Embryogenesis is an extremely complex process. That fertilized eggs are developed to shaped individuals, rather than mounds of pluripotent cells, is largely due to a short period termed gastrulation, which comprises a great many critical events, such as morphogenetic movements, specification of body axes and germ layer, and body plan establishment. By temporal-spatial coordination of cell specification and dynamic cell movement, a three germ layer body plan is established, accompanied by embryonic axis formation, during gastrulation. Primordial organs or tissue rudiments are then formed along the axis and ultimately develop into functional organs or tissues. Thus, gastrulation plays pivotal roles during embryogenesis and organogenesis. During gastrulation, the most dramatic morphogenetic change takes place on mesodermal cells, which are specified and spread between ectoderm and endoderm. To date, research from represented model organisms has revealed that numerous signaling pathways, such as the Wnt (Wingless/Integrated), FGF (Fibroblast growth factor), BMP (Bone morphogenetic protein), Notch, and TGF-β (Transforming growth factor-beta)/Nodal signaling pathways [1
], are involved in mesoderm specification and movement. However, the molecular mechanisms and temporal-spatial orchestrations of these signaling still remain largely obscure.
Although the manifestations of cell movement during gastrulation differ between phyla, some evolutionarily conserved movements, such as epiboly, internalization, convergence, and extension can be characterized [3
]. The amphibian model Xenopus
species contributes greatly to understanding the molecular and cellular mechanisms during gastrulation. In Xenopus
, gastrulation is typically driven by convergent extension, intercalation, and cell migration. Convergent extension plays a pivotal role in elongating the dorsal marginal zone along the anteroposterior axis [4
], which drives the axial and paraxial mesodermal tissues, narrowing (convergence) and lengthening (extension), and also results in blastopore closure and anteroposterior body axis elongation [5
]. The FGF signaling is reported to modulate multiple developmental processes during early embryogenesis [7
]. During Xenopus
gastrulation, FGF signaling plays an integral role in the induction and maintenance of mesoderm [10
], and also regulates morphogenetic movements directly or indirectly [7
]. However, it remains equivocal how FGF signaling interplays with other signaling pathways during the induction, maintenance, and specification of different mesodermal regions.
The Notch signaling pathway is conserved in metazoans, which has usually been shown to modulate cell fate decision and form the boundary between embryonic tissues [11
]. The Notch receptors bind to their adjacent cells’ ligands (Deltas), resulting in cleavage and release of the intracellular domain of Notch (NICD), which then are translocated into the nucleus and interact with CSL (a DNA-binding protein named CBF1 in humans; Su(H) in Drosophila
; LAG1 in C. elegans
) to form a trans-activating complex on the promoters of downstream target genes. The hairy-enhancer of split (Hes)/hairy-enhancer of split related with YRPW motif (Hey) family members [12
] are not all direct effector genes of the Notch signaling pathway. They encode basic helix–loop–helix (bHLH) transcriptional repressors that control cell fate decisions and cell population expansion. For example, mouse Hes1 and Hes5 can inhibit neuronal differentiation while promoting the proliferation of neural progenitors in the embryonic brain [13
]. In Xenopus
, Hes3 [14
] and Hes4 (Hairy2) [15
] mediate the Notch signaling on neural crest induction on the ridge of the neural plate board. Interestingly, there is clear evidence that Notch signaling is active from the beginning of gastrulation in Xenopus
. For example, components of the Notch pathway such as Notch, and Serrate-1 Su(H)1 are present maternally and zygotically [16
], ligands such as Delta-1 and Delta-2 are present in the marginal zone as a ring encircling the blastopore at early gastrula [18
], and more importantly, Notch signaling has been reported to be involved in the segregation and boundary formation of the three germ layers in Xenopus
during gastrulation [21
Intriguingly, on integration with other signaling pathways, gastrulation is cellular context responsive, and delicately fine-tuned by Notch signaling [22
]. The existence has been reported of coordination between FGF and Notch signaling in the establishment of the proper periodicity of vertebrate somite [22
], ear development [25
], and sensory neuron formation [26
], via specific target genes. Thus, whether FGF and Notch signaling coordinate during gastrulation, and what are the key targets, are of great interests for elucidating the molecular events and the underlying mechanism of specification and patterning of the germ layers.
We previously screened the differential expression transcripts under the influence of FGF signaling in frog Xenopus tropicalis gastrula by treating with FGFR (Fibroblast growth factor receptor) inhibitor SU5402. A novel basic helix-loop-helix gene (Hes5.9) has been isolated. In this work, we report the expression and mainly characterize the function of Hes5.9 by microinjection of synthetic Hes5.9 mRNA, and antisense oligonucleotides respectively. In general, our results demonstrate that Hes5.9 may function as a transcriptional factor and be regulated by the FGF and Notch signaling, which is critical for cell fate determination and gastrulation during early embryonic development.
