Heterozygous Embryos Research Articles

Zebrafish etv2 knock-in line labels vascular endothelial and blood progenitor cells.

ETS transcription factor Etv2/Etsrp is one of the earliest markers for vascular and hematopoietic progenitors and functions as a key regulator of hematovascular development in multiple vertebrates, including zebrafish. Therefore, transgenic etv2 reporter lines provide a valuable tool to study vasculogenesis and hematopoiesis. However, previously generated zebrafish reporter lines do not fully recapitulate the endogenous pattern of etv2 expression. Here we used CRISPR/Cas9-mediated homology-independent DNA repair approach to knock-in a Gal4 transcriptional activator into the zebrafish etv2 genomic locus, thus generating etv2 ci32Gt gene trap line. etv2 ci32Gt ; UAS:GFP embryos show GFP expression in vascular endothelial, myeloid and red blood cells. Because gal4 insertion interrupts the etv2 locus, homozygous etv2 ci32Gt embryos display defects in vasculogenesis and myelopoiesis, and enable visualizing etv2-deficient hematovascular progenitors in live embryos. Furthermore, we performed differential transcriptome analysis of sorted GFP-positive cells from heterozygous and homozygous etv2 ci32Gt embryos. Approximately 500 downregulated genes were identified in etv2 ci32Gt homozygous embryos, which include multiple genes expressed in vascular endothelial and myeloid cells. The etv2 ci32Gt gene trap line and the data sets of misregulated genes will be valuable resources to study hematopoietic and vascular development.

Functional characterization of a novel PBX1 de novo missense variant identified in a patient with syndromic congenital heart disease.

Pre-B cell leukemia factor 1 (PBX1) is an essential developmental transcription factor, mutations in which have recently been associated with CAKUTHED syndrome, characterized by multiple congenital defects including congenital heart disease (CHD). During analysis of a whole-exome-sequenced cohort of heterogeneous CHD patients, we identified a de novo missense variant, PBX1:c.551G>C p.R184P, in a patient with tetralogy of Fallot with absent pulmonary valve and extra-cardiac phenotypes. Functional analysis of this variant by creating a CRISPR-Cas9 gene-edited mouse model revealed multiple congenital anomalies. Congenital heart defects (persistent truncus arteriosus and ventricular septal defect), hypoplastic lungs, hypoplastic/ectopic kidneys, aplastic adrenal glands and spleen, as well as atretic trachea and palate defects were observed in the homozygous mutant embryos at multiple stages of development. We also observed developmental anomalies in a proportion of heterozygous embryos, suggestive of a dominant mode of inheritance. Analysis of gene expression and protein levels revealed that although Pbx1 transcripts are higher in homozygotes, amounts of PBX1 protein are significantly decreased. Here, we have presented the first functional model of a missense PBX1 variant and provided strong evidence that p.R184P is disease-causal. Our findings also expand the phenotypic spectrum associated with pathogenic PBX1 variants in both humans and mice.

P313ADGRL2 is an essential surface molecule for cardiac lineage specification and heart development

Abstract Background Specific surface markers that enable monitoring of cell subsets would be valuable for establishing the conditions under which pluripotent stem cells (PSCs) differentiate into cardiac progenitor cells (CPCs) and cardiomyocytes (CMCs). Methods and results To verify whether a specific marker is expressed during heart development, we assessed its expression using the CLARITY technique. After immersion in a solution with a refractive index matching that of the CLARITY hybrid, the mouse embryo became transparent. After immunostaining the cleared embryo sample, Adgrl2 was exclusively observed in cardiac cells expressing α-SA at embryonic day E9.5 and E10.5. Our clarified 3D images and movies show that four chambers of the heart are fully developed at E10.5 but not at E9.5. At E9.5, Adgrl2 is observed at the ventricle and atrium, while Adgrl2 is present in all chambers of the heart at E10.5. Next, we performed LacZ (β-Gal) staining in heterozygous Adgrl2 KO embryos to evaluate Adgrl2 expression. As a result, LacZ staining showed that Adgrl2 was predominantly expressed in the heart during the embryonic developmental stage. Adgrl2 knockout in mice was embryonically lethal because of severe heart, but not vascular, defects. To examine the use of Adgrl2 as a bona fide CPC marker during heart development, we tracked Adgrl2 expression during early embryonic development. The heart of Adgrl2−/− embryos at E10.5 exhibited occlusion of the RV, and the expression levels of Gata4 and Nkx2.5 were not as high as those in wild-type and Adgrl2+/− embryos. Interestingly, the heart of Adgrl2−/− embryos, unlike those of wild-type and Adgrl2+/− embryos between E13.5 and E15.5 had a single ventricle revealing a ventricular septal defect. The specific expression pattern of Adgrl2 in PSC-derived cardiac lineage cells as well as in embryonic heart, adult mice, and human heart tissues. Conclusion We demonstrate that Adgrl2 plays a pivotal and functional role across all strata of the cardiomyogenic lineage, as early as the precursor stage of heart development. These findings shed light on heart development and regeneration. Acknowledgement/Funding Grants from “Strategic Center of Cell and Bio Therapy” (grant number: HI17C2085) and “Korea Research-Driven Hospital” (HI14C1277)

