of 29 amino acid residues with high sequence similarity, and the noncanonical type by a BR1 of more, or less, than 29 amino acid residues with a varying degree of sequence divergence. The canonical GATA family members included proteins encoded by five P. tepidariorum genes (designated Pt-GATA1 to Pt-GATA5, encoded by g20514, g1834, g8336, g25261, and g8337, respectively) and their counterparts from other Araneae species, as well as Drosophila Serpent (Srp), the only Nematostella vectensis GATA (Nv-GATA), two echinoderm GATAs, and mouse GATA-1 to GATA-6 (Fig. 4B–D; Additional file 7: Table S8). Phylogenetically widespread distribution of the specific sequence features suggested that the canonical GATA family members represent the ancestral state for metazoan GATA proteins. The Pt-GATA3 and Pt-GATA5 genes were closely located on the same scaffold (< 100 kb), although the Pt-GATA1, Pt-GATA2, and Pt-GATA4 genes were located on separate scaffolds (Additional file 3: Fig. S3).

The sequence alignment of the noncanonical GATA family members, including Fuchi, guided the identification of at least three groups specifically found within the Araneae lineage, based on gap insertion patterns, cysteine residue spacing, and sequence signatures (Fig. 4C, D; Additional file 7: Table S8). These three groups were designated as GATA-like 1 to 3 (GATAL1, GATAL2, and GATAL3). GATAL1, which included a P. tepidariorum protein encoded by g26871 (Pt-GATAL1), was relatively close to the canonical GATA family members at the sequence level, but they had a BR1 of 34 amino acid residues. GATAL2, which included Fuchi and another P. tepidariorum protein encoded by g26875 (designated Pt-GATAL2b), was specifically identified by a signature sequence QCV(K/R)CG at the N-terminal end of ZF1, varied cysteine residue spacing in ZF1 (CX₂CX_17-20CX₂C), and a highly varied number (23–44) of amino acid residues in BR1. fuchi and the Pt-GATAL2b gene were tandemly located in the same direction on a scaffold, where the Pt-GATAL1 gene was also present, but in the opposite direction (Additional file 3: Fig. S3). GATAL3, which was only found in spiders of RTA clade, was specifically identified by conserved unique sequences and cysteine residue spacing (CX₂CX₁₈CX₂C) in ZF1. The signature sequence unique to GATAL2, QCV(K/R)CG, corresponded to ECVNCG, conserved in most canonical GATA family members (including Pt-GATA1 to 4) and GATAL1, to QC(A/V)(M/V)CG, conserved in Pt-GATA5 and its counterparts, and to ECANCG, conserved in GATAL3.

To investigate the phylogenetic relationship between canonical and noncanonical GATA family proteins, we performed maximum likelihood (ML) analysis using unambiguously aligned 75 amino acid sites from a total of 118 proteins with both ZF1 and ZF2 representing the typical cysteine residue spacing (CX₂CX₁₇CX₂C) (Fig. 4E, F; Additional file 7: Table S8). Results showed Araneae-specific expansions of GATA protein family members, through gene duplication and divergence. ML tree topology supported the orthology of each of the GATA5, GATAL1, and GATAL2 groups. Furthermore, branch lengths indicated that the sequences of the GATAL2 proteins had been evolving much faster than those of the canonical GATA family and GATAL1 proteins (Fig. 4F). Owing to their atypical cysteine residue spacing in ZF1 (CX₂CX_18-20CX₂C), Fuchi and other Araneoidea GATAL2 proteins could not be included in the ML analysis (Fig. 4C; Additional file 7: Table S8). Importantly, GATAL2 proteins with the typical ZF1 (CX₂CX₁₇CX₂C), which were used in the ML analysis, were present in a broader phylogenetic range (including Leptonetidae, Eresidae, and RTA clade) (Fig. 4C, E, F; Additional file 7: Table S8). To confirm the phylogenetic affinity of Fuchi to GATAL2 proteins with the typical ZF1, we conducted a second ML analysis using fewer amino acid sites (63 sites) and more proteins (168 proteins, including Fuchi) (Additional file 3: Fig. S4; Additional file 7: Table S8). This analysis showed that Fuchi and all other GATAL2 proteins were clustered together, with relatively long branches, and separated from the other GATA family subgroups. The combined results from the two ML analyses suggested that fuchi is a lineage-specific divergent gene. It was also suggested that variously divergent sequences in ZF1 of the Araneoidea GATAL2 proteins, except the specific signature sequence, were likely due to rapid sequence evolution from the GATAL2 ancestral state.

Unlike the other two of the three genes judged as positive in the functional screen, fuchi was characterized as a lineage-specific gene. We therefore decided to focus our later analyses on this gene.

fuchi is expressed in endoderm cells originating from both sides of the early embryo

Among the eight GATA family genes in P. tepidariorum, fuchi was the only gene which was transcribed at substantial levels during the germ-disc forming stage (stage 3) (Fig. 3C; Additional file 3: Fig. S5). We investigated the expression of fuchi transcript in early embryos at cellular resolution, using conventional WISH and the combination of FISH with antibody staining for β-catenin, which serves as a marker for regions of cell–cell contact (Fig. 5). Owing to technical limitations in the fixation of stage-1 embryos, the earliest stage of development examined was stage 2, the blastoderm stage (13 h AEL), when there is no morphological sign of asymmetry reflecting the future embryonic axes. At this stage, localized signals for fuchi expression were detected on one side of the embryo (Fig. 5A, A’, B, B') but could not be related with the axis that would emerge a little later (15 h AEL).

At early stage 3 (15 h AEL), the embryo begins to display morphological asymmetries, with an emerging embryonic-abembryonic (Em-Ab) axis (Fig. 1). At this stage and later, the fuchi transcript was detected in a small number of cells condensed at the embryonic pole (Fig. 5I, I’, I'') and in a larger number of cells showing a progressively wider distribution on the abembryonic side of the embryo (Fig. 5J, J’, J"). Using a flat-preparation of a representative early stage 3 (15 h AEL) embryo stained for fuchi transcript, the number of fuchi-expressing cells was counted. There were 8 and 20 fuchi-expressing cells around the embryonic pole and on the abembryonic side, respectively, among a total of approximately 300 cells (Additional file 3: Fig. S6). Notably, signals for fuchi transcript in the nuclei were, in many cases, observed as paired dots (Fig. 5I', J'), indicating that the zygotic transcription was already active at early stage 3. In the equatorial area of stage 3 embryos (15 and 21 h AEL), there was a transition from cells expressing fuchi transcript, on the abembryonic side, to those not expressing it, on the embryonic side (Fig. 5J, J’, J'', K, K', K''). In the embryo at 21 h AEL, the fuchi-expressing cells had larger apical surfaces than the fuchi-negative cells (Fig. 5K').

In embryos at stage 4 (25 and 27 h AEL), most fuchi-expressing cells on the abembryonic side had further enlarged surfaces with less tensed lines of cell–cell contact, which contrasted with a tightly packed organization of fuchi-negative cells constituting the nascent germ disc on the embryonic side (Fig. 5D, L, M, M’, M''). A few fuchi-expressing cells, however, were identified as part of the germ disc at its border (Fig. 5M', M"). At the blastopore, cEND cells and CM cells are internalized (Fig. 1). fuchi expression was observed in all these internalizing and internalized cells (Fig. 5L, L', L'').

