Search

The retinoblastoma tumor suppressor protein (RB) plays a critical role in cell proliferation and differentiation and its inactivation is a frequent underlying factor in tumorigenesis. While the regulation of RB function by phosphorylation is well studied, proteasome-mediated RB protein degradation is emerging as an important regulatory mechanism. Although our understanding of RB turnover is currently limited, there is evidence that the nuclear lamina filament protein Lamin A/C protects RB from proteasomal degradation. Here we show that SUMO1 conjugation of RB and Lamin A/C is modulated by the SUMO protease SENP1 and that sumoylation of both proteins is required for their interaction. Importantly, this SUMO1-dependent complex protects both RB and Lamin A/C from proteasomal turnover.

Background: Pluripotent embryonic stem cells (ESCs) offer great potential for regenerative medicine. However, efficient in vitro generation of specific desired cell types is still a challenge. We previously established that Smad2/3 signaling, essential for endoderm formation, regulates target gene expression by counteracting epigenetic repression mediated by Polycomb Repressive Complex 2 (PRC2). Although this mechanism has been demonstrated during differentiation and reprogramming, little is known of its role in pluripotent cells., Results: Chromatin immunoprecipitation-deep sequencing of undifferentiated mouse ESCs inhibited for Smad2/3 signaling identified Prdm14, important for protecting pluripotency, as a target gene. Although Prdm14 accumulates the normally repressive PRC2 deposited histone modification H3K27me3 under these conditions, surprisingly, expression increases. Analysis indicates that increased H3K27me3 leads to increased binding of PRC2 accessory component Jarid2 and recruitment of RNA polymerase II. Similar increases were found at the Nodal endoderm target gene Eomes but it remained unexpressed in pluripotent cells as normal. Upon differentiation, however, Eomes expression was significantly higher than in cells that had not been inhibited for signaling before differentiation. In addition, endoderm formation was markedly increased., Conclusions: Blocking Smad2/3 signaling in pluripotent stem cells results in epigenetic changes that enhance the capacity for endoderm differentiation. Developmental Dynamics 245:807-815, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

In the early mouse embryo, a specialized population of extraembryonic visceral endoderm (VE) cells called the distal VE (DVE) arises at the tip of the egg cylinder stage embryo and then asymmetrically migrates to the prospective anterior, recruiting additional distal cells. Upon migration these cells, called the anterior VE (AVE), establish the anterior posterior (AP) axis by restricting gastrulation-inducing signals to the opposite pole. The Nodal-signaling pathway has been shown to have a critical role in the generation and migration of the DVE/AVE. The Nodal gene is expressed in both the VE and in the pluripotent epiblast, which gives rise to the germ layers. Previous findings have provided conflicting evidence as to the relative importance of Nodal signaling from the epiblast vs. VE for AP patterning. Here we show that conditional mutagenesis of the Nodal gene specifically within the VE leads to reduced Nodal expression levels in the epiblast and incomplete or failed DVE/AVE migration. These results support a required role for VE Nodal to maintain normal levels of expression in the epiblast, and suggest signaling from both VE and epiblast is important for DVE/AVE migration., (Published by Elsevier Inc.)

Current methods to isolate rare (1:10,000-1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamless BAC mutagenesis, selectable markers need to be removed after isolation of recombinants through counterselection. Here we illustrate founder principle-driven enrichment (FPE), a simple method to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof of principle, we isolated 1:100,000 seamless fluorescent protein-modified Nodal BACs and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1 phage-derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated with <40 PCRs, and we developed a web-based calculator to optimize FPE.

Background: Human PAX-Interacting Protein 1 (PAXIP1)-associated glutamate rich protein 1 (PAGR1, also known as PA1) originally was discovered as part of a complex containing PAXIP1 and histone H3K4 methyltransferases MLL3 and MLL4, suggesting a role in epigenetic gene regulation. Further in vitro studies suggested additional functions in DNA damage repair and transcription. However, in vivo analysis of PAGR1 function has been lacking., Results: Here we show that expression of the cognate mouse gene Pagr1a is found predominately in the extraembryonic and chorionic ectoderm from pregastrulation stages and is up-regulated within the embryo proper after gastrulation. Embryos with a germ line deletion of Pagr1a establish the anterior-posterior axis, and show normal neuroectodermal, mesodermal, and endodermal patterning, but fail to develop beyond the four- to five-somite stage or to undergo axial rotation. Pagr1a(-) (/) (-) embryos also show abnormal development of extraembryonic tissues with defects seen in the amnion, chorion and visceral yolk sac. At the molecular level, Pagr1a(-) (/) (-) embryos have reduced expression of BMP2, a known regulator of extraembryonic development., Conclusions: Loss of mouse Pagr1a function leads to defective extraembryonic development, likely due at least in part to altered BMP signaling, contributing to developmental arrest., (© 2014 Wiley Periodicals, Inc.)