2. Materials and Methods
2.1. Animal Ethics and Embryo Manipulation
Xenopus tropicalis (Nigerian) were purchased from NASCO (USA), then bred and maintained in our lab. All animal procedures were performed in full accordance with the requirements of the Regulation on the Use of Experimental Animals in Zhejiang Province. This work was specifically approved by the Animal Ethics Committee of the School of Medicine, Zhejiang University (ETHICS CODE Permit NO. 14887, issued by the Animal Ethics Committee in the School of Medicine, Zhejiang University). In brief, ovulation was induced by injection of human chorionic gonadotropin (HCG) into the dorsal lymph sac of mature frogs; male and female frogs were injected with 150 and 200 units of HCG, respectively. The embryos were dejellied by 2% cysteine (pH 8.0) and then cultured in 0.1× Marc’s modified ringer solution (MMR). Developmental stages were assessed according to Nieuwkoop and Faber (1994). For drug treatment, we incubated the embryos in 0.1× MMR solution containing 20 μM SU5402 (Santa Cruz) or Dimethyl sulfoxide (DMSO, Sigma) at stage 8, and then removed the solution at stage 11.
2.2. Multiple Sequence Alignment and Phylogenetic Tree Construction
The relative protein sequences were retrieved from NCBI, Xenbase, and Ensemble databases, and then aligned via DNAMAN (Lynnon Biosoft, CA, USA) with default parameters. Phylogenetic trees were constructed by neighbor-joining algorithm, and displayed via DNAMAN. The proximal elements of promoters were obtained as described [27
], and predicted at http://jaspar.genereg.net
2.3. RNA Extraction, Reverse Transcription-PCR, and cDNA Cloning
Different developmental stage embryos were collected. After homogenizing with RNAiso plus (Takara), chloroform was added. The homogenate was then centrifuged and divided into three layers, the total RNA was precipitated from the upper aqueous layer with isopropanol, and impurities removed with 70% ethanol. After that, the RNA degradation and contamination were detected by 1% agarose gel electrophoresis; 1 μg of total RNA was reversed to cDNA by using oligonucleotide (dT)-tailed primer and Reverse Transcriptase M-MLV (Takara), 10 μL reaction volume including l μg total RNA, 50 μM Oligo(dT)12–18 primer, 5× M-MLV Buffer, 10 mM dNTP Mixture, 40 U/μL RNase Inhibitor, 200 U/μL RTase M-MLV, and RNase-free water, the mixture was incubated at 42 °C for 1 h, and then the reaction was stopped by heating at 70 °C at 15 min. The primers with restriction sites for SmaI and NotI were designed to amplify the full length of Hes5.9, forward: 5′-atacccgggACTACAGACACGTGGACTTA-3′; reverse: 5′-attgcggccAACAAACAATTTATTACATG-3′. Simultaneously, the constructs of the PCR products and pCS107 vector were digested with EcoRI and XhoI, then purified and ligated with T4 DNA ligase (Thermo) at 22 °C. The ligation products were transformed into TG1 competent cells and herein the cells were spread on LB plates containing ampicillin (50 μg/mL). The Hes5.9 fragment inserted into a vector was verified by colony PCR, the pCS107-Hes5.9 plasmid was extracted from 2 mL overnight culture by SanPrep Kit (Sangon), and then using SP6 as a primer to sequence, the Hes5.9 from pCS107-Hes5.9 plasmid and the sequences were aligned with NCBI.
2.4. mRNA Synthesis and Microinjection
The plasmid pCS107-Hes5.9 was linearized with ApaI (Takara), then capped mRNAs were synthesized using the mMESSAGE mMACHINE SP6 Kit (Ambion), and purified by MEGAclear Kit (Ambion). In brief, the following transcription reaction was carried out at room temperature: with 2 × NTP/CAP, 10× Reaction buffer, SP6 enzyme Mix, 1 μg linear template, and Nuclease-free water, the compound was incubated at 37 °C for 1 h, in sequent, the RNA was absorbed on the membrane in the filter cartridge, and then contaminants were washed away, lastly, mRNA was resuspended in a low salt buffer. The mRNA was bilaterally injected into the dorsal of the four-cell stage blastomere, meanwhile, the fluorescent dextran was co-injected as a lineage tracer.
2.5. Quantitative Reverse Transcription PCR (RT-qPCR)
Total RNA was extracted from X. tropicalis embryos according to the above-mentioned method, after synthesizing cDNA. RT-qPCR reactions were performed in triplicate for each sample, using a FastStart Universal SYBR Green Master (Roche) in CFX-Connect Real-Time System (BIO-RAD). The relative expression level of each target was normalized to the expression level of ornithine decarboxylase (Odc).