In embryos at stage 5 (32 and 38 h AEL), all cells internalized through the blastopore continued to express fuchi (Fig. 5E, E’, F, F', N, N1, N1', O, O1). Peripheral populations of fuchi-expressing cells appeared to be internalized a little later than the central populations. At early stage 5 (32 h AEL), fuchi-expressing cells on the embryo surface were found not only in the non-germ-disc region but also at the rim of the germ disc (Fig. 5N, N2, N2’). At late stage 5 (38 h AEL), there were fuchi-expressing cells that were internalized/internalizing from or through the rim of the germ disc (Fig. 5F, F’, O, O2, O2'). Using a DNA stain probe, SPY555-DNA, we tracked some cell nuclei in the germ-disc/non-germ-disc transition area from early stage 5 to stage 8 in a live embryo (Additional file 4: Movie S4), which directly visualized two phases of cell internalization. Phase 1 of cell internalization occurred from mid to late stage 5, where cells that were not part of the germ disc were approaching the rim of the germ disc and then internalizing to below the surface cell layer of the germ disc. These were most likely to be fuchi-expressing cells. Phase 2 of cell internalization occurred subsequently, where cells at the rim of the germ disc were internalizing. Double staining for fuchi and Pt-hh transcripts revealed that certain cells at and near the rim of the germ disc at early stage 5 expressed both fuchi and Pt-hh (Fig. 5P, P’). Upon internalization, however, fuchi-expressing cells were exclusively Pt-hh-negative (Fig. 5Q, Q’).

To determine whether fuchi-expressing cells internalized from the peripheral side of the germ disc are endoderm or mesoderm, double staining of late stage 5 and stage 6 embryos for fuchi and Pt-fkh (a marker for pMES plus pEND) [56] or 003_J01 (a marker for pMES) [57] was performed. The use of 003_J01 enabled us to identify pMES cells prior to internalization, which was not the case with Pt-twi, another pMES marker [32, 56, 58]. Observations indicated that all internalized, fuchi-expressing cells were Pt-fkh-positive and most of them were 003_J01-negative (Additional file 3: Fig. S7). It was also indicated that 003_J01-positive cells appeared to be internalized later than fuchi-expressing cells, which presumably corresponded to Phase 2 of cell internalization. fuchi expression in embryos at stages 6 and 7 never showed a spatially periodic pattern along the emerging body axis (Fig. 5G, G’, H, H'), contrasting with mesoderm marker expression [58]. Taken together, these data suggested that fuchi is expressed in endoderm cells originating from both sides of the early embryo. In addition, fuchi expression occurred in association with extraembryonic differentiation (Fig. 5G, G’, H, H'). This fuchi expression was already initiated in the germ disc at late stage 5 (Fig. 5O,O1).

fuchi knockdown prevents the demarcation of a forming germ disc

To investigate the defects following fuchi knockdown, we analyzed the expression of Pt-hh, Pt-fkh, and fuchi transcripts in wild-type and fuchi pRNAi embryos at stage 4 and early stage 5 at cellular resolution. In normal germ-disc development, a line of cells expressing Pt-hh and Pt-fkh was organized to demarcate the boundary of the forming germ disc during stage 4 and early stage 5 (Fig. 6A, A’, C, C', E, E', G, G'). In the recognizable germ-disc/non-germ-disc transition area of fuchi pRNAi embryos at stage 4, the initial Pt-hh expression was strongly suppressed, while Pt-fkh expression was detectably initiated in certain cells with fuchi-positive nuclei and their neighboring cells (Fig. 6B, B’, D, D'). At early stage 5, faint Pt-hh and Pt-fkh expression were detected in small numbers of cells, but they did not contribute to shaping the germ disc (Fig. 6F, F’, H, H'). In fuchi pRNAi embryos at a later stage (late stage 5), there was no clear separation between cell populations on the embryonic and abembryonic sides, with cell internalizations being highly limited (Fig. 6I, I1’, I1", I2', I2"). The only cells situated below the surface layer were cells at or near the embryonic pole (Fig. 6I1', I1"), which were associated with β-catenin concentrations marking the CM cell cluster. These observations suggested that fuchi knockdown prevented the demarcation of a forming germ disc and interrupted the formation of the germ layers.

fuchi is required for zygotic activation of endodermal and patterning genes

To explore the role of fuchi in regulating early P. tepidariorum development, we conducted comparative transcriptome analyses of fuchi pRNAi versus untreated embryos at stages 2 (11 h AEL), 3 (20 h AEL), and early stage 5 (31 h AEL) using RNA-seq. These analyses identified 6, 139, and 785 DEGs for stages 2, 3 and early stage 5, respectively (FDR < 0.01; Fig. 7A, B; Additional file 9: Tables S9-S11). The experiments were validated by detecting lowered levels of fuchi transcript and unaffected levels of the α-catenin (catA) transcript in the fuchi pRNAi samples in comparison to the untreated ones (Fig. 7C). Except for the fuchi gene itself, there was no overlap between the stage-2 and stage-3 DEGs but there was an overlap of 93 genes between the stage-3 and early stage-5 DEGs (Fig. 7B; Additional file 9: Tables S9-S11). According to NCBI’s gene annotation, 26 of the 93 DEGs encoded uncharacterized proteins. Transcripts for the stage-2 DEGs appeared to be maternally supplied (Additional file 3: Fig. S8), which contrasted with those for highly ranked genes from the stage-3 and early stage-5 DEG lists (sorted by FDR value). They tended to show a sharp increase in the transcript level during stages 2 to 4, with no or few transcripts at stage 1, indicating little transcript supply from the mother (Fig. 7C, D). All the top 20 genes from the stage-3 DEG list were included in the stage-5 DEG list (Fig. 7D; Additional file 9: Table S10). Blastx searches against mouse and Drosophila RefSeq protein databases resulted in no hits for seven of the top 10 DEGs (E-value < 1e − 5) (Fig. 7C). Transcripts from 8 of the top 10 DEGs (g1125, g2114, g20850, g17950, g12453, g27539, g486, and g26914) were detected in all or part of the internalized endoderm at late stage 5, and those from 3 of the 8 genes (g17950, g12453, g20850) were detected in cells on the abembryonic side at stage 3 (Additional file 3: Fig. S9). Notably, the g20850 was identified as one of the Group C genes in the initial comparative transcriptome analysis of isolated cells (Fig. 2C; Additional file 3: Fig. S1).

The stage-3 fuchi pRNAi DEG list also included several genes previously characterized as key regulators of embryonic patterning in P. tepidariorum, such as Pt-hh (g4322) [30], Pt-sog (g23966) [29], and Pt-Delta (g25248) [32] (Additional file 3: Fig. S10; Additional file 9: Table S10). Despite the effects of fuchi knockdown on the expression of these patterning genes, certain patterning events involving Pt-msx1 and Pt-Delta expression were initiated around the blastopore in fuchi pRNAi embryos (Additional file 3: Fig. S10). fuchi knockdown led to a lack of circular patterns of Pt-hh and Pt-otd expression associated with anterior patterning, but its effects on the Pt-hh and Pt-otd expression levels in whole embryos at early stage 5 were limited (Additional file 3: Fig. S10).

