STUDY QUESTION What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in... Show moreSTUDY QUESTION What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in comparison to mouse? SUMMARY ANSWER TEAD4 regulates TE lineage differentiation in the human preimplantation embryo acting upstream of caudal-type homeobox protein 2 (CDX2), but in contrast to the mouse in a GATA-binding protein 3 (GATA3)-independent manner. WHAT IS KNOWN ALREADY Tead4 is one of the earliest transcription factors expressed during mouse embryo preimplantation development and is required for the expression of TE-associated genes. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination, have shown that Tead4-targeted embryos have compromised development and expression of the TE-specific Cdx2 and Gata3 is downregulated. Cdx2 and Gata3 act in parallel pathways downstream of Tead4 to induce successful TE differentiation. Downstream loss of Cdx2 expression, compromises TE differentiation and subsequent blastocoel formation and leads to the ectopic expression of inner cell mass (ICM) genes, including POU Class 5 homeobox 1 (Pou5f1) and SRY-box transcription factor (Sox2). Cdx2 is a more potent regulator of TE fate in mouse as loss of Cdx2 expression induces more severe phenotypes compared with loss of Gata3 expression. The role of TEAD4 and its downstream effectors during human preimplantation embryo development has not been investigated yet. STUDY DESIGN, SIZE, DURATION The clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas9) system was first introduced in pronuclei (PN)-stage mouse zygotes aiming to identify a guide RNA (gRNA), yielding high editing efficiency and effective disruption of the Tead4 locus. Three guides were tested (gRNA1-3), each time targeting a distinct region of Exon 2 of Tead4. The effects of targeting on developmental capacity were studied in Tead4-targeted embryos (n = 164-summarized data from gRNA1-3) and were compared with two control groups; sham-injected embryos (n = 26) and non-injected media-control embryos (n = 51). The editing efficiency was determined by next-generation sequencing (NGS). In total, n = 55 (summarized data from gRNA1-3) targeted mouse embryos were analysed by NGS. Immunofluorescence analysis to confirm successful targeting by gRNA1 was performed in Tead4-targeted embryos, and non-injected media-control embryos. The downregulation of secondary TE-associated markers Cdx2 and Gata3 was used as an indirect confirmation of successful Tead4-targeting (previously shown to be expressed downstream of Tead4). Additional groups of gRNA1 Tead4-targeted (n = 45) and media control (n = 36) embryos were cultured for an extended period of 8.5 days, to further assess the developmental capacity of the Tead4-targeted group to develop beyond implantation stages. Following the mouse investigation, human metaphase-II (MII) oocytes obtained by IVM were microinjected with gRNA-Cas9 during ICSI (n = 74) to target TEAD4 or used as media-control (n = 33). The editing efficiency was successfully assessed in n = 25 TEAD4-targeted human embryos. Finally, immunofluorescence analysis for TEAD4, CDX2, GATA3 and the ICM marker SOX2 was performed in TEAD4-targeted (n = 10) and non-injected media-control embryos (n = 29).PARTICIPANTS/MATERIALS, SETTING, METHODS A ribonucleoprotein complex consisting of a gRNA-Cas9 mixture, designed to target Exon 2 of Tead4/TEAD4, was microinjected in mouse PN stage zygotes or human IVM MII oocytes along with sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. In mouse, an additional group of Tead4-targeted and media-control embryos was cultured in vitro for an extended period of 8.5 days. Embryonic development and morphology were assessed daily, during culture in vitro of mouse and human embryos and was followed by a detailed scoring at late blastocyst stage. Targeting efficiency following gRNA-Cas9 introduction was assessed via immunostaining and NGS analysis. MAIN RESULTS AND THE ROLE OF CHANCE NGS analysis of the Tead4-targeted locus revealed very high editing efficiencies for all three guides, with 100% of the mouse embryos (55 out of 55) carrying genetic modifications resulting from CRISPR-Cas9 genome editing. More specifically, 65.22% (15 out 23) of the PN zygotes microinjected with gRNA1-Cas9, which exhibited the highest efficiency, carried exclusively mutated alleles. The developmental capacity of targeted embryos was significantly reduced (data from gRNA1), as 44.17% of the embryos arrested at the morula stage (2.5 days post coitum), coincident with the initiation of TE lineage differentiation, compared with 8.51% in control and 12.50% in sham control groups. High-quality blastocyst formation rates (Grade 3) were 8.97% in the gRNA1-targeted group, compared with 87.23% in the media-control and 87.50% in the sham group. Immunofluorescence analysis in targeted embryos confirmed downregulation of Tead4, Cdx2, and Gata3 expression, which resulted from successful targeting of the Tead4 locus. Tead4-targeted mouse embryos stained positive for the ICM markers Pou5f1 and Sox2, indicating that expression of ICM lineage markers is not affected. Tead4-targeted embryos were able to cavitate