Tbx1 and Foxi3 genetically interact in the pharyngeal pouch endoderm in a mouse model for 22q11.2 deletion syndrome.

We investigated whether Tbx1, the gene for 22q11.2 deletion syndrome (22q11.2DS) and Foxi3, both required for segmentation of the pharyngeal apparatus (PA) to individual arches, genetically interact. We found that all Tbx1+/-;Foxi3+/- double heterozygous mouse embryos had thymus and parathyroid gland defects, similar to those in 22q11.2DS patients. We then examined Tbx1 and Foxi3 heterozygous, null as well as conditional Tbx1Cre and Sox172A-iCre/+ null mutant embryos. While Tbx1Cre/+;Foxi3f/f embryos had absent thymus and parathyroid glands, Foxi3-/- and Sox172A-iCre/+;Foxi3f/f endoderm conditional mutant embryos had in addition, interrupted aortic arch type B and retroesophageal origin of the right subclavian artery, which are all features of 22q11.2DS. Tbx1Cre/+;Foxi3f/f embryos had failed invagination of the third pharyngeal pouch with greatly reduced Gcm2 and Foxn1 expression, thereby explaining the absence of thymus and parathyroid glands. Immunofluorescence on tissue sections with E-cadherin and ZO-1 antibodies in wildtype mouse embryos at E8.5-E10.5, revealed that multilayers of epithelial cells form where cells are invaginating as a normal process. We noted that excessive multilayers formed in Foxi3-/-, Sox172A-iCre/+;Foxi3f/f as well as Tbx1 null mutant embryos where invagination should have occurred. Several genes expressed in the PA epithelia were downregulated in both Tbx1 and Foxi3 null mutant embryos including Notch pathway genes Jag1, Hes1, and Hey1, suggesting that they may, along with other genes, act downstream to explain the observed genetic interaction. We found Alcam and Fibronectin extracellular matrix proteins were reduced in expression in Foxi3 null but not Tbx1 null embryos, suggesting that some, but not all of the downstream mechanisms are shared.

A Long Polymorphic GT Microsatellite within a Gene Promoter Mediates Non-Imprinted Allele-Specific DNA Methylation of a CpG Island in a Goldfish Inter-Strain Hybrid.

It is now widely accepted that allele-specific DNA methylation (ASM) commonly occurs at non-imprinted loci. Most of the non-imprinted ASM regions observed both within and outside of the CpG island show a strong correlation with DNA polymorphisms. However, what polymorphic cis-acting elements mediate non-imprinted ASM of the CpG island remains unclear. In this study, we investigated the impact of polymorphic GT microsatellites within the gene promoter on non-imprinted ASM of the local CpG island in goldfish. We generated various goldfish heterozygotes, in which the length of GT microsatellites or some non-repetitive sequences in the promoter of no tail alleles was different. By examining the methylation status of the downstream CpG island in these heterozygotes, we found that polymorphisms of a long GT microsatellite can lead to the ASM of the downstream CpG island during oogenesis and embryogenesis, polymorphisms of short GT microsatellites and non-repetitive sequences in the promoter exhibited no significant effect on the methylation of the CpG island. We also observed that the ASM of the CpG island was associated with allele-specific expression in heterozygous embryos. These results suggest that a long polymorphic GT microsatellite within a gene promoter mediates non-imprinted ASM of the local CpG island in a goldfish inter-strain hybrid.

