Genetic determinants of micronucleus formation in vivo.

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2.

The yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins [1]. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins [1]. p18(Ubc9), the Xenopus homolog of Ubc9p, associates specifically with p88(RanGAP1) and p340(RanBP2) [2]. Ran-binding protein 2 (p340(RanBP2)) is a nuclear pore protein [3] [4], and p88(RanGAP1) is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran [2]. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier [5-7], and that SUMO-1 conjugation promotes RanGAP1's interaction with RanBP2 [2,5,6]. Here we show that p18(Ubc9) acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. We also show that p18(Ubc9) interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts.

Embryonic induction and muscle gene activation.

Embryonic induction, a process in which the differentiation of a cell is determined by its proximity to other kinds of cells, is of major importance in animal development. We review here what is known of the steps by which a muscle-specific actin gene is first activated by embryonic induction in early amphibian embryos.

A simplified method of generating transgenic Xenopus.

Currently transgenic frog embryos are generated using restriction-enzyme-mediated integration (REMI) on decondensed sperm nuclei followed by nuclear transplantation into unfertilized eggs. We have developed a simplified version of this protocol that has the potential to increase the numbers of normally developing transgenic embryos.

ASPP2 deficiency causes features of 1q41q42 microdeletion syndrome.

Chromosomal abnormalities are implicated in a substantial number of human developmental syndromes, but for many such disorders little is known about the causative genes. The recently described 1q41q42 microdeletion syndrome is characterized by characteristic dysmorphic features, intellectual disability and brain morphological abnormalities, but the precise genetic basis for these abnormalities remains unknown. Here, our detailed analysis of the genetic abnormalities of 1q41q42 microdeletion cases identified TP53BP2, which encodes apoptosis-stimulating protein of p53 2 (ASPP2), as a candidate gene for brain abnormalities. Consistent with this, Trp53bp2-deficient mice show dilation of lateral ventricles resembling the phenotype of 1q41q42 microdeletion patients. Trp53bp2 deficiency causes 100% neonatal lethality in the C57BL/6 background associated with a high incidence of neural tube defects and a range of developmental abnormalities such as congenital heart defects, coloboma, microphthalmia, urogenital and craniofacial abnormalities. Interestingly, abnormalities show a high degree of overlap with 1q41q42 microdeletion-associated abnormalities. These findings identify TP53BP2 as a strong candidate causative gene for central nervous system (CNS) defects in 1q41q42 microdeletion syndrome, and open new avenues for investigation of the mechanisms underlying CNS abnormalities.

Xenopus Smad3 is specifically expressed in the chordoneural hinge, notochord and in the endocardium of the developing heart.

The Smads are intracellular signalling molecules that transduce signals from receptors for members of the TGF-beta superfamily to the nucleus. We have cloned the Xenopus orthologue of Smad3 (XSmad3). It is 94.6% identical to human Smad3 at the amino acid level. It is expressed as a maternal mRNA which disappears after stage 10.5, but reappears at the early tailbud stages. It is much less abundant than XSmad2 at the early developmental stages. From Stage 27 onwards XSmad3 is expressed with XSmad2 throughout the head region and in the somitic region. Strikingly however, XSmad3 alone is specifically expressed in the chordoneural hinge, the notochord and in the developing heart. Closer analysis reveals that XSmad3 is specifically expressed in the endocardium but not in the myocardium or pericardium. The chordoneural hinge staining persists at least until stage 40 whereas the staining in the endocardium peaks at approximately stage 32/33.

Xenopus eHAND: a marker for the developing cardiovascular system of the embryo that is regulated by bone morphogenetic proteins.

