Heterozygous Embryos Research Articles

The mitochondrial citrate carrier SLC25A1 regulates metabolic reprogramming and morphogenesis in the developing heart

The developing mammalian heart undergoes an important metabolic shift from glycolysis towards mitochondrial oxidation that is critical to support the increasing energetic demands of the maturing heart. Here, we describe a new mechanistic link between mitochondria and cardiac morphogenesis, uncovered by studying mitochondrial citrate carrier (SLC25A1) knockout mice. Slc25a1 null embryos displayed impaired growth, mitochondrial dysfunction and cardiac malformations that recapitulate the congenital heart defects observed in 22q11.2 deletion syndrome, a microdeletion disorder involving the SLC25A1 locus. Importantly, Slc25a1 heterozygous embryos, while overtly indistinguishable from wild type, exhibited an increased frequency of these defects, suggesting Slc25a1 haploinsuffiency and dose-dependent effects. Mechanistically, SLC25A1 may link mitochondria to transcriptional regulation of metabolism through epigenetic control of gene expression to promote metabolic remodeling in the developing heart. Collectively, this work positions SLC25A1 as a novel mitochondrial regulator of cardiac morphogenesis and metabolic maturation, and suggests a role in congenital heart disease.

Prenatal and progressive coenzyme Q10 administration to mitigate muscle dysfunction in mitochondrial disease.

ADCK genesencode aarF domain-containing mitochondrial kinases involved in coenzyme Q (CoQ) biosynthesis and regulation. Haploinsufficiency of ADCK2 in humans leads to adult-onset physical incapacity with reduced mitochondrial CoQ levels in skeletal muscle, resulting in mitochondrial myopathy and alterations in fatty acid β-oxidation. The sole current treatment for CoQ deficiencies is oral administration of CoQ10, which causes only partial recovery with postnatal treatment, underscoring the importance of early diagnosis for successful intervention. We used Adck2 heterozygous mice to examine the influence of this gene on muscle structure, function and regeneration throughout development, growth and ageing. This investigation involved techniques including immunohistochemistry, analysis of CoQ levels, mitochondrial respiratory content, muscle transcriptome analysis and functional tests. We demonstrated that Adck2 heterozygous mice exhibit defects from embryonic development, particularly in skeletal muscle (1102 genes deregulated). Adck2 heterozygous embryos were 7% smaller in size and displayed signs of delayed development. Prenatal administration of CoQ10 could mitigate these embryonic defects. Heterozygous Adck2 mice also showed a decrease in myogenic cell differentiation, with more severe consequences in 'aged' mice (41.63% smaller) (P<0.01). Consequently, heterozygous Adck2 mice displayed accelerated muscle wasting associated with ageing in muscle structure (P<0.05), muscle function (less grip strength capacity) (P<0.001) and muscle mitochondrial respiration (P<0.001). Furthermore, progressive CoQ10 administration conferred protective effects on mitochondrial function (P<0.0001) and skeletal muscle (P<0.05). Our work uncovered novel aspects of CoQ deficiencies, revealing defects during embryonic development in mammals for the first time. Additionally, we identified the gradual establishment and progression of the deleterious Adck2 mouse phenotype. Importantly, CoQ10 supplementation demonstrated a protective effect when initiated during development.

Identification of a pathogenic variant and pre-implantation genetic testing for a Chinese family affected with split-hand/foot malformation.

Split-hand/foot malformation is a serious congenital limb malformation characterized by syndactyly and underdevelopment of the phalanges and metatarsals. In this study, we reported a case of a fetus with hand-foot cleft deformity. Whole exome and Sanger sequencing were used to filter out candidate gene mutation sites and provide pre-implantation genetic testing(PGT) for family members. Genetic testing results showed that there was a homozygous mutation c.786G>A (p.Trp262*) in the fetal WNT10B, and both parents were carriers of heterozygous mutations. PGT results showed that out of the two blastocysts, one was a heterozygous mutant and the other was a homozygous mutant. All the embryos had diploid chromosomes. The heterozygous embryo was transferred, and a singleton pregnancy was successfully achieved. This study suggests that homozygous mutations in WNT10B are the likely cause of hand-foot clefts in this family. For families with monogenic diseases, preimplantation genetic testing can effectively prevent the birth of an affected child only after identifying the pathogenic mutation.

