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Biomedical subjects

E J Robertson

Publications and source records attributed to E J Robertson.

At least 19 recordsLinked to original sources
PubMed2000

SPC4/PACE4 regulates a TGFbeta signaling network during axis formation.

In vertebrates, specification of anteroposterior (A/P) and left-right (L/R) axes depends on TGFbeta-related signals, including Nodal, Lefty, and BMPs. Endoproteolytic maturation of these proteins is probably mediated by the proprotein convertase SPC1/Furin. In addition, precursor processing may be regulated by related activities such as SPC4 (also known as PACE4). Here, we show that a proportion of embryos lacking SPC4 develop situs ambiguus combined with left pulmonary isomerism or complex craniofacial malformations including cyclopia, or both. Gene expression analysis during early somite stages indicates that spc4 is genetically upstream of nodal, pitx2, lefty1, and lefty2 and perhaps maintains the balance between Nodal and BMP signaling in the lateral plate that is critical for L/R axis formation. Furthermore, genetic interactions between nodal and spc4, together with our analysis of chimeric embryos, strongly suggest that during A/P axis formation, SPC4 acts primarily in the foregut. These findings establish an important role for SPC4 in patterning the early mouse embryo.

Abnormalities, Multiple↗
PubMed2000

Formation of the definitive endoderm in mouse is a Smad2-dependent process.

TGFbeta growth factors specify cell fate and establish the body plan during early vertebrate development. Diverse cellular responses are elicited via interactions with specific cell surface receptor kinases that in turn activate Smad effector proteins. Smad2-dependent signals arising in the extraembryonic tissues of early mouse embryos serve to restrict the site of primitive streak formation and establish anteroposterior identity in the epiblast. Here we have generated chimeric embryos using lacZ-marked Smad2-deficient ES cells. Smad2 mutant cells extensively colonize ectodermal and mesodermal populations without disturbing normal development, but are not recruited into the definitive endoderm lineage during gastrulation. These experiments provide the first evidence that TGFbeta signaling pathways are required for specification of the definitive endoderm lineage in mammals and identify Smad2 as a key mediator that directs epiblast derivatives towards an endodermal as opposed to a mesodermal fate. In largely Smad2-deficient chimeras, asymmetric nodal gene expression is maintained and expression of pitx2, a nodal target, is also unaffected. These results strongly suggest that other Smad(s) act downstream of Nodal signals in mesodermal populations. We found Smad2 and Smad3 transcripts both broadly expressed in derivatives of the epiblast. However, Smad2 and not Smad3 mRNA is expressed in the visceral endoderm, potentially explaining why the primary defect in Smad2 mutant embryos originates in this cell population.

Animals↗
PubMed2000

Tissue-specific requirements for the proprotein convertase furin/SPC1 during embryonic turning and heart looping.

Furin, the mammalian prototype of a family of serine proteases, is required for ventral closure and axial rotation, and formation of the yolk sac vasculature. Here we show additionally that left-sided expression of pitx2 and lefty-2 are also perturbed in Furin-deficient embryos. These tissue abnormalities are preceded by a marked delay in the expansion of the definitive endoderm during gastrulation. Using a chimera approach, we show that Furin activity is required in epiblast derivatives, including the primitive heart, gut and extraembryonic mesoderm, whereas it is nonessential in the visceral endoderm. Thus, chimeric embryos, derived by injecting wild-type embryonic stem (ES) cells into fur(-/-) blastocysts, develop normally until at least 9.5 d.p.c. In contrast, Furin-deficient chimeras developing in the context of wild-type visceral endoderm fail to undergo ventral closure, axial rotation and yolk sac vascularization. Fur(-/-) cells are recruited into all tissues examined, including the yolk sac vasculature and the midgut, even though these structures fail to form in fur mutants. The presence of wild-type cells in the gut strikingly correlates with the ability of chimeric embryos to undergo turning. Overall, we conclude that Furin activity is essential in both extraembryonic and precardiac mesoderm, and in definitive endoderm derivatives.

Animals↗
PubMed1999

Asymmetric and node-specific nodal expression patterns are controlled by two distinct cis-acting regulatory elements.