Taken together, these results suggested that fuchi is required for the zygotic activation of a set of endodermal genes and of some patterning genes at stage 3 or shortly before. In addition, Pt-GATA4 (g25261), the likely counterpart of Drosophila srp [59], and Pt-GATA5 (g8337) were included in the stage-5 fuchi pRNAi DEG list but not in the stage-3 one (Additional file 9: Tables S9, S10).

fuchi is involved in regulating chromatin accessibility in specific genomic regions in early embryos

Certain GATA family members act as pioneer transcription factors that can influence chromatin structure [60]. Therefore, we considered the possibility that fuchi controls the transcription of downstream genes through chromatin structure regulation. To investigate this possibility, we conducted assay for transposase-accessible chromatin using sequencing (ATAC-seq) of wild-type (untreated), fuchi pRNAi, and Pt-hh pRNAi embryos at stage 3 (18 h AEL), obtaining genome-wide datasets on chromatin accessibility. Read counts in ATAC-seq peaks were subjected to comparative analyses, and 317 genomic regions that were differentially accessible between the fuchi pRNAi and untreated samples but only 3 between the Pt-hh pRNAi and untreated samples were identified (Fig. 8A; Additional file 10; Tables S12, S13; Additional file 11; Tables S14, S15; FDR < 0.05). This indicated that the Pt-hh pRNAi samples served as a negative control for assessing the impact of fuchi knockdown. The two comparisons revealed only one shared genomic region (Fig. 8A; Additional file 10; Table S13; Additional file 11; Table S15), which potentially reflected a nonspecific effect stemming from pRNAi treatment. Therefore, 316 genomic regions where chromatin accessibility was significantly affected following fuchi knockdown in stage-3 embryos were identified (Fig. 8B).

Most of these regions (276/316, 93%) showed suppressed accessibility upon fuchi knockdown (Fig. 8A, B), and 30 of them were found to be located closer to (or within) genes included in the fuchi pRNAi DEG lists (for stage-3 and/or early stage-5) than to any other annotated genes (Fig. 8B; Additional file 10: Table S13). Moreover, motif discovery search revealed that motif sequences RNWGATAAGAVW, similar to typical GATA-binding motif sequences, were significantly enriched among the sequences of the fuchi-dependent chromatin accessibility peaks (Fig. 8C). fuchi-dependent chromatin accessibility peaks containing these motif sequences were actually present within or close to some of the genes identified by the comparative transcriptome analyses of fuchi pRNAi versus untreated embryos (Fig. 8B).

Notable examples of such genes were the top-ranked gene from the stage-3 fuchi pRNAi DEG list (g1125), the key patterning gene Pt-hh (g4322), and the canonical GATA family member Pt-GATA5 (g8337) (Fig. 9A, B, C). g1125 was predicted to encode an uncharacterized product containing two potential transmembrane domains (XP_015915605.1; Fig. 9D). Genes encoding homologous proteins to this product were detected in the genomes of at least three other Araneoidea species, Araneus ventricosus (GBO06802.1), Argiope bruennichi (KAF8764632.1), and Oedothorax gibbosus (KAG8179838.1) (Fig. 9D), but any non-Araneoidea protein sequences that showed similarity to these products (E-value < 1) were not found in the NCBI non-redundant protein sequence collection (March 7, 2022). Pt-GATA5 transcript, like fuchi, was observed in cEND, CM, and pEND cells at stage 5 and widely distributed endoderm cells at stage 7 (Fig. 9E, F). Altogether, these results suggested that fuchi is required for establishing “open” states of chromatin structure in specific genomic regions, some of which are associated with endodermal and patterning genes expressed in the early stages.

Detection of consistent endodermal gene expression based on single-nucleus and single-cell RNA-seq datasets

In a recent separate study, we performed single-nucleus and single-cell RNA-seq analysis of late stage 5 embryos in P. tepidariorum [61]. In this study, clearly isolated endodermal cell clusters, which were marked by a known early endoderm marker 012_A08 [56], were identified (Additional file 3: Fig. S11). Strong fuchi expression was detected in these clusters, with weaker levels of fuchi expression being additionally detected in mesodermal (marked by Pt-twi) [58] and dorsal germ-disc (marked by Pt-GATA1) [61] cell populations. At the same time, the endoderm-specific expression of Pt-GATA4 and Pt-GATA5 was confirmed. In contrast to fuchi, and the two canonical GATA genes, Pt-hh expression was missing or largely reduced in the endodermal cell populations at late stage 5. These data were consistent with the staining data shown in Fig. 5 and Fig. S7 (Additional file 3). The single-nucleus and single-cell RNA-seq datasets further confirmed that most of the high-ranking genes from stage-3 fuchi pRNAi DEG list showed endoderm-specific expression at late stage 5 (Additional file 3: Fig. S12).

Discussion

We conducted genome-based research using the spider P. tepidariorum to explore the variations in the mechanisms of early embryonic development in the phylum Arthropoda. A key initial attempt in this study was to analyze transcriptomes of cells isolated from different regions of the early spider embryo. This allowed for the genome-wide identification of candidate genes whose expression might be locally restricted along the emerging first embryonic axis, without relying on knowledge from other organisms. Although similar approaches to identifying localized transcripts in early animal embryos have been reported in non-arthropod animals [17, 62, 63], early arthropod embryos are rarely studied in such a way.

A benefit of using P. tepidariorum is the applicability of a simple gene knockdown technique, that is, pRNAi. This allowed us to carry out a functional screen of some of the listed genes. Gene-specific embryonic phenotypes were obtained using 3 of the 19 genes tested (15%). Compared with similar screening efforts, we previously made following sequence-based gene selection, this small proportion is not surprising [30]. Although similar RNAi-based gene knockdown is available in certain arthropods, such as Tribolium castaneum, it is important to note that the P. tepidariorum system allows for the easy monitoring of early embryonic developmental processes, even following pRNAi treatment. Time-lapse recording of the phenotypes from early stages gave sharable, rich, objective information on gene functions and the timing of their actions. Early defects often lead to reduced characteristic morphologies at later stages. Exclusion of common spontaneous defects is required for effective functional screening. Direct detection of the earliest developmental defects following knockdown treatments in the initial screening could simplify the structuring of our interests. Despite the small scale of the functional screen we conducted, incomparable with the large-scale, unbiased RNAi screen in Tribolium [23], our results may encourage additional gene-function screens in P. tepidariorum.

Unexpectedly, our pRNAi screen found that the cell movement toward the formation of a germ disc was reversed following the knockdown of a GATA-like transcription factor gene, which we named fuchi nashi (fuchi). This phenotype suggests the presence of a shape-stabilizing phase in the germ-disc development, which likely corresponds to stage 4, during which a line of cells along the border is organized to gain specific gene expressions. fuchi expression, however, is initiated much earlier than this stage, in cells outside a forming germ disc, and later continues in all internalizing and internalized endoderm cells. Our comparative transcriptome and chromatin accessibility analyses of fuchi pRNAi versus untreated embryos suggested that early fuchi activities have broad impacts on zygotic gene activation around stage 3. Lack of the fuchi activities appeared to hinder the specification of endoderm fates. The abnormal reversed cell movements in fuchi pRNAi embryos could be, at least in part, explained if we assume that the different cell types being specified exhibit different mechanical properties, as proposed in a recent mathematical modelling study [64]. The earliest morphological defects of fuchi pRNAi embryos possibly result from a failure to establish the differences in adhesion tension between cell populations.