Integration of extrinsic signals, epigenetic regulators, and intrinsic transcription factors establishes pluripotent stem cell identity. Interplay between these components also underlies the capacity of stem cells to undergo differentiation, and of differentiated cells to re-establish the pluripotent state in direct reprogramming. Polycomb repressive complexes are epigenetic regulators that play key roles in stem cell identity and in differentiated cell fates. Smad2 and Smad3 (Smad2/3), the intracellular mediators of the Nodal/Activin/transforming growth factor (TGF) β cell-cell signaling pathway also are implicated in stem cell pluripotency and in differentiation. Here, we show that Polycomb imposes responses to Smad2/3-mediated signaling to selectively regulate expression of the master pluripotency factor Oct 4 during initiation of differentiation, but not in the self-renewing pluripotent ground state. During reprogramming back to the ground state, we find that the enhancement of reprogramming efficiency stemming from blocking Nodal/Activin/TGFβ signaling also depends on Polycomb. These context-dependent responses to Smad2/3 imposed by Polycomb action provide a mechanism for selective gene regulation that can reconcile the apparently conflicting roles of this signaling pathway in pluripotency, differentiation, and reprogramming., (Copyright © 2013 AlphaMed Press.)

Posttranslational modification with small ubiquitin-like modifier (Sumo) regulates numerous cellular and developmental processes. Sumoylation is dynamic with deconjugation by Sumo-specific proteases (Senps) regulating steady-state levels. Different Senps are found in distinct subcellular domains, which may limit their deconjugation activity to colocalizing Sumo-modified proteins. In vitro, Senps can discriminate between the different Sumo paralogs: Sumo1 versus the highly related Sumo2 and Sumo3 (Sumo2/3), which can form poly-Sumo chains. However, a full understanding of Senp specificity in vivo is still lacking. Here, using biochemical and genetic approaches, we establish that Senp1 has an essential, nonredundant function to desumoylate Sumo1-modified proteins during mouse embryonic development. Senp1 specificity for Sumo1 conjugates represents an intrinsic function and not simply a product of colocalization. In contrast, Senp1 has only a limited role in Sumo2/3 desumoylation, although it may regulate Sumo1-mediated termination of poly-Sumo2/3 chains., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Sonic hedgehog (Shh) signal, mediated by the Gli family of transcription factors, plays an essential role in the growth and patterning of the limb. Through analysis of the early limb bud transcriptome, we identified a posteriorly-enriched gene, Hyaluronic Acid Synthase 2 (Has2), which encodes a key enzyme for the synthesis of hyaluronan (HA), as a direct target of Gli transcriptional regulation during early mouse limb development. Has2 expression in the limb bud is lost in Shh null and expanded anteriorly in Gli3 mutants. We identified an ∼3kb Has2 promoter fragment that contains two strong Gli-binding consensus sequences, and mutation of either site abrogated the ability of Gli1 to activate Has2 promoter in a cell-based assay. Additionally, this promoter fragment is sufficient to direct expression of a reporter gene in the posterior limb mesenchyme. Chromatin immunoprecipitation of DNA-Gli3 protein complexes from limb buds indicated that Gli3 strongly binds to the Has2 promoter region, suggesting that Has2 is a direct transcriptional target of the Shh signaling pathway. We also showed that Has2 conditional mutant (Has2cko) hindlimbs display digit-specific patterning defects with longitudinally shifted phalangeal joints and impaired chondrogenesis. Has2cko limbs show less capacity for mesenchymal condensation with mislocalized distributions of chondroitin sulfate proteoglycans (CSPGs), aggrecan and link protein. Has2cko limb phenotype displays striking resemblance to mutants with defective chondroitin sulfation suggesting tight developmental control of HA on CSPG function. Together, our study identifies Has2 as a novel downstream target of Shh signaling required for joint patterning and chondrogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures., (Copyright © 2011 Wiley Periodicals, Inc.)

Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

Both intercellular signaling and epigenetic mechanisms regulate embryonic development, but it is unclear how they are integrated to establish and maintain lineage-specific gene expression programs. Here, we show that a key function of the developmentally essential Nodal-Smads2/3 (Smad2 and Smad3) signaling pathway is to recruit the histone demethylase Jmjd3 to target genes, thereby counteracting repression by Polycomb. Smads2/3 bound to Jmjd3 and recruited it to chromatin in a manner that was dependent on active Nodal signaling. Knockdown of Jmjd3 alone substantially reduced Nodal target gene expression, whereas in the absence of Polycomb, target loci were expressed independently of Nodal signaling. These data establish a role for Polycomb in imposing a dependency on Nodal signaling for the expression of target genes and reveal how developmental signaling integrates with epigenetic processes to control gene expression.

A number of proteins can be conjugated with both ubiquitin and the small ubiquitin-related modifier (SUMO), with crosstalk between these two post-translational modifications serving to regulate protein function and stability. We previously identified N4BP1 as a substrate for monoubiquitylation by the E3 ubiquitin ligase Nedd4. Here, we describe Nedd4-mediated polyubiquitylation and proteasomal degradation of N4BP1. In addition, we show that N4BP1 can be conjugated with SUMO1 and that this abrogates N4BP1 ubiquitylation. Consistent with this, endogenous N4BP1 is stabilized in primary embryonic fibroblasts from mutants of the desumoylating enzyme SENP1, which show increased steady-state sumoylation levels. We have localized endogenous N4BP1 predominantly to the nucleolus in primary cells. However, a small fraction is found at promyelocytic leukemia (PML) nuclear bodies (NBs). In cells deficient for SENP1 or in wild-type cells treated with the proteasome inhibitor MG132, there is considerable accumulation of N4BP1 at PML NBs. These findings suggest a dynamic interaction between subnuclear compartments, and a role for post-translational modification by ubiquitin and SUMO in the regulation of nucleolar protein turnover.