A Novel Use of Embryonic Gut Organoid Culture to Investigate Duodenal Atresia

BackgroundThe cause of duodenal atresia (DA) is not known. Tandler's “solid cord” hypothesis conflicts with current biological evidence. In humans, a genetic aetiology is supported by the association with Trisomy 21. Interruption of Fgf10 is the strongest genetic link to DA in mice, demonstrating an increased incidence and severity as embryos mature. This project aimed to develop an organoid model to facilitate ex vivo DA research on the FGF10/FGFR2b signalling pathway. We hypothesised that DA morphology represents an evolving spectrum of disease and that Fgf10 knockout organoids would vary in growth pattern compared to wild-type. MethodsOrganoids were cultured from the duodenum of E12.5 Fgf10 knockout, heterozygous and wild-type embryos, using an air-liquid interface with Growth Factor reduced Matrigel. Organoids were photographed every 48 h to observe growth. Organoids were isolated and fixed after 14 days, then stained with DAPI, KI-67, and cytokeratin to demonstrate proliferation and differentiation. ResultsWild-type duodenum developed into crypt-forming organoids. Fgf10 heterozygous duodenum failed to progress beyond the development stage of spheroids. Fgf10 knockout duodenum failed to demonstrate any growth. Wholemount staining showed the greatest cell proliferation and differentiation in wild-type tissue. ConclusionThis research presents a novel concept for the growth of embryonic gastrointestinal tissue to inform normal biology. The small sample numbers and restricted culture duration limit longer-term growth analysis. While this model serves as a potential ex vivo setting for future research, that research should consider organoid models with greater standardisation and other gastrointestinal regions. Level of EvidenceAnimal/laboratory study.

Impact of gallbladder hypoplasia on hilar hepatic ducts in biliary atresia

BackgroundBiliary atresia (BA) is an intractable disease of unknown cause that develops in the neonatal period. It causes jaundice and liver damage due to the destruction of extrahepatic biliary tracts,. We have found that heterozygous knockout mice of the SRY related HMG-box 17 (Sox17) gene, a master regulator of stem/progenitor cells in the gallbladder wall, exhibit a condition like BA. However, the precise contribution of hypoplastic gallbladder wall to the pathogenesis of hepatobiliary disease in Sox17 heterozygous embryos and human BA remains unclear.MethodsWe employed cholangiography and histological analyses in the mouse BA model. Furthermore, we conducted a retrospective analysis of human BA.ResultsWe show that gallbladder wall hypoplasia causes abnormal multiple connections between the hilar hepatic bile ducts and the gallbladder-cystic duct in Sox17 heterozygous embryos. These multiple hilar extrahepatic ducts fuse with the developing intrahepatic duct walls and pull them out of the liver parenchyma, resulting in abnormal intrahepatic duct network and severe cholestasis. In human BA with gallbladder wall hypoplasia (i.e., abnormally reduced expression of SOX17), we also identify a strong association between reduced gallbladder width (a morphometric parameter indicating gallbladder wall hypoplasia) and severe liver injury at the time of the Kasai surgery, like the Sox17-mutant mouse model.ConclusionsTogether with the close correlation between gallbladder wall hypoplasia and liver damage in both mouse and human cases, these findings provide an insight into the critical role of SOX17-positive gallbladder walls in establishing functional bile duct networks in the hepatic hilus of neonates.

LncRNA gene ANTSR coordinates complementary sex determination in the Argentine ant.

Animals have evolved various sex determination systems. Here, we describe a newly found mechanism. A long noncoding RNA (lncRNA) transduces complementary sex determination (CSD) signal in the invasive Argentine ant. In this haplodiploid species, we identified a 5-kilobase hyper-polymorphic region underlying CSD: Heterozygous embryos become females, while homozygous and hemizygous embryos become males. Heterozygosity at the CSD locus correlates with higher expression of ANTSR, a gene that overlaps with the CSD locus and specifies an lncRNA transcript. ANTSR knockdown in CSD heterozygotes leads to male development. Comparative analyses indicated that, in Hymenoptera, ANTSR is an ancient yet rapidly evolving gene. This study reveals an lncRNA involved in genetic sex determination, alongside a previously unknown regulatory mechanism underlying sex determination based on complementarity among noncoding alleles.