The bHLH protein eHAND is a sensitive marker for cardiovascular precursors in the Xenopus embryo. The earliest site of expression is a broad domain within the lateral plate mesoderm of the tailbud embryo. This domain comprises precursors that contribute to the posterior cardinal veins in later stages. Surprisingly, expression is profoundly asymmetric at this stage and is random with respect to embryo side. XeHAND is also expressed in an anterior domain that encompasses the prospective heart region. Within the myocardium and pericardium, transcripts are also asymmetrically distributed, but in these tissues they are localised in a left-sided manner. Later in development XeHAND transcripts are largely restricted to the ventral aorta, aortic arches and venous inflow tract (sinus venosus) which flank the heart itself, but no expression is detected in neural crest derivatives at any stage. This demonstrates that patterns of XeHAND expression differ markedly amongst vertebrates and that in Xenopus, XeHAND expression identifies all of the earliest formed elements of the cardiovascular system. In animal cap explants, expression of XeHAND (but not other markers of cardiogenic differentiation) is strongly induced by ectopic expression of the TGFbeta family members, BMP-2 and BMP-4, but this can be blocked by coexpression of a dominant negative BMP receptor. This suggests that XeHAND expression in the embryo is regulated by the ventralising signals of bone morphogenetic proteins. High levels of expression are also detected in explants treated with high doses of activin A which induces cardiac muscle differentiation. No such effect is seen with lower doses of activin, indicating that a second pathway may regulate the XeHAND gene during cardiogenesis.

Technical note: A comparison of DNA collection methods in cattle and yaks.

A variety of biological materials are suitable for the analysis of bovine DNA. The objective of this study was to evaluate the ease of collection, storage, and cost as well as quality and quantity of DNA samples obtained from Bos taurus (European cattle) and Bos grunniens (yak) using 2 different sample types: whole blood sampling and nasal swabs. Hair follicle DNA samples from yaks were also analyzed. Deoxyribonucleic acid samples were collected from 1 herd of Black Angus yearling bulls (n = 166) and 1 herd of yaks (n = 24). A NanoDrop Bioanalyzer ND1000 was used to quantify DNA. To assess DNA purity, absorbance ratios were determined at wavelengths of 260 nm relative to 280 nm and 260 nm relative to 230 nm. Single nucleotide polymorphism genotyping was performed using a competitive allele-specific PCR (KASP) genotyping system and the call rates to 3 specific SNP were compared. Using a commercially available nonautomated ethanol DNA extraction technique, nasal swabs yielded a greater quantity of DNA than blood (P < 0.0001) and a greater quality DNA sample than blood (P < 0.0001). Blood and nasal swab performance in SNP genotyping assays were similar (P = 0.5). The greater expense of nasal swabs was offset by their ease of use: less time, skill, and equipment was needed to obtain a sample and the storage of samples was more convenient (room temperature). In yaks, accessing the coccygeal vein, which is relatively straightforward in cattle, was difficult. Nasal swabbing and hair follicle sampling in yaks was performed relatively easily. Yak hair follicles were a poor source of DNA. In conclusion, DNA collection using nasal swabs was more convenient and provided a greater quantity of DNA and better quality sample than blood collection in both Angus and yak. Notably, yak hair was a poor source of DNA, and yak blood was difficult to obtain.

An amphibian cytoskeletal-type actin gene is expressed exclusively in muscle tissue.

The complete nucleotide sequence of two Xenopus actin genes encoding cytoskeletal protein isoforms has been determined. Transcripts from these genes are remarkably similar in nucleotide sequence throughout their length and code for type-5 and type-8 cytoskeletal actins. Both share some sequence homology with human gamma-actin mRNA within the 3' untranslated region but none with the equivalent region of any vertebrate beta-actin transcript. The promoter regions of the two Xenopus genes are virtually identical from the cap site to the CCAAT box and show extensive homology further upstream. Despite such similarity, the two genes are divergently expressed during embryonic development. The type-5 actin gene is expressed in all regions of the developing embryo whilst the type-8 gene is coregulated with the muscle-specific skeletal actin gene. In common with mammalian and avian cytoskeletal actin counterparts, the Xenopus genes possess a conserved sequence within their promoter that has previously been identified as a transcription-factor-binding site.

Temporal and tissue-specific expression of the proto-oncogene c-fos during development in Xenopus laevis.

