January 1, 2017;
Identification of new regulators of embryonic patterning and morphogenesis in Xenopus gastrulae by RNA sequencing.
During early vertebrate embryogenesis, cell fate specification is often coupled with cell acquisition of specific adhesive, polar and/or motile behaviors. In Xenopus gastrulae, tissues fated to form different axial structures display distinct motility. The cells in the early organizer
move collectively and directionally toward the animal pole
and contribute to anterior mesendoderm
, whereas the dorsal and the ventral
tissues surrounding the blastopore
embryos undergo convergent extension and convergent thickening movements, respectively. While factors regulating cell lineage specification have been described in some detail, the molecular machinery that controls cell motility is not understood in depth. To gain insight into the gene battery that regulates both cell fates and motility in particular embryonic tissues, we performed RNA sequencing (RNA-seq) to investigate differentially expressed genes in the early organizer
, the dorsal and the ventral marginal zone
of Xenopus gastrulae. We uncovered many known signaling and transcription factors that have been reported to play roles in embryonic patterning during gastrulation. We also identified many uncharacterized genes as well as genes that encoded extracellular matrix
) proteins or potential regulators of actin cytoskeleton. Co-expression of a selected subset of the differentially expressed genes with activin in animal caps revealed that they had distinct ability to block activin-induced animal cap
elongation. Most of these factors did not interfere with mesodermal induction by activin, but an ECM
, inhibited activin signaling and acted downstream of the activated type I receptor. By focusing on a secreted protein kinase PKDCC1
, we showed with overexpression and knockdown experiments that PKDCC1
regulated gastrulation movements as well as anterior
neural patterning during early Xenopus development. Overall, our studies identify many differentially expressed signaling and cytoskeleton regulators in different embryonic regions of Xenopus gastrulae and imply their functions in regulating cell fates and/or behaviors during gastrulation.
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Fig. 1. Schematic representation of the RNA-seq experiment. A) The early organizer from stage 10+ embryos and the dorsal and the ventral marginal zone (DMZ and VMZ) explants from mid-gastrula stage embryos were dissected and subjected to RNA sequencing. Cells in these regions have distinct fates and motile behaviors. Pairwise comparison of differential expressed genes was performed between the organizer and the DMZ, and the DMZ and the VMZ, samples. The ANOVA-like analysis for all three samples sets was also performed, which produced similar results as that in the pairwise comparison. Genes with potential to regulate embryonic patterning and/or movements were especially scrutinized in more detail in this study. B) A list of the selected known and uncharacterized genes with differential expression patterns in different embryonic regions is shown.
Fig. 2. Differential gene expression in the organizer, the DMZ, and the VMZ. RT-PCR was performed to assay for gene expression in different embryonic regions. A) Marker analysis confirmed the expression of known region-specific genes in the dissected tissues. B) Group I genes were enriched in the organizer. C) Group II genes showed highest expression in the DMZ. D) Group III genes were expressed at high levels in both the organizer and the DMZ. E) Group IV genes were expressed at high levels in both the DMZ and the VMZ. F) Group V genes were enriched in the VMZ.
Fig. 3. Distinct activities of the differentially expressed genes to block activin-induced animal cap elongation. RNAs encoding the RNA-seq clones and activin were co-injected into the animal region of 2-cell stage embryos. Animal caps were dissected at blastula stages and cultured to late neurula stages. A) Two GPRC proteins with the highest expression levels in the DMZ, GPRC5C and CXCR7, reduced activin-induced animal cap elongation without interfering with the mesodermal induction by activin. B) The DMZ-enriched GEF, ARHGEF3, efficiently blocked animal cap elongation; but the organizer-enriched GEF, PLEKHG5, or RhoGEF expressed in the DMZ and VMZ, FGD5, could not do so. C) CDC42EP3, which was expressed at high levels in both the organizer and the DMZ, blocked activin-induced animal cap elongation more efficiently than CDC42EP2, a gene enriched in the organizer. D) The transcription factors ATF3 and FOS both reduced the elongation of the animal caps, but KLF10 was ineffective in doing so. E) The ventrally-enriched Ras family proteins, DIRAS and RasL11B, did not block activin-induced animal cap elongation. The doses of RNA used: activin, 2 pg; RNA-seq clones, 0.25–1 ng.