The role of axial structures, especially the notochord, in metanephric kidney development has not been directly examined. Here, we showed that disruption of the notochord and floor plate by diphtheria toxin (DTA)-mediated cell ablation did not disrupt nephrogenesis, but resulted in kidney fusions, resembling horseshoe kidneys in humans. Axial disruptions led to more medially positioned metanephric mesenchyme (MM) in midgestation. However, neither axial disruption nor the ensuing positional shift of the MM affected the formation of nephrons and other structures within the kidney. Response to Shh signaling was greatly reduced in midline cell populations in the mutants. To further ascertain the molecular mechanism underlying these abnormalities, we specifically inactivated Shh in the notochord and floor plate. We found that depleting the axial source of Shh was sufficient to cause kidney fusion, even in the presence of the notochord. These results suggested that the notochord is dispensable for nephrogenesis but required for the correct positioning of the metanephric kidney. Axial Shh signal appears to be critical in conferring the effects of axial structures on kidney positioning along the mediolateral axis. These studies also provide insights into the pathogenesis of horseshoe kidneys and how congenital kidney defects can be caused by signals outside the renal primordia., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

MouseIndelDB is an integrated database resource containing thousands of previously unreported mouse genomic indel (insertion and deletion) polymorphisms ranging from approximately 100 nt to 10 Kb in size. The database currently includes polymorphisms identified from our alignment of 26 million whole-genome shotgun sequence traces from four laboratory mouse strains mapped against the reference C57BL/6J genome using GMAP. They can be queried on a local level by chromosomal coordinates, nearby gene names or other genomic feature identifiers, or in bulk format using categories including mouse strain(s), class of polymorphism(s) and chromosome number. The results of such queries are presented either as a custom track on the UCSC mouse genome browser or in tabular format. We anticipate that the MouseIndelDB database will be widely useful for research in mammalian genetics, genomics, and evolutionary biology. Access to the MouseIndelDB database is freely available at: http://variation.osu.edu/.

We report that NSBP1, a nucleosome binding protein that affects the structure of chromatin, is highly expressed in mouse placenta. In Rcho-1 cells, which recapitulate the differentiation of trophoblast giant cells of living placenta, NSBP1 expression is linked to differentiation. Disregulation of NSBP1 protein levels, by either siRNA treatment or by overexpression, alters the expression of several members of the prolactin gene family without affecting the levels of several transcription factors involved in placental differentiation. Our studies identify NSBP1 as a nucleosome binding protein that modulates the expression of prolactin gene family members most likely by inducing changes in chromatin structure.

Conjugation of the small ubiquitin-like modifier (SUMO) to target proteins regulates numerous biological processes and has been implicated in tumorigenesis and metastasis. The three SUMO isoforms in vertebrates, SUMO1 and the highly similar SUMO2 and SUMO3, can be conjugated to unique as well as overlapping subsets of target proteins. Yet, it is still not clear whether roles for each family member are distinct or whether redundancy exists. Here we describe a mutant mouse line that completely lacks SUMO1, but surprisingly is viable and lacks any overt phenotype. Our study points to compensatory utilization of SUMO2 and/or SUMO3 for sumoylation of SUMO1 targets. The ability of SUMO isoforms to substitute for one another has important implications for rational targeting of the SUMO pathway.

Nodal signaling is a critical regulator of multiple aspects of early vertebrate development including asymmetry along the left/right (LR) axis. To study Nodal function occurring specifically in the postgastrulation embryo, we have used Cre/loxP based conditional mutagenesis. A floxed allele of Nodal was generated and shown to have wild-type function. This allele was then used in conjunction with the T-Cre line, which expresses Cre recombinase broadly in the mesodermal and definitive endodermal lineages posterior to the cranial region. T-Cre activity leads to complete deletion of Nodal before its normal transient expression in the early somite stage lateral plate mesoderm, thereby causing severe LR developmental defects. No other abnormalities were found, suggesting that Nodal signaling has no additional essential functions in developmental patterning within the extensive mesodermal and endodermal domains marked by T-Cre activity.

We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/-) embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo., Competing Interests: The authors have declared that no competing interests exist.

During development, the organizer provides instructive signals to surrounding cells as well as contributing cells to axial structures. To dissect organizer function at different developmental stages, conditional approaches such as the Cre/loxP system for conditional mutagenesis are particularly useful. Here we describe two new Cre transgenic mouse lines, Foxa2 NFP-Cre and Nodal PNC-Cre, with activity in two organizer domains, the posterior notochord (PNC) and notochord. These lines were made using defined regulatory elements from the Foxa2 and Nodal genes that direct Cre expression in overlapping domains of the PNC and notochord. Our detailed analysis of the timing and location of Foxa2 NFP-Cre and Nodal PNC-Cre activity indicates that these lines are appropriate for conditional mutagenesis of genes expressed from early somite stages onward.

Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin-protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73 alpha, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73 alpha for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73 alpha and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1(-/-) cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73 alpha and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy.

In this issue of Developmental Cell, Nacerddine et al. (2005) describe a targeted germ line mutation of the mouse SUMO-specific E2 Ubc9, which abrogates the SUMO conjugation pathway, broadly crippling nuclear function in proliferating cells of the early embryo. This study reveals the wide-ranging roles for this protein modifier in nuclear organization, chromosome segregation, and Ran-driven nucleo-cytoplasmic transport.

Fibroblast growth factors (FGFs) and their receptors have been implicated in limb development. However, because of early post-implantation lethality associated with fibroblast growth factor receptor 1 (FGFR1) deficiency, the role of this receptor in limb development remains elusive. To overcome embryonic lethality, we have performed a conditional knockout of Fgfr1 using the Cre-LoxP approach. We show that Cre-mediated deletion of Fgfr1 in limb mesenchyme, beginning at a time point slightly after the first sign of initial budding, primarily affects formation of the first one or two digits. In contrast, deletion of Fgfr1 at an earlier stage, prior to thickening of limb mesenchyme, results in more severe defects, characterized by malformation of the AER, diminished Shh expression and the absence of the majority of the autopod skeletal elements. We show that FGFR1 deficiency does not affect cell proliferation. Instead, it triggers cell death and leads to alterations in expression of a number of genes involved in apoptosis and digit patterning, including increased expression of Bmp4, Dkk1 and Alx4, and downregulation of MKP3. These data demonstrate that FGF/FGFR1 signals play indispensable roles in the early stages of limb initiation, eliciting a profound effect on the later stages of limb development, including cell survival, autopod formation and digit patterning.

The covalent modification of proteins by the small ubiquitin-like protein SUMO has been implicated in the regulation of numerous biological processes, including nucleocytoplasmic transport, genomic stability, and gene transcription. Sumoylation occurs by a multienzyme process similar to ubiquitination and, in Saccharomyces cerevisiae, is reversed by desumoylating enzymes encoded by the Ulp1 and Smt4/Ulp2 genes. The physiological importance of desumoylation has been revealed by mutations in either gene, which lead to nonoverlapping defects in cell cycle transition and meiosis. Several mammalian Ulp homologues have been identified, but, to date, nothing is known of the phenotypic effects of their loss of function. Here, we describe a random retroviral insertional mutation of one homolog, mouse SENP1/SuPr-2. The mutation causes increased steady-state levels of the sumoylated forms of a number of proteins and results in placental abnormalities incompatible with embryonic development. Our findings provide the first insight into the critical importance of regulating sumoylation in mammals.

We have identified novel transcriptional isoforms of the human and mouse genes encoding muscle type phosphofructokinase-1 (PFK-M). These isoforms are expressed specifically in the testis and in the mid-gestation embryo, and have been termed TE-PFK-M (testis- and embryo-specific PFK-M). The 5'UTR of TE-PFK-M is composed of three newly identified exons that lie much farther upstream of the PFK-M coding region than the previously characterized 5'UTR. In addition, this upstream region encodes a series of small polyadenylated transcripts, some of which share the same exons found in the 5'UTR of TE-PFK-M, and which may play some role in regulating TE-PFK-M expression. These findings indicate an even more complex level of control of PFK-M expression than previously thought.

Nedd4 is a HECT domain-containing ubiquitin ligase that mediates ubiquitylation and proteasome degradation of target proteins. The molecular basis for the interaction of Nedd4 with substrates lies in its WW domains, which can bind proline-rich (PY) domains in target proteins. Nedd4 is a developmentally expressed protein and may have a fundamental role to play in embryonic processes. However, whether Nedd4 has such a function is currently unknown, in part because few developmentally regulated ubiquitylation substrates have been identified or characterized. We have carried out a yeast two-hybrid screen and identified four proteins expressed in the mid-gestation embryo that are able to interact with Nedd4. Characterization of their functional interaction with Nedd4 in vitro and in vivo demonstrated that three of the four are bona fide Nedd4 binding partners, and two have the capacity to be ubiquitylation substrates. One of these is the first identified nonviral substrate for Nedd4-mediated monoubiquitylation. Interestingly, neither of these two ubiquitylated proteins interacts with Nedd4 through PY-mediated mechanisms. For one of the three Nedd4 binding partners, there was no discernable evidence of ubiquitylation. However, this protein clearly associates with Nedd4 through its PY domains and can alter the location of Nedd4 in cells, suggesting a role other than as a ubiquitylation substrate.