Loss of function mutation of Eftud2, the gene responsible for mandibulofacial dysostosis with microcephaly (MFDM), leads to pre-implantation arrest in mouse.

Mutations in EFTUD2 are responsible for the autosomal dominant syndrome named MFDM (mandibulofacial dysostosis with microcephaly). However, it is not clear how reduced levels of EFTUD2 cause abnormalities associated with this syndrome. To determine if the mouse can serve as a model for uncovering the etiology of abnormalities found in MFDM patients, we used in situ hybridization to characterize expression of Eftud2 during mouse development, and used CRISPR/Cas9 to generate a mutant mouse line with deletion of exon 2 of the mouse gene. We found that Eftud2 was expressed throughout embryonic development, though its expression was enriched in the developing head and craniofacial regions. Additionally, Eftud2 heterozygous mutant embryos had reduced EFTUD2 mRNA and protein levels. Moreover, Eftud2 heterozygous embryos were born at the expected Mendelian frequency, and were viable and fertile despite being developmentally delayed. In contrast, Eftud2 homozygous mutant embryos were not found post-implantation but were present at the expected Mendelian frequency at embryonic day (E) 3.5. Furthermore, only wild-type and heterozygous E3.5 embryos survived ex vivo culture. Our data indicate that Eftud2 expression is enriched in the precusor of structures affected in MFDM patients and show that heterozygous mice carrying deletion of exon 2 do not model MFDM. In addition, we uncovered a requirement for normal levels of Eftud2 for survival of pre-implantation zygotes.

Pleomorphic Adenoma Gene 1 Is Needed For Timely Zygotic Genome Activation and Early Embryo Development

Pleomorphic adenoma gene 1 (PLAG1) is a transcription factor involved in cancer and growth. We discovered a de novo DNA motif containing a PLAG1 binding site in the promoters of genes activated during zygotic genome activation (ZGA) in human embryos. This motif was located within an Alu element in a region that was conserved in the murine B1 element. We show that maternally provided Plag1 is needed for timely mouse preimplantation embryo development. Heterozygous mouse embryos lacking maternal Plag1 showed disrupted regulation of 1,089 genes, spent significantly longer time in the 2-cell stage, and started expressing Plag1 ectopically from the paternal allele. The de novo PLAG1 motif was enriched in the promoters of the genes whose activation was delayed in the absence of Plag1. Further, these mouse genes showed a significant overlap with genes upregulated during human ZGA that also contain the motif. By gene ontology, the mouse and human ZGA genes with de novo PLAG1 motifs were involved in ribosome biogenesis and protein synthesis. Collectively, our data suggest that PLAG1 affects embryo development in mice and humans through a conserved DNA motif within Alu/B1 elements located in the promoters of a subset of ZGA genes.

Functional analysis of Niemann-Pick disease type C family protein, NPC1a, in Drosophila melanogaster.

During embryonic gonad coalescence, primordial germ cells (PGCs) follow a carefully choreographed migratory route circumscribed by guidance signals towards somatic gonadal precursor cells (SGPs). In Drosophila melanogaster, SGP-derived Hedgehog (Hh), which serves as a guidance cue for the PGCs, is potentiated by mesodermally restricted HMGCoA-reductase (Hmgcr) and the ABC transporter Multi-drug-resistant-49 (Mdr49). Given the importance of cholesterol modification in the processing and long-distance transmission of the Hh ligand, we have analyzed the involvement of the Niemann-Pick disease type C-1a (NPC1a) protein, a cholesterol transporter, in germ cell migration and Hedgehog signaling. We show that mesoderm-specific inactivation of Npc1a results in germ cell migration defects. Similar to Mdr49, PGC migration defects in the Npc1a embryos are ameliorated by a cholesterol-rich diet. Consistently, reduction in Npc1a weakens the ability of ectopic HMG Coenzyme A reductase (Hmgcr) to induce germ cell migration defects. Moreover, compromising Npc1a levels influences Hh signaling adversely during wing development, a process that relies upon long-range Hh signaling. Last, doubly heterozygous embryos (Mdr49/Npc1a) display enhanced germ cell migration defects when compared with single mutants (Npc1a/+ or Mdr49/+), supporting cooperative interaction between the two.