We showed that early fuchi activities influence the expression of some of the key signaling genes involved in embryonic development and pattern formation. These include Pt-hh, Pt-Delta, and Pt-sog. Pt-hh and Pt-Delta, downregulated in response to fuchi knockdown at stage 3, are known to show expression patterns similar to fuchi in early embryos (stages 3 and 4) [30, 32], and Pt-sog, upregulated oppositely, to be expressed in broad domains of the stage-5 germ disc [29]. Notably, early Pt-hh expression initiated from the abembryonic side contributes to the formation of the global polarity in the germ disc and subsequent anterior patterning. In fuchi pRNAi embryos, however, no patterning events that should occur from the abembryonic side of the egg were identified. These findings suggest that early fuchi activities specify endoderm fates and promote pattern formation in a wide area of the embryo, at the same time. Similar GATA factor-mediated regulation of hh expression has been documented in several mammalian and Drosophila developmental events [65,66,67].

The family of GATA transcription factors is known to regulate cell fate specification and cell differentiation during various developmental processes in a wide range of animals [69]. Our functional and phylogenetic characterization of fuchi provides the opportunity to reconsider the relationship between the evolution of GATA family members and their developmental roles. We defined “canonical” members of the GATA family, as they have perfectly alignable, close sequences to each other throughout the double zinc finger DNA-binding domain regardless of the phylogenetic distance of the species. The canonical members include all six GATA paralogs in vertebrates, GATA-1 to GATA-6, the only Nematostella GATA, Drosophila Serpent (Srp), and five P. tepidariorum GATA paralogs, Pt-GATA1 to Pt-GATA5. The vertebrate GATAs are phylogenetically classified into two subgroups: GATA-1/2/3 and GATA-4/5/6, while Srp is closer to GATA-4/5/6 [55, 70]. srp functions for endoderm specification in early Drosophila embryos [71], and vertebrate GATA-4/5/6 genes have developmental roles in endodermal cell lineages [72,73,74,75]. Notably, in mammals, GATA-6, in cooperation with GATA-4, is essential in the specification of the primitive and definitive endoderm, through chromatin accessibility regulation [76, 77]. Based on these lines of evidence, the idea that the roles of GATA-4/5/6 genes in endoderm specification and development are conserved across bilaterians has been widely accepted. Consistent with this idea, single-cell and single-nucleus transcriptomes indicated that the likely srp ortholog Pt-GATA4 (Pt-srp) appears to be expressed in an endoderm-specific manner at least at late stage 5 of P. tepidariorum development (Additional file 3: Fig. S11), although a previous report mentioned that “Pt-srp expression starts at stage 8 in the presumptive extraembryonic cells.” [59] Evidently, developmental transcriptomes gave a quantitative indication that there is little Pt-GATA4 expression at stage 4 and earlier but substantial expression at late stage 5 and later (Additional file 3: Fig. S5). We suggested that instead of Pt-GATA4, fuchi and Pt-GATA5 are key GATA family genes contributing to endoderm specification and development in the earlier stages of P. tepidariorum development. Although Pt-GATA5 is a canonical member of the GATA family, it appears to have no orthologous counterpart outside the Araneae. Fuchi is not a canonical GATA factor, having a highly diverged sequence with retaining the dual zinc fingers. fuchi appears to act on Pt-GATA5, possibly through chromatin accessibility regulation. The fuchi-dependent ATAC-seq peak detected in the Pt-GATA5 locus is located in an intron between exons encoding the two zinc fingers. Sequential regulation of multiple GATA factors in early endoderm specification and the subsequent differentiation process is a shared feature among distantly related bilaterians, including mouse, C. elegans and Drosophila [77,78,79]. However, the genetic components responsible for this regulation exhibit no orthologous relationships between the species, even within the Arthropoda, suggesting independent modifications of the GATA-mediated regulatory system for endoderm specification and development in the respective animal lineages.

fuchi and its orthologs (GATAL2) were found in sub lineages of the Araneae, including many Araneomorphae taxa but not non-Araneomorphae. These GATA-like genes, regardless of the cysteine spacing patterns in ZF1, have been rapidly evolving their sequences compared to most other GATA and GATA-like genes. Despite this divergence trend, we could define the likely orthologous GATAL2 group using the conservative signature sequence QCV(K/R)CG. Because the amino acid residues homologous to this sequence are involved in binding to specific cofactors, such as the vertebrate Friend of GATA (FOG) and Drosophila U-shaped (Ush), in canonical GATA factors [80,81,82], Fuchi possibly binds to a unique cofactor via this sequence. In relation with GATA cofactors, one of two P. tepidariorum homologs of Drosophila Ush (g14866; the other homolog is g16893) was included in both the gene lists obtained from the comparative RNA-seq and ATAC-seq analyses using stage-3 fuchi pRNAi embryos (Fig. 8; Additional file 9: Table S10; Additional file 10: Table S13). Certain GATA cofactors are known to exert an inhibitory effect to complicate the GATA factor-mediated transcriptional regulation [65, 66, 83]. Such an inhibitory effect of a GATA cofactor potentially accounts for the transient Pt-hh expression in the early abembryonic cell population. High degrees of sequence divergence in the DNA-binding domain of Fuchi and other GATAL2 proteins may be an indication of evolving interactions with cofactors and DNA-binding sites. Previous studies have suggested that N-terminal zinc fingers (ZF1) influence selectivity of DNA-binding sequences [82, 84]. Alternatively, the sequence divergences might have simply resulted from reducing degrees of functional constrains. Future studies will focus on testing these possibilities.

The phylogenetic origin of the fuchi activity in early endoderm specification and those of the regulatory connection between fuchi and Pt-GATA5, is an intriguing issue that provides molecular clues regarding the diversification mechanism of the process of early embryonic development. The formation of a sharply demarcated germ disc is not a common feature of early spider development. In Theridiidae spider embryos, the formation of the germ disc involves an appearance of two distinct endoderm or endoderm-like cell populations derived from both polar regions of the early embryo, and the formed sharp boundary of the germ disc serves as sites of endoderm and mesoderm cell internalization, as well as sites of sending patterning signals [30, 32, 41, 44]. These developmental strategies have potential merits of gaining parallel processes, which may allow for a more rapid development. In the embryos of many other spider species, including three distinct examples from outside the Araneoidea, Cupiennius salei [45], Pholcus phalangioides [46], and Hasarius adansoni [85], cell movements causing the germ layers only occur from one of the polar regions, which corresponds to the embryonic pole or the center of the germ disc in the P. tepidariorum embryo. The technical term “germ disc” results in potential confusions in documenting the Theridiidae embryonic development. This term is, in certain cases, used to indicate the multicell-layered region of the embryo that results from germ layer formation in spiders and other arthropods [86]. The initial germ disc in Theridiidae embryos, however, is a form of a single epithelial cell layer that precedes the formation of the germ layers [26, 27, 37]. fuchi serves as a molecular marker for the cells outside the forming germ disc. fuchi homologs may aid in identifying the existence of an evolutionarily conserved abembryonic cell type in other spider species. Complicatedly, the P. tepidariorum genome has a paralog of fuchi that was presumably generated by recent tandem gene duplication. No corresponding paralog was detected in other Theridiidae and non-Theridiidae spiders. The fuchi paralog, Pt-GATAL2b, shows little expression at least during the early and mid-embryogenesis of P. tepidariorum. Studies on the expression and function of fuchi/Pt-GATAL2b and Pt-GATA5 orthologs in the early embryos of other spider species will enable us to further address the problems on the early developmental variations among the Araneae lineages.