2.6. Whole-Mount in situ Hybridization
For the hybridization studies, the digoxigenin labeled antisense RNA probe of Hes5.9 was prepared by linearizing the pCS107-Hes5.9 plasmid with SmaI (Takara), and transcribing with T7 RNA polymerase (Promega). The different stages of embryos were collected and fixed in MEMFA (0.1 M MOPS, 2 mM EGTA, 1 mM MgSO4, 3.7% formaldehyde) for 2 h at room temperature, then these embryos were permeabilized by incubating them for about 15 min at room temperature in proteinase K (Roche; final concentration, 2.8 μg/mL), when the process of acetylation, fixation, and pre-hybridization was finished, the embryos were incubated in fresh hybridization buffer containing 0.5 ug/mL probe, and hybridized overnight at 60 °C. The embryos were washed with 2× saline sodium citrate (SSC), and 0.2× SSC at 60 °C, to remove the excess probe, then the embryos were washed twice with maleic acid buffer (MAB), MAB was replaced with blocking reagent (MAB, 2% Boehringer Mannheim blocking reagent, and 10% inactivated sheep serum), incubated for 2 h at room temperature; embryos were then incubated with antibody solution (Roche; anti-digoxigenin alkaline phosphatase (AP) antibody, 1:2000) overnight at 4 °C. The free antibody was removed by washing 3 × 30 min in MAB, before chromogenic reaction. We first washed the embryos 2 × 5 min at room temperature in alkaline phosphatase (AP) buffer, then incubated the embryos with BM purple (Roche), and when staining becomes apparent, embryos were fixed with MEMFA for 2 h at room temperature, then bleached with 30% hydrogen peroxide solution. Finally, the embryos were stored in 1× phosphate-buffered saline (PBS) for photographing.
2.7. Animal Cap and Dorsal Marginal Zone (DMZ) Elongation Assays
The embryos were injected with mRNA into the dorsal blastomeres, or MOs into ventral blastomeres at the four-cell stage embryos. Animal cap explants were excised at stage 8–9, and were cultured in 1× MBS with antibiotic, or together with 25 pg/mL recombinant human activin A (R&D) protein, until stage 17. DMZ explants were excised at stage 10.25 then cultured in 1× MBS with the antibiotic until stage 17.
2.8. RNA-Sequencing and Data Analyses
RNA sequencing was performed on the Illumina HiSeq2000 platform, and paired-end reads were mapped to the reference Xenopus tropicalis
transcriptome annotation. HTSeq v0.6.1 was used to count the reads number mapped to each gene. Differential expression analysis of WT and Hes5.9
overexpression was performed by using the DEGSeq R package (1.20.0). The P values were adjusted using the Benjamini and Hochberg method. Corrected P
-value of 0.005 and log2 (fold change) of 1 were set as the threshold for significantly differential expression. Gene ontology (GO) enrichment analysis of differentially expressed genes was implemented by the GOseq R package. GO terms with corrected P-value less than 0.05 were considered significantly enriched by differentially expressed genes. KOBAS software was used to test the statistical enrichment of the differential expression genes in KEGG pathways (http://www.genome.jp/kegg/
). The DEGs, GO, and KEGG were collected in Supplemental Excel files.
4. Discussion and Conclusions
In this study, we characterized the roles of Hes5.9 in regulating cell fate decisions and cell migration in gastrulating Xenopus tropicalis embryos, which were regulated by the FGF and Notch signaling pathways during gastrulation. Results indicated the coordination of the FGF and Notch signaling pathways through fine-tuning of the expression pattern of Hes5.9 during gastrulation.
We originally isolated a novel helix–loop–helix DNA binding domain protein (LOC733709), now suggested as Hes5.9
, by screening the possible target genes of the FGF signaling during gastrulation. We characterized the roles of Hes5.9
in embryogenesis, focusing on gastrulation in particular. Compared with humans, Xenopus
has the same subfamilies (Hes1–Hes7
), but more Hes
genes, about 37. These Hes
genes are involved in neurogenesis, somitogenesis (Hes1, Hes5, Hes7, etc
], and midbrain-hindbrain boundary formation (Hes7.1
genes are located on the same chromosome, especially Hes5.3–Hes5.10
located in the Hes5.3
gene cluster. Most of these genes are downstream targets of the Notch signaling. As a Hes5
gene member, Hes5.9
contains conserved Notch signaling regulating promoter sequences. Moreover, the mRNA expression levels of Hes5.9
were significantly influenced by either chemical or genetic interference of the Notch signaling (Figure 2
). This evidence strongly indicates that Hes5.9
would also be a target of the Notch signaling.