EFTUD2, The Gene Responsible For Mandibulofacial Dysostosis With Microcephaly (MFDM), Is Required For Implantation And Craniofacial Development In Mouse

Haploinsufficiency of EFTUD2 is associated with MFDM (mandibulofacial dysostosis with microcephaly), but the etiology of this syndrome remains unknown. Our goal is to determine the tissue and temporal specific expression and requirement for Eftud2 during craniofacial development. We used RT‐PCR and in situ hybridization to examine expression of Eftud2 during embryogenesis. Using CRISPR/Cas9 we designed guide RNAs to generate mice with deletion (Eftud2 del) and conditional mutation of exon 2 of Eftud2 (Eftud2 flox). At embryonic day (E) 7.5 and 8.5 of mouse development, Eftud2 was highly expressed in both ectodermal and mesodermal components of the developing head and craniofacial region, by E9.5 Eftud2 was more broadly expressed, in the body wall and developing heart. Eftud2 del heterozygous mice and embryos were viable and fertile and showed a 38% and a 30% reduction of Eftud2 mRNA and protein expression, respectively. Before the onset of organogenesis, Eftud2 del heterozygous embryos had reduced number of somites when compared to their wild type litter mates, indicating a delay in development. Noticeably, RNA sequencing revealed that Eftud2 was the only transcript significantly affected in these heterozygous mice. Eftud2 del homozygous mutant embryos did not implant and failed to grow and hatch ex vivo. Next, we used the Wnt1‐Cre2 transgenic line to delete exon 2 of Eftud2 specifically in the neural crest cells. Eftud2 flox heterozygous embryos carrying the Wnt‐Cre2 transgene were normal. However, Eftud2 flox homozygous mutant embryos that also carry the Wnt1‐Cre2 transgene displayed hypoplasia of the midbrain and pharyngeal arches starting as early as E9.5. By E11.5, most of these embryos showed an open neural tube, all embryos showed exencephaly at E14.5. Cartilage preparations revealed an absence of cartilage in the head, reduced/or absence of Meckel's cartilage, and abnormal inner ear development. Crosses with the Rosa26R mice reporter line, revealed reduced neural crest cell migration into the pharyngeal region at E10.5. Since the mutation designed is predicted to generate a truncated protein with partial function, our data suggest that normal levels of Eftud2 is crucial during embryogenesis. Future studies are focused on determining the molecular and transcriptional basis of MFDM using this mouse model.Support or Funding InformationCanadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Roles for Notch‐Sox9 in biliary epithelial cells‐to‐hepatocytes differentiation during liver regeneration

Liver regeneration after most forms of injury is mediated through proliferation of hepatocytes. However, when hepatocyte proliferation is impaired, such as during chronic liver disease, liver progenitor cells (LPCs) from the biliary epithelial cell (BEC) compartment can give rise to hepatocytes to mediate hepatic repair. Promotion of LPC‐to‐hepatocyte differentiation in patients with chronic liver disease could serve as a potential new therapeutic option, but first requires the identification of the molecular mechanisms driving this process. Notch signaling has been identified as an important signaling pathway promoting the BEC fate during development, and has also been implicated in regulating LPC differentiation during regeneration. SRY‐related HMG box transcription factor 9 (Sox9) is a direct target of Notch signaling in the liver, and Sox9 has also been shown to promote the BEC fate during development. We have recently shown that inhibition of Hdac1 activity during liver regeneration in both zebrafish and mouse models enhances sox9 expression in LPCs and impairs LPC‐to‐hepatocyte differentiation. Therefore, we hypothesized that inhibition of Notch signaling would promote LPC‐to‐hepatocyte differentiation by repressing sox9 expression. To this end, we blocked Notch activation during liver regeneration through treatment with γ‐secretase inhibitor LY411575 in a zebrafish LPC‐driven regeneration model and demonstrated enhanced induction of Hnf4a in LPCs. Alternatively, enhancing Notch signaling via Notch3 intracellular domain (N3ICD) overexpression impaired Hnf4a induction. Hepatocyte ablation in sox9b heterozygous mutant embryos enhanced Hnf4a induction, while BEC‐specific Sox9b overexpression impaired LPC‐to‐hepatocyte differentiation. Moreover, LY411575 treatment promoted the differentiation of murine LPCs into hepatocytes in vitro. Lastly, we administrated LY411575 to mice with hepatocyte‐specific deletion of β‐catenin, which induces differentiation of LPCs into hepatocytes following a choline‐deficient, ethionine‐supplemented (CDE) diet. We also examined the effect of liver‐specific Sox9 ablation upon CDE diet liver injury. Our results will elucidate the detailed roles for Notch‐Sox9 signaling in liver regeneration and LPC‐to‐hepatocyte differentiation.Support or Funding InformationThe work was supported by NIH grants to D.S. (DK101426), S.P.M. (DK62277, DK100287, CA204586), and J.O.R. (T32EB0010216, 1F31DK115017). S.P.M. is an Endowed Chair for Experimental Pathology.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Notch Inhibition Promotes Differentiation of Liver Progenitor Cells into Hepatocytes via sox9b Repression in Zebrafish.