and form a blastocoel without being able to hatch. Extended embryo culture following zona pellucida removal, revealed that the targeted embryos can attach and form egg-cylinder-like structures in the absence of trophoblast giant cells. In human embryos, Exon 2 of TEAD4 was successfully targeted by CRISPR-Cas9 (n = 74). In total, 25 embryos from various developmental stages were analysed by NGS and 96.00% (24 out of 25) of the embryos carried genetic modifications because of gRNA-Cas9 editing. In the subgroup of the 24 edited embryos, 17 (70.83%) carried only mutant alleles and 11 out of these 17 (64.70%) carried exclusively frameshift mutations. Six out of 11 embryos reached the blastocyst stage. In contrast to mice, human-targeted embryos formed blastocysts at a rate (25.00%) that did not differ significantly from the control group (23.81%). However, blastocyst morphology and TE quality were significantly compromised following TEAD4-targeting, showing grade C TE scores, with TE containing very few cells. Immunofluorescence analysis of TEAD4-targeted embryos (n = 10) confirmed successful editing by the complete absence of TEAD4 and its downstream TE marker CDX2, but the embryos generated retained expression of GATA3, which is in contrast to what we have observed and has previously been reported in mouse. In this regard, our results indicate that GATA3 acts in parallel with TEAD4/CDX2 towards TE differentiation in human. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION CRISPR-Cas9 germline genome editing, in some cases, induces mosaic genotypes.These genotypes are a result of inefficient and delayed editing, and complicate the phenotypic analysis and developmental assessment of the injected embryos. We cannot exclude the possibility that the observed differences between mouse and human are the result of variable effects triggered by the culture conditions, which were however similar for both mouse and human embryos in this study. Furthermore, this study utilized human oocytes obtained by IVM, which may not fully recapitulate the developmental behaviour of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS Elucidation of the evolutionary conservation of molecular mechanisms that regulate the differentiation and formation of the trophoblast lineage can give us fundamental insights into early implantation failure, which accounts for similar to 15% of human conceptions. STUDY FUNDING/COMPETING INTEREST(S) The research was funded by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01) and Ghent University (BOF.BAS.2018.0018.01). G.C. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 11L8822N). A.B. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 1298722 N). We further thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. Show less
Stamatiadis, P.; Boel, A.; Cosemans, G.; Popovic, M.; Bekaert, B.; Guggilla, R.; ... ; Heindryckx, B. 2021
STUDY QUESTIONWhat is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model?SUMMARY ANSWERPOU5F1 is required for successful... Show moreSTUDY QUESTIONWhat is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model?SUMMARY ANSWERPOU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation.WHAT IS KNOWN ALREADYClustered regularly interspaced short palindromic repeats—CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage.STUDY DESIGN, SIZE, DURATIONThe efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain—B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos.PARTICIPANTS/MATERIALS, SETTING, METHODSA gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing.MAIN RESULTS AND THE ROLE OF CHANCEGenomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts.LIMITATIONS, REASONS FOR CAUTIONOne of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes.WIDER IMPLICATIONS OF THE FINDINGSComparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis. Show less
AbstractBackground: Mutations of cysteine residues in von Willebrand factor are known to reduce its storage and secretion, thus leading to reduced antigen levels. However, one cysteine mutation, p... Show moreAbstractBackground: Mutations of cysteine residues in von Willebrand factor are known to reduce its storage and secretion, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in type 2A(IID) von Willebrand disease patients who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies.Design and Methods: The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection in human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced with phorbol 12-myristate 13-acetate. Results: p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser was stored normally. As expected, wild type von Willebrand factor formed proteinaceous tubules that displayed as longitudinal striations in pseudo Weibel-Palade bodies under electron microscopy. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but it formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser. Conclusions: We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect the storage in Weibel-Palade bodies and secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds will lead to reduced von Willebrand factor storage and secretion due to ER retention. Show less