This study has produced genome-wide datasets related to molecular and genetic regulation of early embryonic development in P. tepidariorum, which can be integrated with single-nucleus and single-cell RNA-seq datasets established in another study [61]. These data resources, as well as the research strategies presented, will facilitate our efforts to dissect the early developmental processes at molecular resolution and establish an independent paradigm of genetic programs for early arthropod development. Importantly, many of the genes revealed in this study encode proteins not yet characterized, thus underscoring the importance of taking genome-based approaches to studying the evolution of animal development.

Conclusions

Using a Theridiidae spider empowered by genome sequencing, we combined comparative transcriptomes of isolated cells from different regions of early embryos and a pRNAi-based functional screen of genes. This research strategy allowed us to identify a lineage-specific, fast-evolving GATA-like gene, fuchi, that is essential for early embryonic development in the spider. fuchi is expressed in future endodermal cell populations in the early embryo. Further genome-wide analyses suggest that fuchi regulates chromatin state and zygotic gene activation to promote endoderm specification and pattern formation. The presented datasets have rich information about the molecular regulation of early embryonic development in the Theridiidae spider. The presence of many uncharacterized genes under the control of fuchi has been revealed. Our genome-based research using a chelicerate arthropod phylogenetically distant from Drosophila provides the foundation for molecular and genetic exploration of the variations in early development across Arthropoda.

Methods

Spiders

Laboratory stocks of P. tepidariorum were maintained at 25 ℃ in 16 h light/8 h dark cycles. Developmental stages of the embryo were determined according to the previous descriptions [28, 58]. To determine the precise time of egg laying, female behaviors were recorded every 5 min using a trail camera.

Cell isolation

Embryos at stage 3 were dechorionated with 50% commercial bleach for 1 min and rinsed several times with distilled water. After removing water, the embryos were placed on a glass slide with double-sticky tape and immediately covered with halocarbon oil 700 (Sigma-Aldrich). Glass capillaries (2-000-075; Drummond) were prepared in advance by pulling with a puller (PN-3; Narishige). The capillary tip was broken using forceps, just before use, and filled with lysis buffer supplied in Dynabeads mRNA DIRECT Kit (Ambion) by capillary action. Upon the manual manipulation of the capillary under the stereomicroscope, approximately 10–30 cells were sucked from a central, an intermediate, or a peripheral region of the stage-3 embryo and transferred into 0.2-ml tubes containing 10 µl of lysis buffer. The samples were stored at − 80 °C until they were retrieved for RNA extraction.

RNA-seq of isolated cells

Poly(A) mRNA was extracted using the Dynabeads mRNA DIRECT Kit (Ambion). After fragmentation of the extracted RNA for 5 min at 94 °C, first-strand cDNA synthesis and subsequent cDNA amplification by 22 cycles of polymerase chain reaction (PCR) were performed using the SMARTer stranded RNA-seq kit (Takara). The synthesized first-strand cDNA and amplified cDNA were purified using Ampure XP Beads (Beckman Coulter). Constructed libraries for sequencing were quantified using the Agilent 2100 BioAnalyzer high sensitivity DNA kit (Agilent). Two or three libraries for each region of the stage-3 embryo were sequenced in single-end runs in the antisense direction using the MiSeq reagent kit V3 (150 cycles) on the Illumina MiSeq platform. Over 10 million reads were obtained from each library. Following essentially the same procedure, RNA-seq of cells from central and peripheral regions of stage 4 and 5 embryos was performed.

Processing of RNA-seq data

The adapter and primer sequences, as well as the first three bases derived from the SMARTer Stranded Oligo, were trimmed from the MiSeq raw reads using CLC Genomics Workbench 7.0.3 (Qiagen). Quality trimming was also performed at the following parameter settings: quality score, limit = 0.05; trim ambiguous nucleotides, maximum number of ambiguities = 2; and filter on length, discard reads below length = 30. Trimmed reads were aligned to the Parasteatoda tepidariorum genome (Ptep_1.0, GCA_000365465.1) using the BLAT algorithm [87] in the DNA Data Bank of Japan (DDBJ) Read Annotation Pipeline [88]. Output alignments were filtered using the PERL script filterPSL.pl, which is accessible from the AUGUSTUS 3.0.1 scripts folder (http://github.com/Gaius-Augustus/Augustus) [89], with the following settings: 60% coverage; 90% identity; uniqueness threshold 0.96. The filtered alignments (pslx-format) were converted to sam-format. Based on these sam-format data, the number of mapped reads was counted against the AUGUSTUS gene models (aug3.1; https://i5k.nal.usda.gov/content/data-downloads) [31, 90] using htseq-count v.0.6.1p1 [91] with default settings. The RNA-seq datasets, including the gene count matrix, were deposited in the NCBI Gene Expression Omnibus (GEO) database (GSE193511) [92].

Comparative analyses of RNA-seq gene count datasets

To assess the consistency of RNA-seq gene count datasets between biological replicates of each sample group, the coefficients of determination were calculated using the gene count matrix and confirmed to exceed 0.85. Out of the 27,990 annotated genes, 10,862 genes showed one or more counts per million (cpm) for at least one of the eight samples (p1-p3, i1-i3, c1, and c2). Count data matrices on these genes (10,862 genes × 8 samples) were used for comparative analyses. Differentially expressed genes (DEGs) were identified using a Bioconductor package edgeR (version 3.8.6) [49]. The following three types of comparisons were set up: comparison I, c1 and c2 vs. p1-p3 and i1-i3; comparison II, p1-p3 vs. c1, c2, and i1-i3; and comparison III, i1-i3 vs. c1, c2, and p1-p3. Two criteria were applied to determine the high-priority candidate genes: first, log₂FC < − 10 in comparisons I and II, and log₂FC > 10 in comparison III; and second, lower FDR values. As the FDR values were only used to rank the DEGs, the genes selected from each comparison for the functional screen included genes with FDR > 0.05.

cDNA cloning

Full-length or partial cDNAs of newly identified genes were obtained from our collections of expressed sequence tag clones previously described [32, 39] or were isolated by PCR amplification. cDNA fragments cloned in pBluescript II SK( +) (Agilent), pTriplEx2 (Takara), or pZL1 (Invitrogen) were used to synthesize dsRNA for pRNAi and/or RNA probes for in situ hybridization. The primers and cDNA clones used are summarized in Table S16 (Additional file 12).