Liver regeneration after most forms of injury is mediated through the proliferation of hepatocytes. However, when hepatocyte proliferation is impaired, such as during chronic liver disease, liver progenitor cells (LPCs) arising from the biliary epithelial cell (BEC) compartment can give rise to hepatocytes to mediate hepatic repair. Promotion of LPC-to-hepatocyte differentiation in patients with chronic liver disease could serve as a potentially new therapeutic option, but first requires the identification of the molecular mechanisms driving this process. Notch signaling has been identified as an important signaling pathway promoting the BEC fate during development and has also been implicated in regulating LPC differentiation during regeneration. SRY-related HMG box transcription factor 9 (Sox9) is a direct target of Notch signaling in the liver, and Sox9 has also been shown to promote the BEC fate during development. We have recently shown in a zebrafish model of LPC-driven liver regeneration that inhibition of Hdac1 activity through MS-275 treatment enhances sox9b expression in LPCs and impairs LPC-to-hepatocyte differentiation. Therefore, we hypothesized that inhibition of Notch signaling would promote LPC-to-hepatocyte differentiation by repressing sox9b expression in zebrafish. We ablated the hepatocytes of Tg(fabp10a:CFP-NTR) larvae and blocked Notch activation during liver regeneration through treatment with γ-secretase inhibitor LY411575 and demonstrated enhanced induction of Hnf4a in LPCs. Alternatively, enhancing Notch signaling via Notch3 intracellular domain (N3ICD) overexpression impaired Hnf4a induction. Hepatocyte ablation in sox9b heterozygous mutant embryos enhanced Hnf4a induction, while BEC-specific Sox9b overexpression impaired LPC-to-hepatocyte differentiation. Our results establish the Notch-Sox9b signaling axis as inhibitory to LPC-to-hepatocyte differentiation in a well-established in vivo LPC-driven liver regeneration model.

Folic acid modifies the shape of epithelial cells during morphogenesis via a Folr1 and MLCK dependent mechanism.

ABSTRACTFolic acid supplementation can prevent neural tube defects, but the specific molecular mechanisms by which it does have not been elucidated. During neural plate morphogenesis, epithelial cell apical constriction cooperates with other events to drive tissue-bending, and when defective, can result in neural tube defects. A Rho-kinase deficient binding mutant of the apical constriction regulating protein, Shroom3 (Shroom3R1838C), is one of only a handful of mouse mutant lines with neural tube defects that can be rescued by folic acid supplementation. This provided a unique opportunity to probe the functional rescue of a protein linked to neural tube development by folic acid. Utilizing an epithelial cell culture model of apical constriction, it was observed that treatment with exogenous folic acid, as well as co-expression of the folic acid receptor Folr1, can rescue the function of the Rho-kinase binding deficient mutant of Shroom3 in vitro. It was also determined that the rescuing ability of folic acid is RhoA and Rho-kinase independent but myosin light chain kinase (MLCK) and Src-kinase dependent. Inhibition of Rho-kinase-dependent apical constriction in chick embryo neural epithelium was also observed to be rescued by exogenous folic acid and that treatment with folic acid is accompanied by elevated activated myosin light chain and MLCK. Furthermore, doubly heterozygous mouse embryos lacking one copy each of Shroom3 and Folr1 exhibit a low rate of neural tube defects and also have lower levels of activated myosin light chain and MLCK. These studies suggest a novel mechanism by which folic acid modifies epithelial cell shape during morphogenesis, shedding light onto how folic acid may prevent neural tube defects.