Nodal has been thought to be an embryo-specific factor that regulates development, but nodal is also expressed in the mouse placenta beginning at midgestation, specifically in the spongiotrophoblasts. In an insertional null nodal mutant, not only is embryonic development disrupted, but mouse placental development is also grossly altered with the loss of the diploid spongiotrophoblasts and labyrinth and an expansion of the polyploid giant cell layer. A hypomorphic mutation in nodal results in an expansion of the giant cell and spongiotrophoblast layers, and a decrease in labyrinthine development. Expression of nodal in trophoblast cell cultures is sufficient to inhibit trophoblast giant cell differentiation, demonstrating that nodal can act directly on trophoblasts. The mechanism of nodal action includes the inhibition of junB gene transcription. These results suggest that nodal may be involved in redirecting trophoblast fate towards the midgestational expansion of the labyrinth region while maintaining the thin layer of trophoblast giant cells and the underlying layer of spongiotrophoblasts that form the boundary between the maternal and extraembryonic compartments., (Copyright 2001 Academic Press.)

Loss-of-function analysis has shown that the transforming growth factor-like signaling molecule nodal is essential for mouse mesoderm development. However, definitive proof of nodal function in other developmental processes in the mouse embryo has been lacking because the null mutation blocks gastrulation. We describe the generation and analysis of a hypomorphic nodal allele. Mouse embryos heterozygous for the hypomorphic allele and a null allele undergo gastrulation but then display abnormalities that fall into three distinct mutant phenotypic classes, which may result from expression levels falling below critical thresholds in one or more domains of nodal expression. Our analysis of each of these classes provides conclusive evidence for nodal-mediated regulation of several developmental processes in the mouse embryo, beyond its role in mesoderm formation. We find that nodal signaling is required for correct positioning of the anteroposterior axis, normal anterior and midline patterning, and the left-right asymmetric development of the heart, vasculature, lungs and stomach.

Nodal, a member of the transforming growth factor beta (TGF-beta) superfamily, is implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification. Here we define the intracellular signaling pathway induced by recombinant nodal protein treatment of P19 embryonal carcinoma cells. Nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and TGF-beta. However, nodal is unable to induce pTlx2-Lux, a reporter specifically responsive to bone morphogenetic proteins. We also demonstrate that nodal induces p(CAGA)(12), a reporter previously shown to be specifically activated by Smad3. Expression of a dominant negative Smad2 significantly reduces the level of luciferase reporter activity induced by nodal treatment. Finally, we show that nodal signaling rapidly leads to the phosphorylation of Smad2. These results provide the first direct biochemical evidence that nodal signaling is mediated by both activin-TGF-beta pathway Smads, Smad2 and Smad3. We also show here that the extracellular cripto protein is required for nodal signaling, making it distinct from activin or TGF-beta signaling.

Background: Normal development of the left/right (L/R) body axis leads to the characteristic sidedness of asymmetric body structures, e.g., the left-sided heart. Several genes are now known to be expressed with L/R asymmetry during embryogenesis, including nodal, a member of the transforming growth factor-beta (TGF-beta) family. Mutations or experimental treatments that affect L/R development, such as those that cause situs inversus (reversal of the sidedness of asymmetric body structures), have been shown to alter or abolish nodal's asymmetric expression., Methods: In the present study, we examined the effects on nodal expression of alpha(1)-adrenergic stimulation, known to cause a 50% incidence of situs inversus in rat embryos grown in culture, using reverse transcription-polymerase chain reaction assay and whole-mount in situ hybridization assay., Results: In embryos cultured with phenylephrine, an alpha(1)-adrenergic agonist, nodal's normal asymmetric expression only in the left lateral plate mesoderm was altered. In some treated embryos, nodal expression was detected in either the left or right lateral plate mesoderm. However, most treated embryos lacked lateral plate mesoderm expression. In addition, the embryos that did show expression were at a later stage than when nodal expression is normally found., Conclusions: Our results demonstrate that alpha(1)-adrenergic stimulation delays the onset and perturbs the normal asymmetric pattern of nodal expression. Either of these effects might contribute to situs inversus., (Copyright 2000 Wiley-Liss, Inc.)

Mice mutant for the TGF-beta family member, nodal, lack mesoderm and die between E8.5 and E9.5. The short ear-lethal (se(l) ) mutation, a deletion that eliminates Bmp-5, causes a strikingly similar gastrulation defect. Here we analyze se(l);nodal compound mutants and find a dosage effect. Embryos homozygous for one mutation show distinct gastrulation stage defects that depend on whether they are heterozygous or homozygous for the other mutation. Embryos mutant for nodal or se(l);nodal compound mutants fail to execute an antigenic shift indicative of mesoderm differentiation and ectoderm cells are shunted into an apoptotic pathway. Furthermore, we find a novel phenotype in se(l);nodal double mutant litters, in which two to four genetically different embryos are contained within the same deciduum. Both the gastrulation and implantation phenotypes can also arise in short ear-viable (se(v) ) and se(v); nodal mutant mice. These data indicate that loss of Bmp-5 may underlie the se(l) gastrulation phenotype and suggest that nodal and Bmp-5 interact during murine mesoderm formation. Our data also reveal an unsuspected role for Bmp-5 in implantation and the decidual response in the mouse.