We describe the isolation and complete sequence of the Xenopus c-fos proto-oncogene. c-fos expression throughout Xenopus development was analysed using a homologous probe derived from the cloned gene. c-fos RNA is accumulated during oogenesis to reach a plateau of 2 x 10(5) transcripts per stage VI oocyte, suggesting an unusual stability of the c-fos message. The amount of RNA per embryo decreases substantially after fertilisation to reach a level corresponding to less than 0.1 molecule per cell at the tailbud stage. Subsequently, at the swimming tadpole stage, the amount of c-fos mRNA increases; an increase that is correlated with the start of skeleton formation. In the newly metamorphosed froglet, c-fos mRNA shows a marked tissue-specific distribution, with the highest level in intestine and lowest in gall bladder, lung and spleen. We also demonstrate that the Xenopus c-fos gene is serum-inducible in Xenopus cultured cells, a property attributable to a promoter sequence known as the Serum Response Element (SRE). A protein activity (indistinguishable from Serum Response Factor) in both whole cell and nuclear Xenopus embryo extracts binds specifically to the SRE and is present at an approximately constant level throughout early development. Our results suggest roles for c-fos in aspects of both the rapid cell proliferation and cell differentiation characteristic of early Xenopus development.

Upstream sequences required for tissue-specific activation of the cardiac actin gene in Xenopus laevis embryos.

The entire DNA sequence of the Xenopus laevis cardiac actin gene was determined. A recombinant plasmid comprising the cardiac actin gene promoter fused to the bacterial chloramphenicol acetyl transferase (CAT) gene is correctly regulated after introduction into fertilized Xenopus eggs. The fusion gene shows a temporal and tissue-specific pattern of expression in the early embryo which is indistinguishable from that of the endogenous cardiac actin gene. The fusion gene is also activated in cultured embryo fragments that are induced by cell interactions to form embryonic muscle tissue. Tissue-specific expression of the recombinant requires sequences between 217 and 416 nucleotides upstream from the transcription initiation site. In contrast, both the chimaeric gene and the entire cardiac actin gene are expressed at a basal level after microinjection into Xenopus oocytes, requiring only the presence of a TATA box upstream from the cap site.

Region-specific regulation of the actin multi-gene family in early amphibian embryos.

The actin multi-gene family shows both spatial and temporal regulation during early embryogenesis in the amphibian Xenopus laevis. Both muscle-specific and ubiquitous cytoskeletal actin genes are activated at the end of gastrulation; transcription of the alpha-cardiac and alpha-skeletal actin genes is restricted to the somitic mesoderm and its muscle-forming derivatives providing a convenient molecular marker for this early embryonic tissue.

A role for BMP signalling in heart looping morphogenesis in Xenopus.

The heart develops from a linear tubular precursor, which loops to the right and undergoes terminal differentiation to form the multichambered heart. Heart looping is the earliest manifestation of left-right asymmetry and determines the eventual heart situs. The signalling processes that impart laterality to the unlooped heart tube and thus allow the developing organ to interpret the left-right axis of the embryo are poorly understood. Recent experiments in zebrafish led to the suggestion that bone morphogenetic protein 4 (BMP4) may impart laterality to the developing heart tube. Here we show that in Xenopus, as in zebrafish, BMP4 is expressed predominantly on the left of the linear heart tube. Furthermore we demonstrate that ectopic expression of Xenopus nodal-related protein 1 (Xnr1) RNA affects BMP4 expression in the heart, linking asymmetric BMP4 expression to the left-right axis. We show that transgenic embryos overexpressing BMP4 bilaterally in the heart tube tend towards a randomisation of heart situs in an otherwise intact left-right axis. Additionally, inhibition of BMP signalling by expressing noggin or a truncated, dominant negative BMP receptor prevents heart looping but allows the initial events of chamber specification and anteroposterior morphogenesis to occur. Thus in Xenopus asymmetric BMP4 expression links heart development to the left-right axis, by being both controlled by Xnr1 expression and necessary for heart looping morphogenesis.

Vimentin expression in oocytes, eggs and early embryos of Xenopus laevis.