The ubiquitin ligase HECTD1 promotes retinoic acid signaling required for development of the aortic arch.

ABSTRACTThe development of the aortic arch is a complex process that involves remodeling of the bilaterally symmetrical pharyngeal arch arteries (PAAs) into the mature asymmetric aortic arch. Retinoic acid signaling is a key regulator of this process by directing patterning of the second heart field (SHF), formation of the caudal PAAs and subsequent remodeling of the PAAs to form the aortic arch. Here, we identify the HECTD1 ubiquitin ligase as a novel modulator of retinoic acid signaling during this process. Hectd1opm/opm homozygous mutant embryos show a spectrum of aortic arch abnormalities that occur following loss of 4th PAAs and increased SHF marker expression. This sequence of defects is similar to phenotypes observed in mutant mouse models with reduced retinoic acid signaling. Importantly, HECTD1 binds to and influences ubiquitination of the retinoic acid receptor, alpha (RARA). Furthermore, reduced activation of a retinoic acid response element (RARE) reporter is detected in Hectd1 mutant cells and embryos. Interestingly, Hectd1opm/+ heterozygous embryos exhibit reduced retinoic acid signaling, along with intermediate increased expression of SHF markers; however, heterozygotes show normal development of the aortic arch. Decreasing retinoic acid synthesis by reducing Raldh2 (also known as Aldh1a2) gene dosage in Hectd1opm/+ heterozygous embryos reveals a genetic interaction. Double heterozygous embryos show hypoplasia of the 4th PAA and increased incidence of a benign aortic arch variant, in which the transverse arch between the brachiocephalic and left common carotid arteries is shortened. Together, our data establish that HECTD1 is a novel regulator of retinoic acid signaling required for proper aortic arch development.

Generation of bicistronic reporter knockin mice for visualizing germ layers.

Knockout mouse models are commonly used in developmental biology to investigate the functions of specific genes, and the knowledge obtained in such models has yielded insights into the molecular mechanisms underlying developmental processes. Gastrulation is the most dynamic process in embryogenesis during which differentiation into three germ layers occurs. However, the functions of genes involved in gastrulation are not completely understood. One major reason for this is the technical difficulty of embryo analysis to understand germ layer location. We have generated three reporter mouse strains in which the germ layers are distinguished by different fluorescent reporters. Using CRISPR/Cas9 genome editing in mouse zygotes, the fluorescent reporter genes, EGFP, tdTomato, and TagBFP including 2A peptide sequences were knocked into the appropriate sites before the stop codon of the Sox17 (endoderm marker), Otx2 (ectoderm marker), and T (mesoderm marker) genes, respectively. Founder mice were successfully generated in the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter strains. Further, homozygous knockin mice of all strains appeared morphologically normal and were fertile. On stereomicroscopic analysis, fluorescent signals were detected in a germ layer-specific manner from heterozygous embryos at embryonic day (E) 6.5–8.5 in all strains, and were immunohistochemically demonstrated to match their respective germ layer-specific marker protein at E7.5. Taken together, these observations suggest that the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter mice may be useful for comprehensive analysis of gene function in germ layer formation.

Macrophage Protease-Activated Receptor 2 Regulates Fetal Liver Hematopoiesis in Mice