Vertebrates develop distinct asymmetries along the left-right axis, which are consistently aligned with the anteroposterior and dorsoventral axes. The mechanisms that direct this handed development of left-right asymmetries have been elusive, but recent studies of mutations that affect left-right development have shed light on the molecules involved. One molecule implicated in left-right specification is left-right dynein (LRD), a microtubule-based motor protein. In the LRD protein of the inversus viscerum (iv) mouse, there is a single amino acid difference at a conserved position, and the lrd gene is one of many genes deleted in the legless (lgl) mutation. Both iv and lgl mice display randomized left-right development. Here we extend the analysis of the lrd gene at the levels of sequence, expression and function. The complete coding sequence of the lrd gene confirms its classification as an axonemal, or ciliary, dynein. Expression of lrd in the node at embryonic day 7.5 is shown to be symmetric. At embryonic day 8.0, however, a striking asymmetric expression pattern is observed in all three germ layers of the developing headfold, suggesting roles in both the establishment and maintenance of left-right asymmetries. At later times, expression of lrd is also observed in the developing floorplate, gut and limbs. These results suggest function for LRD protein in both ciliated and non-ciliated cells, despite its sequence classification as axonemal. In addition, a targeted mutation of lrd was generated that deletes the part of the protein required for ATP binding, and hence motor function. The resulting left-right phenotype, randomization of laterality, is identical to that of iv and lgl mutants. Gross defects in ciliary structure were not observed in lrd/lrd mutants. Strikingly, however, the monocilia on mutant embryonic node cells were immotile. These results prove the identity of the iv and lrd genes. Further, they argue that LRD motor function, and resulting nodal monocilia movement, are required for normal left-right development.

The establishment of the main body axis and the determination of left-right asymmetry are fundamental aspects of vertebrate embryonic development. A link between these processes has been revealed by the frequent finding of midline defects in humans with left-right anomalies. This association is also seen in a number of mutations in mouse and zebrafish, and in experimentally manipulated Xenopus embryos. However, the severity of laterality defects accompanying abnormal midline development varies, and the molecular basis for this variation is unknown. Here we show that mouse embryos lacking the early-response gene SIL have axial midline defects, a block in midline Sonic hedgehog (Shh) signalling and randomized cardiac looping. Comparison with Shh mutant embryos, which have axial defects but normal cardiac looping, indicates that the consequences of abnormal midline development for left-right patterning depend on the time of onset, duration and severity of disruption of the normal asymmetric patterns of expression of nodal, lefty-2 and Pitx2.

Left-right asymmetry in vertebrates is controlled by activities emanating from the left lateral plate. How these signals get transmitted to the forming organs is not known. A candidate mediator in mouse, frog and zebrafish embryos is the homeobox gene Pitx2. It is asymmetrically expressed in the left lateral plate mesoderm, tubular heart and early gut tube. Localized Pitx2 expression continues when these organs undergo asymmetric looping morphogenesis. Ectopic expression of Xnr1 in the right lateral plate induces Pitx2 transcription in Xenopus. Misexpression of Pitx2 affects situs and morphology of organs. These experiments suggest a role for Pitx2 in promoting looping of the linear heart and gut.

Patterning along the left/right axes helps establish the orientation of visceral organ asymmetries, a process which is of fundamental importance to the viability of an organism. A linkage between left/right and axial patterning is indicated by the finding that a number of genes involved in left/right patterning also play a role in anteroposterior and dorsoventral patterning. We have recovered a spontaneous mouse mutation causing left/right patterning defects together with defects in anteroposterior and dorsoventral patterning. This mutation is recessive lethal and was named no turning (nt) because the mutant embryos fail to undergo embryonic turning. nt embryos exhibit cranial neural tube closure defects and malformed somites and are caudally truncated. Development of the heart arrests at the looped heart tube stage, with cardiovascular defects indicated by ballooning of the pericardial sac and the pooling of blood in various regions of the embryo. Interestingly, in nt embryos, the direction of heart looping was randomized. Nodal and lefty, two genes that are normally expressed only in the left lateral plate mesoderm, show expression in the right and left lateral plate mesoderm. Lefty, which is normally also expressed in the floorplate, is not found in the prospective floor plate of nt embryos. This suggests the possibility of notochordal defects. This was confirmed by histological analysis and the examination of sonic hedgehog, Brachyury, and HNF-3 beta gene expression. These studies showed that the notochord is present in the early nt embryo, but degenerates as development progresses. Overall, these findings support the hypothesis that the notochord plays an active role in left/right patterning. Our results suggest that nt may participate in this process by modulating the notochordal expression of HNF-3 beta.