Optical coherence tomography-guided Brillouin microscopy highlights regional tissue stiffness differences during anterior neural tube closure in the Mthfd1l murine mutant.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

Evaluating neural crest cell migration in a Col4a1 mutant mouse model of ocular anterior segment dysgenesis

The periocular mesenchyme (POM) is a transient migratory embryonic tissue derived from neural crest cells (NCCs) and paraxial mesoderm that gives rise to most of the structures in front of the eye. Morphogenetic defects of these structures can impair aqueous humor outflow, leading to elevated intraocular pressure and glaucoma. Mutations in collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause Gould syndrome – a multisystem disorder often characterized by variable cerebrovascular, ocular, renal, and neuromuscular manifestations. Approximately one-third of individuals with COL4A1 and COL4A2 mutations have ocular anterior segment dysgenesis (ASD), including congenital glaucoma resulting from abnormalities of POM-derived structures. POM differentiation has been a major focus of ASD research, but the underlying cellular mechanisms are still unclear. Moreover, earlier events including NCC migration and survival defects have been implicated in ASD; however, their roles are not as well understood. Vascular defects are among the most common consequences of COL4A1 and COL4A2 mutations and can influence NCC survival and migration. We therefore hypothesized that NCC migration might be impaired by COL4A1 and COL4A2 mutations. In this study, we used 3D confocal microscopy, gross morphology, and quantitative analyses to test NCC migration in Col4a1 mutant mice. We show that homozygous Col4a1 mutant embryos have severe embryonic growth retardation and lethality, and we identified a potential maternal effect on embryo development. Cerebrovascular defects in heterozygous Col4a1 mutant embryos were present as early as E9.0, showing abnormal cerebral vasculature plexus remodeling compared to controls. We detected abnormal NCC migration within the diencephalic stream and the POM in heterozygous Col4a1 mutants whereby mutant NCCs formed smaller diencephalic migratory streams and POMs. In these settings, migratory NCCs within the diencephalic stream and POM localize farther away from the developing vasculature. Our results show for the first time that Col4a1 mutations lead to cranial NCCs migratory defects in the context of early onset defective angiogenesis without affecting cell numbers, possibly impacting the relation between NCCs and the blood vessels during ASD development.

Multifaceted effects on even-skipped transcriptional dynamics upon Krüppel dosage changes.

Although fluctuations in transcription factor (TF) dosage are often well tolerated, TF dosage modulation can change the target gene expression dynamics and result in significant non-lethal developmental phenotypes. Using MS2/MCP-mediated quantitative live imaging in early Drosophila embryos, we analyzed how changing levels of the gap gene Krüppel (Kr) affects transcriptional dynamics of the pair-rule gene even-skipped (eve). Halving the Kr dosage leads to a transient posterior expansion of the eve stripe 2 and an anterior shift of stripe 5. Surprisingly, the most significant changes are observed in eve stripes 3 and 4, the enhancers of which do not contain Kr-binding sites. In Kr heterozygous embryos, both stripes 3 and 4 display narrower widths, anteriorly shifted boundaries and reduced mRNA production levels. We show that Kr dosage indirectly affects stripe 3 and 4 dynamics by modulating other gap gene dynamics. We quantitatively correlate moderate body segment phenotypes of Kr heterozygotes with spatiotemporal changes in eve expression. Our results indicate that nonlinear relationships between TF dosage and phenotypes underlie direct TF-DNA and indirect TF-TF interactions.