Deficiencies in many coagulation factors and protease-activated receptors (PARs) affect mouse embryonic development, but phenotypes of single PAR-deficiencies are often obscured by compensation by other receptors. Although most PAR1/PAR2 double deficient embryos do not survive to birth, PAR2 single-deficient embryos exhibit an unresolved partial preterm lethality. We have observed that PAR2-/- embryos at day 13.5 to 15.5 p.c. from timed mating of homozygous PAR2-/- parents display an increased embryonic death and growth retardation as well as variable signs of anemia in comparison to WT embryos of the same gestational age. The pale appearance suggested that PAR2 might play a role during fetal hematopoiesis. To investigate whether this phenotype is merely a consequence of a previously demonstrated placental defect arising from PAR2 deficiency in the mother, we compared heterozygous PAR2+/- embryos produced by timed-mating of WT mothers mated with PAR2-/- fathers or of PAR2-/- mother mated with WT father. Although reduced embryo weights in PAR2-/- relative to WT mothers indicated a developmental delay due to maternal PAR2 deficiency, anemia was not observed in these PAR2+/- embryos.The fetal liver is the site of hematopoiesis during embryonic development. We therefore evaluated fetal hematopoiesis at day 13.5 to 15.5 p.c. in PAR2+/- and PAR2-/- embryos from timed mated heterozygous PAR2+/- mothers mated with PAR2-/- fathers. Macroscopic phenotypic abnormalities, including signs of anemia, were more prevalent in PAR2-/- versus PAR2+/- embryos, demonstrating an embryonic phenotype that develops independent from maternal PAR2-deficiency. Gene expression analyses of fetal liver at day 13.5 p.c. showed a reduction in marker genes for erythropoiesis, i.e. beta-globin, GATA-1, BCL-11a and for macrophages, i.e. F4/80, Id3, Nr1h3, specifically in PAR2-/- relative to PAR2+/- embryos. These differences were also seen at day 15.5 p.c. in homozygous PAR2 R38E cleavage resistant relative to PAR2 R38E/WT heterozygous embryos in the same litter, confirming PAR2 proteolytic signaling as the major regulator of fetal hematopoiesis.Since macrophage markers in the fetal liver were reduced in PAR2-/- embryos, we next evaluated the role of macrophage PAR2 signaling by cell-type specific deletion in PAR2flfl-LysMcre mice. Macrophage PAR2-deficient mice had reduced erythroblast islands, and erythropoiesis and macrophage marker genes were reduced in the fetal liver at day 15.5 p.c. to the same extent as seen in completely PAR2-deficient embryos. Blood island macrophages play pivotal roles in erythrophagocytosis and clearance of red blood cell expelled nuclei. Deficiency in red blood cell nuclei clearance, e.g. in DNase I deficient mice, causes macrophage inflammatory activation and upregulation of the interferon regulated gene Hamp-1. PAR2flfl-LysMcre fetal livers showed increased Hamp-1 expression relative to cre-negative controls from the same pregnancy, further indicating that macrophage PAR2 signaling directly participates in the regulation of erythropoiesis. These data identify a novel function for proteolytic PAR2 activation in the supporting fetal hematopoiesis and raise the possibility that PAR2 signaling may also regulate adult hematopoiesis under stress conditions or aging. DisclosuresNo relevant conflicts of interest to declare.

Sf3b1 Regulation of Jak/Stat Signaling Is Essential for Hematopoietic Stem Cell Formation

Hematopoietic stem cells (HSCs) maintain the hematopoietic system throughout the lifetime of an organism. During embryonic development, HSCs emerge through an endothelial-to-hematopoietic transition (EHT) from specialized hemogenic endothelial (HE) cells in the dorsal aorta. HSC fate specification depends on gene expression, which is the culmination of coordinated transcription, RNA splicing, and translation. Although transcriptional regulation of HSC fate choice is well studied, the regulatory role of RNA splicing in this process is poorly understood. Using zebrafish loss-of-function mutants for the spliceosomal component splicing factor 3b, subunit 1 (sf3b1), we identified that impaired splicing hindered HSC production. Surprisingly, we found that this constitutive splicing factor selectively regulates the fate of hemogenic endothelium while leaving the identity of closely-related non-hemogenic endothelium unperturbed. To identify Sf3b1-regulated transcripts important in EHT, we performed RNA-sequencing on purified kdrl:gfp+ endothelial cells from sf3b1 mutant and wild-type siblings at 24 hpf. Approximately 900 genes were mis-spliced, 144 of which were differentially expressed. Ingenuity Pathway Analysis identified Janus Kinase (Jak)/Signaling Transducer and Activator of Transcription (Stat) signaling, in particular Stat3, as one of the top perturbed pathways in the mis-spliced gene set. Stat3 is a transcription factor activated in response to several cytokine and inflammatory signals. To determine if altered splicing of stat3 was critical for HSC formation, we injected antisense splice-blocking morpholinos (MO) targeting the Sf3b1-sensitive stat3 exon19 into wild-type and sf3b1 heterozygous embryos, which normally generate equivalent levels of HSCs. We observed an impairment of HSC production in stat3 morpholino-injected sf3b1 heterozygotes, but not wild-type siblings, indicating a synthetic lethal interaction between sf3b1 and stat3. We also found that overexpression of a constitutively active form of Stat3 significantly suppressed the HSC defects in sf3b1 homozygous mutants. Together, these data indicate that Sf3b1-mediated splicing regulation of the Jak/Stat pathway is critical for HSC emergence. DisclosuresNo relevant conflicts of interest to declare.