Parental RNAi

For dsRNA synthesis, DNA templates with the T7 promoter sequence at both ends were prepared by PCR using appropriate primers depending on the plasmid type used. dsRNAs were synthesized using a Megascript Kit (Ambion) as described previously [29]. One to two microliters of the dsRNA solution at a concentration of 2.0 µg/µl was injected into the abdomen of adult females four times at 2–3-day intervals using pulled glass capillary tubes. In the pilot pRNAi screen, dsRNA against each gene was injected into two or four females. When egg laying took place, more than 10 eggs were randomly selected from each egg sac and transferred into halocarbon oil 700, which allowed us to nondestructively monitor the embryonic development through cleared chorion.

For three genes (g26874, g7720, g4238) that were judged as positive in the pilot screen, 2 or 3 additional dsRNAs were prepared from non-overlapping regions of each of the cDNAs, and each dsRNA was injected into at least 2 females. Development of eggs laid by the injected females were monitored using time-lapse recording, or with occasional visual inspection, under the stereo microscope. gfp dsRNA was used as the control. In cases where we examined RNAi-treated embryos at the molecular and cellular level, they were randomly selected from egg sacs, and aliquots of their siblings were confirmed to exhibit severe morphological phenotypes at high frequencies (> 80%).

Time-lapse microscopy of live embryos

Embryos dechorionated with 50% commercial bleach were placed on a glass slide with double-sticky tape and covered with halocarbon oil 700. When the observation was started from stage 1, the dechorionation step was omitted to prevent possible effect on the development. Images were taken every 5 or 10 min using a stereo microscope (SZX12, Olympus) equipped with a color CCD camera (C7780-10, Hamamatsu Photonics) controlled by AquaCosmos software (Hamamatsu Photonics) or other stereo microscopes (M165C, Leica; or SZX7, Olympus) equipped with a color CMOS camera (WRAYCAM-G200 or -NF300, WRAYMER).

For live imaging of nuclei around the germ-disc/non-germ-disc transition area, dechorionated stage-4 embryos were placed on a glass slide with double-sticky tape, and an embryo appropriately oriented was selected and skewered with a thin glass needle to prevent the embryo from rotating during later development. A 1000 × stock solution of a cell-permeable dye SPY555-DNA (Spirochrome), which was prepared in 50 µl of dimethylsulfoxide, was microinjected into the perivitelline space of the skewered egg. The injecting volume should be low to avoid affecting the development of the embryo from being affected. Approximately 1 h after dye injection, the embryo started to be examined under an Olympus BX50 fluorescence microscope equipped with a cooled CCD camera (CoolSNAP HQ, Roper Scientific) controlled by MetaMorph.version 6.1 (Universal Imaging). Using × 10 objective lens (UPlanAPO 10 × /0.40, Olympus), bright-field and fluorescence 500 × 500 pixel images were acquired every 5 min at exposure times of 10 and 100 ms, respectively. To reduce the excitation light, two neutral density filters (U-ND6-2 and U-ND25-2, Olympus) were used. A single focal plane, which was manually adjusted during approximately 30 h of observation, was recorded at each time point. The observed embryo was checked for its normal development. Time-lapse images were analyzed using Imaris version 7.6.5 and ImageJ version 1.51d. On the Imaris software, cells were manually tracked.

Molecular phylogenetic analysis of GATA family members

To collect amino acid sequences containing GATA-type DNA-binding domain from many arachnid species and other arthropod and non-arthropod metazoan representative species, we used nucleotide sequence resources presented in Table S7 (Additional file 7), which included coding sequences from sequenced genomes, transcriptome assemblies, and RNA-seq raw data, retrieved from GenBank (NCBI) or GigaScience database. RNA-seq of Hasarius adansoni mid-stage embryos was performed in this study, as described below. The RNA-seq data were subjected to de novo assembling using Trinity (version 2.11.0). To identify amino acid sequences coding for GATA-type DNA-binding domains in the retrieved or assembled nucleotide sequences, blastp searches were performed using the Fuchi amino acid sequence as a query with a cut-off e-value of 1e − 2, followed by the systematic selection of sequences that contained at least one GATA-type zinc finger motif sequence (CX₂CX_16-20CX₂C). The identified amino acid sequences were manually aligned and classified based on the detection of signature sequences, excluding GATA-like proteins that had only one GATA-type zinc finger motif. For molecular phylogenetic characterization, a total of 211 amino acid sequences were manually aligned based on unambiguous detection of conserved amino acids (Additional file 7: Table S8; Additional file 13). The molecular phylogeny was inferred using the Maximum Likelihood (ML) method and JTT matrix-based model [93] on the software MEGA11 [94]. For the first ML analysis, 75 amino acid sites from 118 sequences were used (Additional file 14). For the second ML analysis, 63 amino acid sites from 168 sequences were used (Additional file 15). None of the amino acid sites contained gaps.

RNA-seq of whole embryos

For RNA-seq analyses of fuchi pRNAi embryos, mated adult females were injected with approximately 1–2 µl of fuchi dsRNA solution (2 µg/µl) four times at 2- to 3-day intervals. For RNA extraction, 50–100 embryos at stage 2 (11 h AEL), 3 (20 h AEL), and early stage 5 (31 h AEL) that were derived from egg sacs produced by the injected females 2 days before (untreated) and 18–19 days after the first injection of the dsRNA were used. Two biological replicates were prepared for each sample type. The mRNA extraction and library construction for sequencing were performed as previously described [36]. The libraries were quantified using the Library quantification kit (Takara) and Thermal cycler Dice Realtime TP800 (Takara) and sequenced in single-end runs (150 cycles) in the antisense direction on the Illumina MiSeq platform. The sequence reads were processed as previously described [36], and the datasets were deposited in the GEO database (GSE193650) [95]. Estimates of DEGs between untreated and fuchi pRNAi embryos at each stage were performed using EdgeR (version 3.8.6).

For the H. adansoni RNA-seq, poly(A) mRNA was extracted using a QuickPrep Micro mRNA purification kit (GE Healthcare) from 20 sibling mid-stage embryos with extending limbs. Library construction for sequencing was performed as previously described [36]. The library was sequenced in a paired-end run (150 × 2 cycles) on the Illumina Miseq platform, and the raw reads (SRR17326784) and the H. adansoni transcriptome assembly (GJQJ00000000) were deposited in GenBank [96].

ATAC-seq of whole embryos

ATAC-seq was performed following the procedure described by Buenrostro et al. [97] with modifications considering the procedure reported by Haines and Eisen [98]. In brief, 40 whole embryos at stage 3 (18 h AEL) were homogenized using disposable pellet pestles (12–141-368; Fisher Scientific) and 1.5-mL Eppendorf tubes (0030125150) in 50 µl lysis buffer (10 mM Tris, 10 mM NaCl, 3 mM MgCl₂, adjusted to pH 7.4 with HCl) containing 0.15 mM spermidine, after which 50 µl of lysis buffer and 1 µl of 10% Nonidet P-40 were added as the pestle was rinsed. After a 10-min incubation, nuclei were spun down at 800 g and resuspended in 17.5 µl water, followed by the addition of 25 µl of 2 × TD buffer (Illumina) and 7.5 µl of Nextra Tn5 transposase (Illumina). Transposed DNA was purified using MinElute Reaction Cleanup Kit (Qiagen) and amplified with Illumina Nextera Transposase Adaptors index PCR primers, and the amplified DNA was purified using Ampure XP Beads (Beckman Coulter). Constructed libraries were paired-end sequenced (151 bp reads × 2) on the Illumina HiSeq X Ten system.