Immunocytochemical studies using a monoclonal anti-porcine vimentin antibody reveal a well-organized pattern of staining in Xenopus laevis oocytes, eggs and early embryos. The positions of Xenopus vimentin and desmin in two-dimensional (2D) polyacrylamide gels were first established by immunoblotting of muscle Triton extracts with anti-intermediate filament antibodies (anti-IFA), which cross-react with all intermediate filament proteins (IFPs). The anti-porcine vimentin reacts with vimentin and desmin in muscle 2D immunoblots, but only reacts with one polypeptide in oocyte blots in the position predicted for vimentin (Mr 55 x 10(3), pI 5.6). Using an anti-sense probe derived from a Xenopus vimentin genomic clone in RNase protection assays, we show that expression of vimentin begins in previtellogenic oocytes. The level of expression remains constant throughout oogenesis and in unfertilized eggs. These data suggest that vimentin is expressed in oocytes and eggs. Most interestingly, the immunocytochemical results also show that vimentin is present in the germ plasma of oocytes, eggs and early embryos. It is therefore possible that vimentin has an important role in the formation or behaviour of early germ line cells.

Transcription of muscle-specific actin genes in early Xenopus development: nuclear transplantation and cell dissociation.

Cloned cDNA probes that recognize muscle-specific alpha-actin gene transcripts have been used to analyze two kinds of experimental embryos in Xenopus. In one, genetically marked nuclei of larval muscle cells were transplanted to wild-type enucleated eggs; alpha-actin genes became transcriptionally inactive in the resulting blastulae but were reactivated when these embryos reached the normal stage of alpha-actin expression (late gastrula). In the other, blastula embryos reared from fertilized eggs were separated into animal, vegetal, and equatorial regions, and their cells dissociated and reaggregated. alpha-Actin RNA was synthesized at the normal time in development, but only by equatorial cells. We conclude that alpha-actin gene transcription is normally regulated in nuclear-transplant embryos and is undisturbed by the absence of cell contacts during cleavage.

Cell type-specific activation of actin genes in the early amphibian embryo.

Muscle actin genes are the earliest yet described to show cell type-specific activation in amphibian embryos. Gene-specific probes show that alpha-skeletal and alpha-cardiac actin genes start to be transcribed simultaneously at the end of gastrulation, but only in those regions of the mesoderm that subsequently form embryonic muscle. Their expression provides a molecular marker for early cell determination.

Xom: a Xenopus homeobox gene that mediates the early effects of BMP-4.

Bone morphogenetic protein-4 (BMP-4) is thought to play an important role in early Xenopus development by acting as a "ventralizing factor' and as an epidermal determinant: local inhibition of BMP-4 function in whole embryos causes the formation of an additional dorsal axis, and inhibition of BMP-4 function in isolated ectodermal cells causes the formation of neural tissue. In this paper we describe a homeobox-containing gene whose expression pattern is similar to that of BMP-4, whose expression requires BMP-4 signalling and which, when over-expressed, causes a phenotype similar to that caused by over-expression of BMP-4. We suggest that this gene, which we call Xom, acts downstream of BMP-4 to mediate its effects.

The secretion of proteins in vitro from Xenopus oocytes and their accessory cells: a biochemical and morphological study.

Protein secretion by Xenopus laevis oocytes and their surrounding follicular cells in vitro has been investigated using two-dimensional gel electrophoresis. Viable oocytes, devoid of follicle layers, were prepared by treatment with collagenase; they retain in full their capacity to synthesize, sequester and export secretory proteins following microinjection with heterologous messenger RNA. Both RNA-injected and normal cells export a large number of endogenous oocyte proteins and, as with heterologous secretory translation products, these proteins are found within the oocyte in a vesicle fraction. Electron microscopy indicates that secretion involves exocytotic release of cortical vesicle contents. The follicular cells themselves also seem to contribute a number of proteins to the incubation medium surrounding isolated oocytes, but the presence of follicle layers is not required for the export of endogenous oocyte proteins.

Actin genes in Xenopus and their developmental control.

The results summarized here have established the temporal and regional activation of three kinds of Xenopus actin genes. The cardiac and skeletal muscle actin genes are among the first cell-type-specific genes to be expressed in early development. The first transcripts to be synthesized by these genes appear to be correctly initiated, spliced, and at once translated into proteins. Both cardiac and skeletal actin genes are strongly transcribed in the axial skeletal muscle of embryos. The mechanism by which the cardiac actin gene is first transcribed in only the somite region of an embryo depends, at least in part, on materials already localized in the subequatorial region of a fertilized but uncleaved egg. Cells which acquire this material seem able to activate their cardiac actin genes without requiring normal contact with other cells.