The TGFbeta-related molecule Nodal is required for establishment of the anterior-posterior (A-P) and left-right (L-R) body axes of the vertebrate embryo. In mouse, several discrete sites of nodal activity closely correlate with its highly dynamic expression domains. nodal function in the posterior epiblast promotes primitive streak formation, whereas transient nodal expression in the extraembryonic visceral endoderm is essential for patterning the rostral central nervous system. Asymmetric nodal expression in the developing node and at later stages in left lateral plate mesoderm has been implicated as a key regulator of L-R axis determination. We have analyzed the cis-regulatory elements controlling nodal expression domains during early development. We show that the regulatory sequences conferring node-specific expression are contained in an upstream region of the locus, whereas early expression in the endoderm and epiblast and asymmetric expression at later stages on the left side of the body axis are controlled by a 600-bp intronic enhancer. Targeted deletion of a 100-bp subregion of this intronic enhancer eliminates nodal expression in the early epiblast and visceral endoderm and disrupts asymmetric expression in the node and lateral plate mesoderm. Thus, developmentally regulated nodal expression at distinct tissue sites during A-P and L-R axis formation is potentially controlled by common transcriptional activators.

Animals↗
PubMed1999

Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme.

Nephrogenesis in the mouse kidney begins at embryonic day 11 and ends approximately 10 days postpartum. During this period, new nephrons are continually being generated from a stem-cell population-the nephrogenic mesenchyme-in response to signals emanating from the tips of the branching ureter. Relatively little is known about the mechanism by which the nephrogenic mesenchyme cell population is maintained at the tips of the ureter in the presence of signals promoting tubulogenesis. Previous studies have shown that a loss of Bmp7 function leads to kidney defects that are a likely result of progressive loss of nephrogenic mesenchyme by apoptosis. The studies presented here demonstrate that BMP7 signaling can prevent apoptosis in explants of metanephric mesenchyme. The surviving mesenchyme cell population, however, is not competent to respond to signals promoting tubulogenesis. In conjunction with FGF2, BMP7 promotes growth and maintains competence of the mesenchyme in vitro. In addition, FGF2 and BMP7 signaling, both independently and in combination, inhibit tubulogenesis. Interestingly, FGF2 and BMP7 also promote expansion of the stromal progenitor cell population in whole kidney culture. Because BMP7 is not produced by stromal progenitor cells, these data suggest a novel interaction between the nephrogenic mesenchyme and stromal progenitor cell populations. A model for the regulation of nephrogenesis by FGF and BMP signaling is presented.

Animals↗
PubMed1999

BMP7 acts in murine lens placode development.

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation.

Animals↗
PubMed1999

Regulation of bone morphogenetic protein activity by pro domains and proprotein convertases.

Bone morphogenetic proteins (BMPs) are derived from inactive precursor proteins by endoproteolytic cleavage. Here we show that processing of Nodal and Myc-tagged BMP4 is significantly enhanced by SPC1/Furin or SPC4/PACE4, providing direct evidence that regulation of BMP signaling is likely to be controlled by subtilisin-like proprotein convertase (SPC) activities. Nodal processing is dramatically enhanced if two residues adjacent to the precursor cleavage site are substituted with amino acids found at the equivalent positions of Activin, demonstrating that structural constraints at the precursor cleavage site limit the processing efficiency. However, in transfection assays, mature Nodal is undetectable either in culture supernatants or in cell lysates, despite efficient cleavage of the precursor protein, suggesting that mature Nodal is highly unstable. Domain swap experiments support this conclusion since mature BMP4 or Dorsalin are also destabilized when expressed in conjunction with the Nodal pro domain. By contrast, mature Nodal is stabilized by the Dorsalin pro domain, which mediates the formation of stable complexes. Collectively, these data show that the half-life of mature BMPs is greatly influenced by the identity of their pro regions.

Amino Acid Sequence↗
PubMed1999

Efficient gene-specific expression of cre recombinase in the mouse embryo by targeted insertion of a novel IRES-Cre cassette into endogenous loci.