Comparative analyses of ATAC-seq datasets from three different sample types (wild-type, fuchi pRNAi, and Pt-hh pRNAi) were performed basically following the procedure described by Reske et al. [99] with slight modifications based on guidelines provided by Gasper [100] and Delisle et al. [101]. Two biological replicates were obtained from each sample type. ATAC-seq raw reads were trimmed for adaptor and primer sequences and low-quality sequences using Trim Galore! with default settings. Trimmed reads were then aligned to the P. tepidariorum genome (Ptep_2.0) using bowtie2 v2.2.5 with default settings. Reads mapped with low-quality scores (less than 30) were removed using samtools (version 1.9). PCR duplicates were removed using Picard MarkDuplicates (version 2.23.4). The alignments of filtered reads to the genome were saved as bam files. Peak calling was performed using the ATAC-seq mode of Genrich (version 6.0). Differential accessibility analysis was performed using a Bioconductor package, csaw [102]. Peak regions called in both replicates of each sample type were extracted, and the peak regions extracted from the three sample types were merged. Reads were counted in peaks, with low abundance peaks (log CPM < − 3) removed. Read counts were subject to normalization with the trimmed mean of M values (TMM) method [103]. Differential accessibility was estimated using the glmQLFit and glmQLFTest functions in EdgeR (version 3.32.0). Peaks with FDR < 0.05 were considered as significant. To discover motif sequences in the identified differential peak regions, STREME [104] was used.

Wild-type whole embryos at stages 1 (9 h AEL) and 2 (12 h AEL) were similarly analyzed by ATAC-seq to detect genomic regions differentially accessible between the different stages (stages 1 to 3), but no clear results were obtained. All the ATAC-seq datasets were deposited in the GEO database (GSE193870) [105].

Staining of embryos

Antisense RNA probes for in situ hybridization were prepared by in vitro transcription using Digoxigenin- or Dinitrophenyl-conjugated UTP (DIG, Roche 11,277,073,910; DNP, PerkinElmer NEL555001EA) as previously described [56]. Whole-mount in situ hybridization with chromogenic substrate was performed in the previously described manner [29] with the exception of an additional tyramide-based signal amplification (TSA) process described previously [56]. In brief, after washing DIG-conjugated RNA probes, the samples were incubated with anti-DIG-POD antibody (11,633,716,001, Roche; 1:500 dilution) and then reacted with DNP-tyramide (NEL747, PerkinElmer). In the detection step, anti-DNP-AP antibody (MB-3100, Vector; 1:50) and NBT/BCIP were used. Most samples were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich).

Double-color FISH was performed as previously described [56]. To visualize the DIG- and DNP-conjugated probes, anti-DIG-POD (Roche 11,633,716,001; 1:500) and anti-DNP-HRP (PerkinElmer FP1129; 1:100) were used in combination with DyLight488-tyramide and DyLight680-tyramide, respectively.

Single- and double-color FISH were combined with immunostaining for β-catenin in certain cases (Figs. 5 and 6), where Proteinase K treatment prior to hybridization was omitted. After completing the FISH procedure, the embryos were incubated with rabbit anti-β-catenin antibody (C2206, Sigma-Aldrich 1:500) and then with Cy3-conjugated anti-rabbit IgG antibody (AP187C, Chemicon; 1:200), followed by counterstaining with DAPI.

Image acquisition and processing

Images for chromogenically stained WISH samples were obtained using a stereo microscope (SZX12, Olympus), a fluorescence unit BT-ExSMOP (Biotools), and a CCD camera (C7780-10, Hamamatsu Photonics). For confocal microscopy of fluorescently stained embryos, the whole embryos were individually mounted on glass slides with spacers of approximately 100–500 µm, which were adjusted depending on the sample using sticky tape (CT-18S, Nichiban) and silicone vacuum grease (335,148, Beckman). To view different sides of a single embryo, it was rotated after being mounted on the glass slide. The embryos were examined using a Leica TCS SPE confocal system equipped with four laser sources (405, 488, 532, and 635 nm), which were used to excite DAPI, DyLight488, Cy3, and DyLight680, respectively. Acquired confocal stacks were processed and analyzed using Imaris version 7.6.5 (Bitplane) and ImageJ version 1.51d. The Imaris snapshot feature and oblique slicer tool were used to obtain images capturing the embryonic regions of interest. Linear signals of cell–cell adherens junctions visualized by β-catenin staining were traced in Adobe Photoshop CS5 ver. 12.0.4.

Availability of data and materials

Raw and processed data for the comparative RNA-seq and ATAC-seq analyses are accessible from NCBI GEO (GSE193511, GSE193650, and GSE193870) [92, 92, 92]. The data aligned to the reference genome can be viewed on our web database (https://www.brh2.jp) [106]. The nucleotide sequences determined for fuchi and Pt-GATA5 are available under the accessions LC671429 and LC671430 in the DDBJ/EMBL/GenBank International Nucleotide Sequence Databases. The sources of sequence data used in the molecular phylogenetic analysis are listed in Additional file 7: Tables S7 and S8. Supplementary movies showing the pilot pRNAi screen and the pRNAi specificity validation are available on figshare [50].

Change history

17 December 2022
A Correction to this paper has been published: https://doi.org/10.1186/s12915-022-01494-x

Abbreviations

AEL:: After egg laying
ATAC-seq:: Assay for transposase-accessible chromatin using sequencing
BR:: Basic region
CPM:: Counts per million
DAPI:: 4’,6-Diamidino-2-phenylindole
DDBJ:: DNA Data Bank of Japan
DEG:: Differentially expressed gene
dsRNA:: Double-stranded RNA
FC:: Fold-change
FDR:: False discovery rate
GEO:: Gene Expression Omnibus
ML:: Maximum likelihood
NCBI:: National Center for Biotechnology Information
PCR:: Polymerase chain reaction
pRNAi:: Parental RNA interference
RNA-seq:: RNA sequencing
RPKM:: Reads per kilobase of transcript, per million mapped reads
WISH:: Whole-mount in situ hybridization
ZF:: Zinc finger

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Acknowledgements

We thank Matthias Pechmann for sharing data on Pt-Ets4 before publication, Akiko Noda for technical assistance, and other members of JT Biohistory Research Hall for discussion.

Funding

This work was supported by JSPS KAKENHI (26440130, 17K07418, 20K06676 to YA; 15K07139 to HO), JST PRESTO (JPMJPR2041 to YA), and the core budget of JT Biohistory Research Hall.

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Authors and Affiliations

Laboratory of Evolutionary Cell and Developmental Biology, JT Biohistory Research Hall, Takatsuki, Osaka, 569-1125, Japan
Sawa Iwasaki-Yokozawa, Ryota Nanjo, Yasuko Akiyama-Oda & Hiroki Oda
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Japan
Ryota Nanjo & Hiroki Oda
PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan
Yasuko Akiyama-Oda
Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Japan
Yasuko Akiyama-Oda

Authors

Sawa Iwasaki-Yokozawa
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Ryota Nanjo
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Yasuko Akiyama-Oda
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Hiroki Oda
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Contributions

S.I., Y.A., and H.O. designed the study. S.I. created all the RNA-seq datasets. S.I. analyzed the RNA-seq datasets with help from Y.A. S.I. performed the pRNAi screen and the gene expression and phenotype analyses. S.I. and H.O. performed the phylogenetic analysis. R.N. created the ATAC-seq datasets. R.N. analyzed the ATAC-seq datasets with help from Y.A. and H.O. S.I. wrote the first draft of the manuscript. H.O. wrote the final version of the manuscript with input from all authors. All authors read and approved the final manuscript.