Recently, a pathway of genes which are part of a cascade regulating the side on which the heart forms during chick development was characterized (M. Levin et al., 1995, Cell 82, 1-20). Here we extend these previous studies, showing that manipulation of at least one member of the cascade, Sonic hedgehog (Shh), can affect the situs of embryonic rotation and of the gut, in addition to the heart. Bilateral expression of Shh, which is normally found exclusively on the left, does not result in left isomerism (a bilaterally symmetrical embryo having two left sides) nor in a complete situs inversus phenotype. Instead, misexpression of Shh on the right side of the node, which in turn leads to bilateral nodal expression, produces a heterotaxia-like condition, where different aspects of laterality are determined independently. Heart situs has previously been shown to be altered by ectopic Shh and activin. However, the most downstream gene identified in the LR pathway, nodal, had not been functionally linked to heart laterality. We show that ectopic (right-sided) nodal expression is able to affect heart situs, suggesting that the randomization of heart laterality observed in Shh and activin misexpression experiments is a result of changes in nodal expression and that nodal is likely to regulate heart situs endogenously. The first defined asymmetric signal in the left-right patterning pathway is Shh, which is initially expressed throughout Hensen's node but becomes restricted to the left side at stage 4(+). It has been hypothesized that the restriction of Shh expression may be due to repression by an upstream activin-like factor. The involvement of such an activin-like factor on the right side of Hensen's node was suggested because ectopic activin protein is able to repress Shh on the left side of the node, as well as to induce ectopic expression of a normally right-sided marker, the activin receptor cAct-RIIa. Here we provide further evidence in favor of this model. We find that a member of this family, Activin betaB, is indeed expressed asymmetrically, only on the right side of Hensen's node, at the correct time for it to be the endogenous asymmetric activin signal. Furthermore, we show that application of follistatin-loaded beads eliminates the asymmetry in Shh expression, consistent with an inhibition of an endogenous member of the activin-BMP superfamily. This combined with the previous data on exogenous activin supports the model that Activin betaB functions in the chick embryo to initiate Shh asymmetry. While these data extend our understanding of the early signals which establish left-right asymmetry, they leave unanswered the interesting question of how the bilateral symmetry of the embryo is initially broken to define a consistent left-right axis. Analysis of spontaneous chick twins suggests that, whatever the molecular mechanism, left-right patterning is unlikely to be due to a blastodermal prepattern but rather is initiated in a streak-autonomous manner.

Vertebrates have characteristic and conserved left-right (L-R) visceral asymmetries, for example the left-sided heart. In humans, alterations of L-R development can have serious clinical implications, including cardiac defects. Although little is known about how the embryonic L-R axis is established, a recent study in the chick embryo revealed L-R asymmetric expression of several previously cloned genes, including Cnr-1 (for chicken nodal-related-1), and indicated how this L-R molecular asymmetry might be important for subsequent visceral morphogenesis. Here we show that nodal is asymmetrically expressed in mice at similar stages, as is Xnr-1 (for Xenopus nodal related-1) in frogs. We also examine nodal expression in two mouse mutations that perturb L-R development, namely situs inversus viscerum (iv), in which assignment of L-R asymmetry is apparently random and individuals develop either normally or are mirror-image-reversed (situs inversus), and inversion of embryonic turning (inv), in which all individuals develop with situs inversus. In both, nodal expression is strikingly affected, being reversed or converted to symmetry. These results further support a key role for nodal and nodal-related genes in interpreting and relaying L-R patterning information in vertebrates. To our knowledge, our results provide the first direct evidence that iv and inv normally function well before the appearance of morphological L-R asymmetry.

Mouse embryos homozygous for a null mutation in nodal arrest development at early gastrulation and contain little or no embryonic mesoderm. Here, two Xenopus nodal-related genes (Xnr-1 and Xnr-2) are identified and shown to be expressed transiently during embryogenesis, first within the vegetal region of late blastulae and later in the marginal zone during gastrulation, with enrichment in the dorsal lip. Xnrs and mouse nodal function as dose-dependent dorsoanterior and ventral mesoderm inducers in whole embryos and explanted animal caps. Using a plasmid vector to produce Xnr proteins during gastrulation, we show that, in contrast to activin and other TGF beta-like molecules, Xnr-1 and Xnr-2 can dorsalize ventral marginal zone explants and induce muscle differentiation. Xnr signalling also rescues a complete embryonic axis in UV-ventralized embryos. The patterns of Xnr expression, the activities of the proteins and the phenotype of mouse nodal mutants, all argue strongly that a signaling pathway involving nodal, or nodal-related peptides, is an essential conserved element in mesoderm differentiation associated with vertebrate gastrulation and axial patterning.

One of the first intercellular signalling events in the vertebrate embryo leads to mesoderm formation and axis determination. In the mouse, a gene encoding a new member of the TGF-beta superfamily, nodal, is disrupted in a mutant deficient in mesoderm formation (Zhou et al., 1993, Nature 361, 543). nodal mRNA is found in prestreak mouse embryos, consistent with a role in the development of the dorsal axis. To examine the biological activities of nodal, we have studied the action of this factor in eliciting axis determination in the zebrafish, Danio rerio. Injection of nodal mRNA into zebrafish embryos caused the formation of ectopic axes that included notochord and somites. Axis duplication was preceded by the generation of an apparent ectopic shield (organizer equivalent) in nodal-injected embryos, as indicated by the appearance of a region over-expressing gsc and lim1; isolation and expression in the shield of the lim1 gene is reported here. These results suggest a role for a nodal-like factor in pattern formation in zebrafish.