A previous study suggested that all 37 Hes
genes, except Hes2
, are zygotically expressed in early embryonic stages, and peak from the late gastrula to the late neurula stages (stage 12–20) [37
]. Here, we found that Hes5.9
was another maternally expressed Hes
gene, which was highly expressed before the mid blastula transformation (MBT), and reached a peak during the neurula stages (Figure 3
). Intriguingly, the expression patterns of Hes5.9
are very different from Hes5.7;
based on the Transcriptome Database (http://jason.chuang.ca/research/xenopus/refseq.html
). Therefore, compared with other Hes
genes, even Hes5.7
performs relatively unique roles during embryogenesis, especially during gastrulation.
The temporal expression pattern of Hes5.9
mRNA revealed that Hes5.9
was a maternally expressed gene, which was expressed throughout embryonic development and reached a plateau during the gastrula stage and the neurula stage. We also examined its spatial distribution by WISH, which suggested that Hes5.9
asymmetrically localized along with the animal–vegetal axis, and was mainly detected at the animal pole. During gastrulation, Hes5.9
is expressed predominantly throughout the mesoderm. As development proceeds, Hes5.9
is detected in the neural tube, somites, tailbud, brain, neural crest, otic vesicle, and eyes (Figure 3
and Figure S2
). The FGF signaling has been implicated during several phases of early embryogenesis [7
], which contributes to the establishment of distinct types of mesoderm [38
]. Meanwhile, the Notch signaling is involved early in the induction of the three germ layers [2
], and later, playing important roles in somitogenesis and neural system development [39
]. We further investigated the association of Hes5.9
and the FGF signaling pathway during embryonic development by inhibiting FGFR via SU5402 [40
] or overexpression of Fgf8b
]. Here, we found evidence that the FGF signaling pathway is essential for the transcription of Hes5.9
during gastrulation and neurulation (Figure 4
and Figure 5
It has been reported that the FGF signaling pathway regulated both mesoderm migration and convergent extension movements [42
]. The embryos treated with the SU5402 at the gastrula stage significantly decreased the expression level of Hes5.9
, which was accompanied by delayed gastrulation and ultimately open blastopore (Figure 4
and Figure 5
). Therefore, our results indicate that Hes5.9
is regulated both by FGF and Notch signaling during gastrulation, and maybe an important gene for coordinating the FGF and Notch signaling during the gastrula and even the neurula stages. Although treatment with SU5402 caused abnormal embryonic development, we cannot exclude the possibility that the observed phenotype may be caused by the concomitant ectopic expression of Hes5.9
. To investigate the character of Hes5.9
during embryogenesis, we specifically knocked down Hes5.9
by microinjecting with the morpholino antisense nucleotides that were selectively designed to block Hes5.9
translation and splicing, respectively (Supplementary Figure S3
). It showed that both gastrulation and neurulation were impaired. The embryos injected with Hes5.9
-MO exhibit delayed mesoderm involution and failed to close the blastopore, which ultimately results in blastopore and neural tube open, and shorter axis embryos at later development (Figure 6
). Similar phenotypes were obtained from ectopic expression of Hes5.9
) that both mesoderm convergent extension and mesoderm migration were impaired by dorsally microinjecting Hes5.9
mRNA (Figure 8
). Meanwhile, we also found that downregulated genes were significantly enriched in adherence, and cytoskeletal remolding (Figure 9
C and Figure S3
). Consistently, Hes
genes could also be required to control genes involved in cytoskeletal remodeling and the cell shape change, which are needed for initiating the migration itself [45
]. That is also represented by the expression changes of genes, which are involved in the migration process, such as some extracellular matrix molecules and their receptors, cell adhesion molecules, and guide molecules. And the expression levels of those markers were also regulated by Hes5.9
, either in whole embryos or explant tissues, further suggesting that Hes5.9
plays important roles in cell fate specification. In general, these results suggest a crucial role of Hes5.9
on gastrulation, that is Hes5.9
may coordinate the FGF and Notch signaling to fine-tune the cell fate specification and morphogenetic movements [2
Unexpectedly, we also found that the expression level of Xnr3, a target of the maternal Wnt/β-catenin pathway, was significant upregulated after overexpression of Hes5.9
H). It seems contradictory to the previous report that Xnr3 was supposed to be inhibited by the Notch signaling [47
]. However, Xnr3 is also reported to require the FGF signaling to induce cell elongation movements and thus allocating cells from the organizer [48
]. Thus, it is possible that Xnr3 and Hes5.9
coordinate gastrulation in different regions of the mesoderm, which, however, needs further investigation.
In conclusion, we characterized a novel Hes gene (Hes5.9) in Xenopus tropicalis. Our data suggest that Hes5.9 plays important roles in gastrulation and neurulation, through regulating cell fate determination and convergent extension. Further exploration of the possible roles of Hes5.9 in the coordination of the FGF and Notch pathways will bring new insights into the regulation of embryogenesis and organogenesis in future investigations.