The Role of SHROOM3 in Congenital Heart Disease

Background and Hypothesis: Congenital heart defects (CHD) are the most common, and most frequently fatal birth defects, but most etiology remains unknown. We identified a patient with CHD and implicated a gene called SHROOM3. SHROOM3 binds Dishevelled2 which is the central cytoplasmic component of both canonical and noncanonical Wnt/planar cell polarity (PCP) signaling pathways. PCP drives cell movement and is important to embryogenesis and disruption causes CHD. We hypothesize CHD can result from SHROOM3-loss-of-function due to PCP disruption.&#x0D; Project Methods: To interrogate SHROOM3’s role in CHD and PCP we utilized an established in vivo SHROOM3-loss-of-function model, Shroom3 gene trap mice (Shroom3gt). We also utilized a loss-of-function model for PCP membrane component VANGL2, (Vangl2+/-). We assayed genetic interaction between Shroom3 and Vangl2 during cardiac development by crossing singly heterozygous null mice to produce compound heterozygous embryos, harvested embryos, and performed histologic analysis for cardiac defects. We also utilized a human in vitro SHROOM3-loss-of-function model, a CRISPR-Cas9 edited SHROOM3 knockout HELA cell line. We assayed cell movement using a scratch assay.&#x0D; Results: Compound heterozygous Shroom3+/gt;Vangl2+/- embryos had a three fold increase in heart defects compared to singly heterozygous Shroom3+/gt;Vangl2+/+ or Shroom3+/+;Vangl2+/- embryos (3 of 19 or 15.7%, versus 1 of 17 or 5.2%, and 1 of 19 or 4.8%, respectively), demonstrating a trend towards genetic interaction between SHROOM3 and VANGL2/PCP during cardiac development. The scratch assays demonstrated cell movement defects due to SHROOM3-loss-of-function consistent with increased cell movement.&#x0D; Conclusion and Potential Impact: We demonstrate SHROOM3 interacts with Wnt/PCP during cardiac development. Further interrogation of SHROOM3’s role in Wnt signaling will provide insight into the mechanisms by which a novel CHD candidate participates in cardiogenesis and will improve CHD diagnosis, management, and therapeutic development.

Functional analysis of the Drosophila eve locus in response to non-canonical combinations of gap gene expression levels

Transcription factor combinations play a key role in shaping cellular identity. However, the precise relationship between specific combinations and downstream effects remains elusive. Here, we investigate this relationship within the context of the Drosophila eve locus, which is controlled by gap genes. We measure spatiotemporal levels of four gap genes in heterozygous and homozygous gap mutant embryos and correlate them with the striped eve activity pattern. Although changes in gap gene expression extend beyond the manipulated gene, the spatial patterns of Eve expression closely mirror canonical activation levels in wild type. Interestingly, some combinations deviate from the wild-type repertoire but still drive eve activation. Although in homozygous mutants some Eve stripes exhibit partial penetrance, stripes consistently emerge at reproducible positions, even with varying gap gene levels. Our findings suggest a robust molecular canalization of cell fates in gap mutants and provide insights into the regulatory constraints governing multi-enhancer gene loci.

Sf3b4 regulates chromatin remodeler splicing and Hox expression

SF3B proteins form a heptameric complex in the U2 small nuclear ribonucleoprotein, essential for pre-mRNA splicing. Heterozygous pathogenic variants in human SF3B4 are associated with head, face, limb, and vertebrae defects. Using the CRISPR/Cas9 system, we generated mice with constitutive heterozygous deletion of Sf3b4 and showed that mutant embryos have abnormal vertebral development. Vertebrae abnormalities were accompanied by changes in levels and expression pattern of Hox genes in the somites. RNA sequencing analysis of whole embryos and somites of Sf3b4 mutant and control litter mates revealed increased expression of other Sf3b4 genes. However, the mutants exhibited few differentially expressed genes and a large number of transcripts with differential splicing events (DSE), predominantly increased exon skipping and intron retention. Transcripts with increased DSE included several genes involved in chromatin remodeling that are known to regulate Hox expression. Our study confirms that Sf3b4 is required for normal vertebrae development and shows, for the first time, that like Sf3b1, Sf3b4 also regulates Hox expression. We propose that abnormal splicing of chromatin remodelers is primarily responsible for vertebral defects found in Sf3b4 heterozygous mutant embryos.