FGF10 and the Mystery of Duodenal Atresia in Humans.

Background: Duodenal atresia (DA) is a congenital obstruction of the duodenum, which affects 1 in 7000 pregnancies and requires major surgery in the 1st days of life. Three morphological DA types are described. In humans, the association between DA and Down syndrome suggests an underlying, albeit elusive, genetic etiology. In mice, interruption of fibroblast growth factor 10 (Fgf10) gene signaling results in DA in 30–50% of embryos, supporting a genetic etiology. This study aims to validate the spectrum of DA in two novel strains of Fgf10 knock-out mice, in preparation for future and translational research.Methods: Two novel CRISPR Fgf10 knock-out mouse strains were derived and embryos generated by heterozygous plug-mating. E15.5–E19.5 embryos were genotyped with respect to Fgf10 and micro-dissected to determine the presence and type of DA.Results: One twenty seven embryos (32 wild-type, 34 heterozygous, 61 null) were analyzed. No wild-type or heterozygous embryos had DA. However, 74% of Fgf10 null embryos had DA (49% type 1, 18% type 2, and 33% type 3).Conclusion: Our CRISPR-derived strains showed higher penetrance of DA due to single-gene deletion of Fgf10 in mice than previously reported. Further, the DA type distribution in these mice more closely reiterated that observed in humans. Future experiments will document RNA and protein expression of FGF10 and its key downstream signaling targets in normal and atretic duodenum. This includes exploitation of modern, high-fidelity developmental tools, e.g., Fgf10flox/+–tomatoflox/flox mice.

Correction of the Marfan Syndrome Pathogenic FBN1 Mutation by Base Editing in Human Cells and Heterozygous Embryos.

There are urgent demands for efficient treatment of heritable genetic diseases. The base editing technology has displayed its efficiency and precision in base substitution in human embryos, providing a potential early-stage treatment for genetic diseases. Taking advantage of this technology, we corrected a Marfan syndrome pathogenic mutation, FBN1T7498C. We first tested the feasibility in mutant cells, then successfully achieved genetic correction in heterozygous human embryos. The results showed that the BE3 mediated perfect correction at the efficiency of about 89%. Importantly, no off-target and indels were detected in any tested sites in samples by high-throughput deep sequencing combined with whole-genome sequencing analysis. Our study therefore suggests the efficiency and genetic safety of correcting a Marfan syndrome (MFS) pathogenic mutation in embryos by base editing.

The Ubx Polycomb response element bypasses an unpaired Fab-8 insulator via cis transvection in Drosophila.

Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. Here, we studied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of the transgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights a role of homologous chromosome pairing in the regulation of gene expression in the Drosophila genome.

The concept of type 2 segmental mosaicism, expanding from dermatology to general medicine.

In autosomal dominant skin disorders, the well-known type 1 segmental mosaicism reflects heterozygosity for a postzygotic new mutation. By contrast, type 2 segmental mosaicism originates in a heterozygous embryo from an early postzygotic mutational event giving rise to loss of the corresponding wild-type allele, which results in a pronounced segmental involvement being superimposed on the ordinary, non-segmental phenotype. Today, this concept has been proven by molecular analysis in many cutaneous traits. The purpose of this review was to seek publications of cases suggesting an extracutaneous manifestation of type 2 segmental mosaicism. Case reports documenting a pronounced extracutaneous segmental involvement were collected from the literature available in PubMed and from personal communications to the author. Pertinent cases are compared to the description of cutaneous segmental mosaicism of type 1 or type 2 as reported in a given trait. In total, reports suggesting extracutaneous type 2 segmental mosaicism were found in 14 different autosomal dominant skin disorders. In this way, clinical evidence is accumulated that extracutaneous type 2 segmental mosaicism does likewise occur in many autosomal dominant skin disorders. So far, however, molecular proof of this particular form of mosaicism is lacking. The present review may stimulate readers to inform colleagues of other specialties on this new concept, in order to initiate further research in this particular field of knowledge that has important implications for diagnosis, treatment and genetic counselling.