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Correspondence to Hiroki Oda.

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The animal experimentation described was approved by the institutional committee for animal care and use (No. 2020–1) and conducted according to JT Biohistory Research Hall Regulation on Animal Experimentation.

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The authors declare that they have no competing interests.

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Supplementary Information

Additional file 1: Movie S1. Time-lapse video of sibling Parasteatoda tepidariorum embryos. The left embryo is the same as shown in Fig. 1. Time after egg laying (AEL) is indicated. Scale bar, 100 µm.

Additional file 2: Table S1.

List of DEG candidates identified by comparative transcriptome analysis of c versus i/p cells from stage-3 embryo (comparison I). Table S2. List of DEG candidates identified by comparative transcriptome analysis of p versus c/i cells from stage-3 embryo (comparison II). Table S3. List of DEG candidates identified by comparative transcriptome analysis of i versus c/p cells from stage-3 embryo (comparison III). Table S4. List of DEG candidates indentified by comparative transcriptome analysis of c versus p cells from stage-4 embryo. Table S5. List of DEG candidates identified by comparative transcriptome analysis of c versus p cells from early stage-5 embryo.

Additional file 3: Figure S1.

Chromogenic WISH of stage-3 embryos using probes for 19 selected DEG candidates. Figure S2. Validation of the specificity of the RNAi effects for g26874, g7720, and g4238. Figure S3. Location of P. tepidariorum GATA and GATA-like genes on scaffolds of the genome assembly Ptep_3.0. Figure S4. ML tree of 168 canonical and non-canonical GATA family members from Araneae, non-Araneae chelicerates, non-chelicerate arthropods, non-arthropod bilaterians, and cnidarian, using 63 amino acid sites. Figure S5. Developmental transcript profiling of P. tepidariorum GATA family genes in wild-type embryos. Figure S6. Cell counting in a stage-3 embryo stained for fuchi. Figure S7. fuchi expression in pEND cells. Figure S8. Characterization of 5 DEG candidates from the comparative transcriptome analysis of stage-2 fuchi pRNAi versus untreated embryos. Figure S9. Characterization of the top-10 DEG candidates from the comparative transcriptome analysis of stage-3 fuchi pRNAi versus untreated embryos. Figure S10. Effects of fuchi RNAi on expression of selected genes. Figure S11. Single-nucleus transcriptome data showing endodermal and mesodermal cell populations in late stage-5 embryos. Figure S12. Single-nucleus and single-cell transcriptome data showing cell populations expressing the genes regulated by fuchi.

Additional file 4: Table S6.

Summary of a pilot pRNAi screen of 19 selected genes.

Additional file 5: Movie S2. Time-lapse video showing phenotypes gfp, g26874, g7720, and g4238 pRNAi embryos. Time after egg laying (AEL) and time after start of recording are indicated. Movie starts are adjusted by the timing of blastoderm formation. Some of the embryos are the same as those shown in Fig. 3A. Scale bar, 100 µm.

Additional file 6: Movie S3. Tracking of cells in wild-type and g26874 pRNAi embryos. In the g26874 pRNAi embryo, cells on the abembryonic side initially shift toward forming a germ disc but, later, they return back. Time after start of recording is indicated.

Additional file 7: Table S7.

List of bioinformatic resources used for phylogenetic characterization of Fuchi and other GATA family members in spiders. Table S8. Amino acid sequence alignment and classification of GATA family members from spiders and other metazoans.

Additional file 8: Movie S4. Time-lapse video showing cell internalizations taking place around the germ-disc rim. Nuclei labeled by SPY555-DNA are presented in white. The germ disc on the embryonic side is to the lower right, while the non-germ-disc region on the abembryonic side is to the upper left. Internalizing cells are tracked. Cells from the non-germ-disc region are moving to below the germ-disc epithelium. Later, cells at the germ-disc rim internalize. Time [d : h : m] after start of recording is indicated.

Additional file 9: Table S9.

List of DEGs identified by comparative transcriptome analysis of fuchi pRNAi versus untreated embryos at stage 2. Table S10. List of DEGs identified by comparative transcriptome analysis of fuchi pRNAi versus untreated embryos at stage 3. Table S11. List of DEGs identified by comparative transcriptome analysis of fuchi pRNAi versus untreated embryos at early stage 5.

Additional file 10: Table S12.

Data from comparative analysis of read counts in extracted ATAC-seq peaks between fuchi pRNAi and wild-type embryos at stage 3 using edgeR. Table 13. List of differential ATAC-seq peaks between fuchi pRNAi and wild-type embryos at stage 3 (FDR < 0.05).

Additional file 11: Table S14.

Data from comparative analysis of read counts in extracted ATAC-seq peaks between Pt-hh pRNAi and wild-type embryos at stage 3 using edgeR. Table S15. List of differential ATAC-seq peaks between Pt-hh pRNAi and wild-type embryos at stage 3 (FDR < 0.05).

Additional file 12: Table S16.

Primers and cDNA clones used for RNAi and in situ hybridization.

Additional file 13.

Multi-fasta format file of 212 protein sequences used for molecular phylogenetic characterization.

Additional file 14.

Multi-fasta format file of 118 sequences (75 amino acid sites) used for the first ML analysis.

Additional file 15.

Multi-fasta format file of 168 sequences (63 amino acid sites) used for the second ML analysis.

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Iwasaki-Yokozawa, S., Nanjo, R., Akiyama-Oda, Y. et al. Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider. BMC Biol 20, 223 (2022). https://doi.org/10.1186/s12915-022-01421-0

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Received: 20 July 2022
Accepted: 27 September 2022
Published: 06 October 2022
DOI: https://doi.org/10.1186/s12915-022-01421-0

Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

Abstract

Background

Results

Conclusions

Background

Results

Comparative transcriptome analyses of cells isolated from different regions of early P. tepidariorum embryos

Pilot functional screen identifies three genes required for germ-disc formation and/or axis formation

Molecular characterization of the three identified genes

fuchi is a lineage-specific, fast-evolving GATA-like gene

fuchi is expressed in endoderm cells originating from both sides of the early embryo

fuchi knockdown prevents the demarcation of a forming germ disc

fuchi is required for zygotic activation of endodermal and patterning genes

fuchi is involved in regulating chromatin accessibility in specific genomic regions in early embryos

Detection of consistent endodermal gene expression based on single-nucleus and single-cell RNA-seq datasets

Discussion

Conclusions

Methods

Spiders

Cell isolation

RNA-seq of isolated cells

Processing of RNA-seq data

Comparative analyses of RNA-seq gene count datasets

cDNA cloning

Parental RNAi

Time-lapse microscopy of live embryos

Molecular phylogenetic analysis of GATA family members

RNA-seq of whole embryos

ATAC-seq of whole embryos

Staining of embryos

Image acquisition and processing

Availability of data and materials

Change history

17 December 2022

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Keywords

BMC Biology

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