SOX7 deficiency causes ventricular septal defects through its effects on endocardial-to-mesenchymal transition and the expression of Wnt4 and Bmp2.

SOX7 is a transcription factor-encoding gene located in a region on chromosome 8p23.1 that is recurrently deleted in individuals with ventricular septal defects (VSDs). We have previously shown that Sox7-/- embryos die of heart failure around E11.5. Here, we demonstrate that these embryos have hypocellular endocardial cushions with severely reduced numbers of mesenchymal cells. Ablation of Sox7 in the endocardium also resulted in hypocellular endocardial cushions, and we observed VSDs in rare E15.5 Sox7flox/-;Tie2-Cre and Sox7flox/flox;Tie2-Cre embryos that survived to E15.5. In atrioventricular explant studies, we showed that SOX7 deficiency leads to a severe reduction in endocardial-to-mesenchymal transition (EndMT). RNA-seq studies performed on E9.5 Sox7-/- heart tubes revealed severely reduced Wnt4 transcript levels. Wnt4 is expressed in the endocardium and promotes EndMT by acting in a paracrine manner to increase the expression of Bmp2 in the myocardium. Both WNT4 and BMP2 have been previously implicated in the development of VSDs in individuals with 46,XX sex reversal with dysgenesis of kidney, adrenals and lungs (SERKAL) syndrome and in individuals with short stature, facial dysmorphism and skeletal anomalies with or without cardiac anomalies 1 (SSFSC1) syndrome, respectively. We now show that Sox7 and Wnt4 interact genetically in the development of VSDs through their additive effects on endocardial cushion development with Sox7+/-;Wnt4+/- double heterozygous embryos having hypocellular endocardial cushions and perimembranous and muscular VSDs not seen in their Sox7+/- and Wnt4+/- littermates. These results provide additional evidence that SOX7, WNT4 and BMP2 function in the same pathway during mammalian septal development and that their deficiency can contribute to the development of VSDs in humans.

LACK OF EMBRYONIC HOMOZYGOUS OR ADULT HETEROZYGOUS LYMPHATIC PHENOTYPES FOR A Sos1 MUTATION AND LACK OF LYMPHATIC EMBRYONIC PHENOTYPES FOR A HOMOZYGOUS Cx47 MUTATION IN MICE

We have studied the lymphatic phenotypes of 2 mutations, known to cause abnormalities of lymphatics in humans, in mice. The Cx47 R260C mutation (variably penetrant in humans heterozygous for it and causing limb lymphedema) had an adult mouse phenotype of hyperplasia and increased lymph nodes only in homozygous condition but we did not find any anatomical phenotype in day 16.5 homozygous embryos. Mice harboring the Sos1 mutation E846K (causing Noonan's in man which occasionally shows lymphatic dysplasia) had no adult heterozygous phenotype in lymphatic vessel appearance and drainage (homozygotes are early embryonic lethals) while day 16.5 heterozygous embryos also had no detectable anatomical phenotype.

Primary neurons lacking the SNAREs vti1a and vti1b show altered neuronal development