Fig. 4. The ECM protein EFEMP2 inhibits activin signaling downstream of the activated receptor. A) The ECM protein EFEMP2, but not SPARC, inhibited activin-induced mesodermal formation in animal caps. B) EFEMP2 interfered with mesodermal induction by both activin and the activated type I activin receptor ALK4 (CA-ALK4), but did not inhibit mesodermal induction by Smad2 or Smad2-induced animal cap elongation. The doses of RNA used: activin, 2 pg; CA-ALK4, 1 ng; Smad2, 1 ng; EFEMP2, 1 ng.
Fig. 5. PKDCC1, a dorsally enriched gene, induced gastrulation defects when ectopically expressed. A) RT-PCR showed that PKDCC1 was first expressed during early gastrulation, and its expression persisted until at least tailbud stages. B) In situ hybridization showed that PKDCC1 was expressed in the organizer at the dorsal lip region in early gastrula embryos. Its expression was then up-regulated in the anterior neural tissues during mid-gastrula to neurula stages. At tailbud stages, PKDCC1 transcripts were seen in the eyes, the lateral plate mesoderm, and the heart primordium. C) PKDCC1 reduced activin-induced animal cap elongation without affecting mesodermal cell fates. D) Ectopic expression of PKDCC1 in early Xenopus embryos induced gastrulation defects, with the resulting embryos displaying reduced body axis, smaller head, and failure in neural tube closure. E) Overexpression of PKDCC1 did not inhibit mesodermal formation in early embryos, as indicated by normal expression of brachyury (Bra), but delayed involution and/or migration of dorsal mesodermal cells marked by chordin (Chd) and goosecoid (Gsc).
Fig. 6. Knockdown of PKDCC1 resulted in gastrulation defects. A) A translational blocking antisense PKDCC1-MO was designed to hybridize to the 5′-UTR sequence of the PKDCC transcript. B) Expression of PKDCC1-MO led to embryos with reduced body axis and smaller head, which were greatly rescued by co-expression of low doses of PKDCC1 RNA that did not contain the 5′-UTR MO-target sequence. C) Knockdown of PKDCC1 resulted in delay in internalization of dorsal mesodermal cells marked by Chd and Gsc.
Fig. 7. PKDCC1 regulates gastrulation movements. A) Both ectopic expression and knockdown of PKDCC1 led to reduction of convergent extension of trunk mesodermal tissues derived from the DMZ. The length over width ratio of the explants decreased significantly by enhanced or reduced expression of PKDCC1. B) While altered levels of PKDCC1 did not prevent collective cell sheet migration on fibronectin, prolonged culture of the explants resulted in more pronounced cell dissociation in the absence of PKDCC1, suggesting that PKDCC1 modulated cell cohesion in the anterior mesendoderm.
Fig. 8. PKDCC1 regulates anterior neural patterning. Knockdown of PKDCC1 did not change the expression of the axial and the paraxial mesodermal markers Chd and MyoD or the neural marker Sox2, but reduced the expression domain of the forebrain marker Otx2 and shifted the midbrain marker engrailed (En) anteriorly, indicating that PKDCC1 modulated anterior-posterior neural patterning.
Differential gene expression in the organizer, the DMZ, and the VMZ. RT-PCR was performed to assay for gene expression in different embryonic regions. A) Genes enriched in the organizer. B) Genes with the highest expression in the DMZ.
Distinct activities of the differentially expressed genes to block activin-induced animal cap elongation. RNAs encoding the RNA-seq clones were injected into the animal region of 2-cell stage embryos. Animal caps were dissected at blastula stages and incubated with 8–10 ng/ml Activin B protein (R&D Systems) until late neurula stages. The morphology of the animal caps was then imaged.