During gastrulation, the three germ layers of the embryo are formed and organized along the anterior-posterior body axis. In the mouse, gastrulation involves the delamination of ectodermal cells through the primitive streak and their differentiation into mesoderm. These processes do not occur in embryos homozygous for a retrovirally induced recessive prenatal lethal mutation, the strain 413-d insertional mutation. Instead of giving rise to mesoderm, embryonic ectoderm in 413-d mutants overproliferates and then rapidly degenerates, although extraembryonic lineages remain viable. Here we isolate a candidate for the mutated gene which encodes a new member of the transforming growth factor-beta (TGF-beta) superfamily. Expression is first detected in primitive streak-stage embryos at about the time of mesoderm formation. It then becomes highly localized in the node at the anterior of the primitive streak. This region is analogous to chick Hensen's node and Xenopus dorsal lip (Spemann's organizer), which can induce secondary body axes when grafted into host embryos (reviewed in refs 5 and 6). Our findings suggest that this gene, named nodal, encodes a signalling molecule essential for mesoderm formation and subsequent organization of axial structures in early mouse development.

We examined whether superovulation can be used to improve the efficiency of donor embryo collection in embryonic stem cell injection chimera experiments. Superovulation of prepuberal C57BL/6 mice was compared with spontaneous ovulation of mature mice with respect to production and survival of blastocysts. Our results indicate that, compared with spontaneous ovulation, superovulation results in an increased number of blastocysts per female with no difference in viability when transferred to outbred Swiss Webster foster mothers. The advantage of using superovulated females, in terms of maintaining smaller numbers of both females and stud males, is discussed.

A transgenic mouse strain derived from embryonic stem (ES) cells infected with multiple copies of a retroviral vector carries a recessive insertional mutation resulting in prenatal lethality. A detailed histological analysis of developing embryos has shown that the mutation results in hyperplasia of both embryonic and extraembryonic ectoderm and failure of mesoderm formation in the egg cylinder stage embryo. The number of cells in each lineage of normal and mutant embryos was estimated using stereological analysis of serial sections taken from implantation sites. We observed a 2-fold increase in the number of embryonic ectoderm cells in mutant embryos at 7.5 days postcoitum (dpc). In addition, we found that mutant embryonic ectoderm cells are only 0.6 times as large as normal cells. The number of extraembryonic ectoderm cells in mutant embryos at 7.5 dpc is also increased, by almost 4-fold. Mutant extraembryonic ectoderm cells are also smaller than normal, being only two-thirds the size of wild-type cells. The mutant phenotype suggests that the gene identified by this insertional mutation plays an important role in the growth control of early embryonic lineages.

Two transgenic strains of mutant mice lacking hypoxanthine-guanidine phosphoribosyltransferase (HPRT) activity were examined behaviorally and neurochemically for phenotypic similarity to the human Lesch-Nyhan syndrome. In this syndrome, male children markedly deficient in the enzyme HPRT develop self-mutilation and severe motoric difficulties, and exhibit a pronounced deficiency of dopamine in the basal ganglia. The HPRT-deficient mice showed no evidence of self-mutilation, no detectable motor impairments on tests selected for sensitivity to basal ganglia dysfunction, and no differences in response to apomorphine. Biochemical analyses revealed significantly lower levels of striatal dopamine in the HPRT-deficient mice than in HPRT normal littermates, but the depletion was only of the order of 19%. The results suggest that mice lacking HPRT activity do not phenotypically resemble children born with the same enzymatic deficiency in part because mutant mouse striatal dopamine levels are not as low as those seen in clinical cases with Lesch-Nyhan disease. In contrast to Lesch-Nyhan children, mice may be able to utilize alternative pathways more effectively to maintain purine and neurotransmitter levels within the ranges required for normal brain development and function.

The integrity of forebrain monoamine systems has been assessed both biochemically and immunohistochemically in transgenic mice carrying the mutant hprt-bm2 gene, an animal model of Lesch-Nyhan syndrome. The mutant mice manifested 20-30% depletions of forebrain dopamine, and corresponding increases in dopamine turnover. By contrast, the mutant mice manifested normal tyrosine hydroxylase immunostaining of catecholamine cell bodies and terminals throughout the forebrain, and cell counts revealed no detectable loss of ventral mesencephalic dopamine neurones. Serotonin concentrations were also depleted, whereas no significant changes were found in noradrenaline or adrenaline, methylhydroxyphenylglycol (MHPG) or 5-hydroxyindoleacetic acid. The results indicate that a primary genetic deficiency in purine salvage pathways is associated with additional changes in forebrain monoamine metabolism in mouse as in man, although these changes are less pronounced in the animal model than in the human syndrome. The biochemical changes were not associated with explicit degeneration of the associated populations of neurones.

The human Lesch-Nyhan syndrome is a rare neurological and behavioural disorder, affecting only males, which is caused by an inherited deficiency in the level of activity of the purine salvage enzyme hypoxanthine-guanosine phosphoribosyl transferase (HPRT). How the resulting alterations in purine metabolism lead to the severe symptoms characteristic of Lesch-Nyhan patients is still not understood. No mutations at the Hprt locus leading to loss of activity have been described in laboratory animals. To derive an animal model for the Lesch-Nyhan syndrome, we have used cultured mouse embryonic stem cells, mutagenized by retroviral insertion and selected for loss of HPRT activity, to construct chimaeric mice. Two clonal lines carrying different mutant Hprt alleles have given rise to germ cells in chimaeras, allowing the derivation of strains of mutant mice having the same biochemical defect as Lesch-Nyhan patients. Male mice carrying the mutant alleles are viable and analysis of their cells shows a total lack of HPRT activity.