BackgroundNeurons are highly specialized cells with a complex morphology generated by various membrane trafficking steps. They contain Golgi outposts in dendrites, which are formed from somatic Golgi tubules. In trafficking membrane fusion is mediated by a specific combination of SNARE proteins. A functional SNARE complex contains four different helices, one from each SNARE subfamily (R-, Qa, Qb and Qc). Loss of the two Qb SNAREs vti1a and vti1b from the Golgi apparatus and endosomes leads to death at birth in mice with massive neurodegeneration in peripheral ganglia and defective axon tracts.MethodsHippocampal and cortical neurons were isolated from Vti1a−/−Vti1b−/− double deficient, Vti1a−/−Vti1b+/−, Vti1a+/−Vti1b−/− and Vti1a+/−Vti1b+/− double heterozygous embryos. Neurite outgrowth was determined in cortical neurons and after stimulation with several neurotrophic factors or the Rho-associated protein kinase ROCK inhibitor Y27632, which induces exocytosis of enlargeosomes, in hippocampal neurons. Moreover, postsynaptic densities were isolated from embryonic Vti1a−/−Vti1b−/− and Vti1a+/−Vti1b+/− control forebrains and analyzed by western blotting.ResultsGolgi outposts were present in Vti1a−/−Vti1b+/− and Vti1a+/−Vti1b−/− dendrites of hippocampal neurons but not detected in the absence of vti1a and vti1b. The length of neurites was significantly shorter in double deficient cortical neurons. These defects were not observed in Vti1a−/−Vti1b+/− and Vti1a+/−Vti1b−/− neurons. NGF, BDNF, NT-3, GDNF or Y27632 as stimulator of enlargeosome secretion did not increase the neurite length in double deficient hippocampal neurons. Vti1a−/−Vti1b−/− postsynaptic densities contained similar amounts of scaffold proteins, AMPA receptors and NMDA receptors compared to Vti1a+/−Vti1b+/−, but much more TrkB, which is the receptor for BDNF.ConclusionThe absence of Golgi outposts did not affect the amount of AMPA and NMDA receptors in postsynaptic densities. Even though TrkB was enriched, BDNF was not able to stimulate neurite elongation in Vti1a−/−Vti1b−/− neurons. Vti1a or vti1b function as the missing Qb-SNARE together with VAMP-4 (R-SNARE), syntaxin 16 (Qa-SNARE) and syntaxin 6 (Qc-SNARE) in induced neurite outgrowth. Our data show the importance of vti1a or vti1b for two pathways of neurite elongation.

Abstract LB559: The X-linked gene for the helicase DDX3X is required for lymphoid differentiation and MYC-driven lymphomagenesis

Abstract The X-linked gene DDX3X encodes an RNA helicase and is mutated at high frequencies in several types of human B-cell lymphoma. Females have two active DDX3X alleles and males carry a DDX3Y homolog on the Y chromosome. We show here that pan-hematopoietic, homozygous deletion of Ddx3x in female mice perturbs erythropoiesis causing early developmental arrest. However, both hemizygous male and heterozygous female embryos develop normally, suggesting that one Ddx3x allele is sufficient for fetal hematopoietic development in females and that the Ddx3y allele can compensate for the loss of Ddx3x in males. In adult mice, loss of DDX3X affects hematopoietic progenitors, early lymphoid development, marginal zone, and germinal center B-cells as well as lymphomagenesis driven by an Eμ-Myc or λ-Myc transgene in a sex-dependent manner. A B-cell-specific depletion of both Ddx3x alleles induces a delay in tumorigenesis in female mice while Ddx3x deletion in male mice does not impact tumorigenesis. However, male Eμ-Myc mice lacking Ddx3x on a pan-hematopoietic way remained for the majority lymphoma-free. The few tumors that appeared in these male mice showed upregulated expression of DDX3Y indicating a requirement of DDX3 activity for lymphomagenesis. Our data reveal sex-specific roles of DDX3X in erythro- and lymphopoiesis as well as in MYC-driven lymphomagenesis, which are important when considering inhibition of DDX3 as a treatment of Myc dependent B-cell lymphoma. Citation Format: Marion Lacroix, Hugues Beauchemin, Julie Ross, Jennifer Fraszczak, Peiman Shooshtarizadeh, Riyan Chen, Tarik Moroy. The X-linked gene for the helicase DDX3X is required for lymphoid differentiation and MYC-driven lymphomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB559.

Snrpb is required in murine neural crest cells for proper splicing and craniofacial morphogenesis.

ABSTRACTHeterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for cerebrocostomandibular syndrome (CCMS). We show that Snrpb heterozygous mouse embryos arrest shortly after implantation. Additionally, heterozygous deletion of Snrpb in the developing brain and neural crest cells models craniofacial malformations found in CCMS, and results in death shortly after birth. RNAseq analysis of mutant heads prior to morphological defects revealed increased exon skipping and intron retention in association with increased 5′ splice site strength. We found increased exon skipping in negative regulators of the P53 pathway, along with increased levels of nuclear P53 and P53 target genes. However, removing Trp53 in Snrpb heterozygous mutant neural crest cells did not completely rescue craniofacial development. We also found a small but significant increase in exon skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53 activity and craniofacial-specific genes contributes to craniofacial malformations. This article has an associated First Person interview with the first author of the paper.

The X-linked gene for the helicase DDX3X is required for lymphoid differentiation and MYC-driven lymphomagenesis

Abstract The X-linked gene DDX3X encodes an RNA helicase and is mutated at high frequencies in several types of human B-cell lymphoma. Females have two active DDX3X alleles and males carry a DDX3Y homolog on the Y chromosome. We show here that pan-hematopoietic, homozygous deletion of Ddx3x in female mice perturbs erythropoiesis causing early developmental arrest. However, both hemizygous male and heterozygous female embryos develop normally, suggesting that one Ddx3x allele is sufficient for fetal hematopoietic development in females and that the Ddx3y allele can compensate for the loss of Ddx3x in males. In adult mice, loss of DDX3X affects hematopoietic progenitors, early lymphoid development, marginal zone and germinal center B-cells and lymphomagenesis driven by an Em-Myc transgene in a sex-dependent manner. Loss of one Ddx3x allele slightly accelerated MYC-driven lymphomagenesis in female mice, while male Em-Myc mice lacking Ddx3x remained almost lymphoma-free. However, the few tumors that appeared in male mice showed upregulated expression of DDX3Y indicating a minimal requirement for DDX3 activity for lymphomagenesis. Our data reveal sex specific roles of DDX3X in erythro- and lymphopoiesis as well as in MYC-driven lymphomagenesis, which are important when considering inhibition of DDX3 as a treatment of B-cell lymphoma. Tarik Möröy holds a Canada Research Chair (Tier 1), a Foundation grant from the CIHR (FDN-148372) and support from the Cancer Research Society (CRS). Marion Lacroix is supported by fellowships from the Cole Foundation and the IRCM.

Deletion of Sf3b4 in Neural Crest Cells Encapsulates the Variable Expressivity of Craniofacial Abnormalities Observed in Nager and Rodriguez Syndrome Patients

Recently, both Nager syndrome and Rodriguez syndrome patients were identified to carry rare mutations in SF3B4. Both disorders affect the face as well as the limb including both hands and feet. Nager syndrome patients survive and exhibit smaller jaw bones, smaller cheek bones, cleft palate, hearing problems as well as downward slanted eyelids. However, Rodriguez syndrome is much more severe, and patients often die before or soon after birth. We hypothesized that craniofacial abnormalities associated with reduced SF3B4 levels is due to tissue‐specific expression and requirement of this gene in neural crest cells. To generate a conditional mutant mouse line for Sf3b4, we used CRISPR/Cas9 to insert LoxP sequences in intron 1 and 3 of this gene. To test our hypothesis, we mated Sf3b4conditional mutant mice to Wnt1‐Cre2 transgenic mice to delete exons 2 and 3 specifically in neural crest cells. Heterozygous mutant embryos from these matings were normal. Sf3b4L/L;Wnt1‐Cre2 embryos have brain and craniofacial abnormalities, including abnormal forebrain, midbrain and hindbrain, hypoplasia of the maxillomandibular region and midline cleft. Majority of these embryos die before birth and the few mutants that are born are indistinguishable from their wildtype littermates. Additionally, we generated Sf3b4L/‐;Wnt1‐Cre2 mutants with homozygous deletion of Sf3b4 in neural crest cells and heterozygous deletion of Sf3b4 in the body. These mutant embryos also had unfused frontonasal prominence and unfused and severely hypoplastic mandibular region. Sf3b4L/‐;Wnt1‐Cre2begin to die at E13.5 and none survive to birth. Thus, neural crest specific deletion of Sf3b4 in mice is able to model the craniofacial abnormalities presented in patients as well as capture the variable expressivity observed. Our model can further be used to uncover the splicing and molecular events regulated by SF3B4 during craniofacial development.

Differential Etv2 threshold requirement for endothelial and erythropoietic development.

SUMMARYEndothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.