Site specific recombinases have provided the experimental strategy necessary to modulate the expression of gene products in the mouse embryo. In this study we have exploited Cre recombinase to develop a widely applicable cell marking system which functions efficiently even at early post-implantation embryonic stages. Importantly, the techniques and reagents derived in this study are generally applicable to any recombinase driven approach, including strategies to temporally and spatially modulate endogenous or ectopic gene expression in the embryo. The cell marking scheme has two essential components which were derived as separate mouse lines. The first line carries a universal conditional lacZ reporter (UCR) locus which was prepared by using gene targeting in a novel approach to modify a ubiquitously expressed retroviral lacZ promoter trap insertion. The UCR locus is silent until it undergoes a Cre mediated DNA rearrangement to restore lacZ expression. To generate the Cre expressing allele, we outline a flexible strategy which requires the introduction of a novel IRES-Cre cassette into exon sequence of an endogenous locus by gene targeting. We successfully demonstrate this approach by generating a Cre expressing allele of the EphA2 gene, an Eph receptor protein tyrosine kinase expressed early in development. Analysis of double heterozygote embryos clearly demonstrates that Cre recombinase is expressed in vivo from the EphA2 IRES-Cre allele, and that the conditional reporter locus is efficiently restored in EphA2-expressing cells as early as 7.5 dpc. This cell marking experiment establishes the feasibility of expressing Cre recombinase from a single copy allele in the embryo and demonstrates the utility of the conditional reporter mouse which can be used in the analysis of any Cre expressing allele.

Animals↗
PubMed1999

Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.

Activin Receptors, Type II↗
PubMed1999

The distinctive roles of five different ARC genes in the chloroplast division process in Arabidopsis.

ARC (accumulation and replication of chloroplasts) genes control different aspects of the chloroplast division process in higher plants. In order to establish the hierarchy of the ARC genes in the chloroplast division process and to provide evidence for their specific roles, double mutants were constructed between arc11, arc6, arc5, arc3 and arc1 in all combinations and phenotypically analysed. arc11 is a new nuclear recessive mutant with 29 chloroplasts compared with 120 in wild type. All the phenotypes of the double mutants are unambiguous. ARC1 down-regulates proplastid division but is on a separate pathway from ARC3, ARC5, ARC6 and ARC11. ARC6 initiates both proplastid and chloroplast division. ARC3 controls the rate of chloroplast expansion and ARC11 the central positioning of the final division plane in chloroplast division. ARC5 facilitates separation of the two daughter chloroplasts. ARC5 maps to chromosome 3 and ARC11 and ARC6 map approximately 60 cM apart on chromosome 5.

Arabidopsis↗
PubMed1999

Early embryonic lethality in Bmp5;Bmp7 double mutant mice suggests functional redundancy within the 60A subgroup.

Members of the BMP family of signaling molecules display a high conservation of structure and function, and multiple BMPs are often coexpressed in a variety of tissues during development. Moreover, distinct BMP ligands are capable of activating common pathways. Here we describe the coexpression of two members of the 60A subfamily of BMPs, Bmp5 and Bmp7, at a number of different sites in the embryo from gastrulation onwards. Previous studies demonstrate that loss of either Bmp5 or Bmp7 has negligible effects on development, suggesting these molecules functionally compensate for each other at early stages of embryonic development. Here we show this is indeed the case. Thus we find that Bmp5;Bmp7 double mutants die at 10.5 dpc and display striking defects primarily affecting the tissues where these factors are coexpressed. The present analysis also uncovers novel roles for BMP signaling during the development of the allantois, heart, branchial arches, somites and forebrain. Bmp5 and Bmp7 do not appear to be involved in establishing pattern in these tissues, but are instead necessary for the proliferation and maintenance of specific cell populations. These findings are discussed with respect to potential mechanisms underlying cooperative signaling by multiple members of the TGF-beta superfamily.

Allantoin↗
PubMed1999

Role of BMP family members during kidney development.

Members of the Bone morphogenetic protein (BMP) family have been shown to be important signaling molecules throughout mouse development. Accordingly, many BMPs are also expressed during organogenesis of the metanephric kidney. However, only BMP7 has been shown to be absolutely required for proper formation of the kidney, thus the majority of information known involves this family member. BMP7 is expressed in both the ureteric epithelium and the mesenchyme throughout embryonic development and has been shown to function as a survival factor for the nephrogenic mesenchyme. However, there has been some controversy over the role of BMP7 as an inducing molecule for the metanephric mesenchyme. Recent studies have shown that BMP7 functions as an anti-differentiation factor for this mesenchyme cell population. The function of BMPs in the ureter and in the more differentiated epithelial structures of the nephron is less well understood.

Animals↗
PubMed1998

Synthesis of medium-chain-length polyhydroxyalkanoates in arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the beta-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the beta-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

Journal Article↗
PubMed1998

Pitx2 determines left-right asymmetry of internal organs in vertebrates.

The handedness of visceral organs is conserved among vertebrates and is regulated by asymmetric signals relayed by molecules such as Shh, Nodal and activin. The gene Pitx2 is expressed in the left lateral plate mesoderm and, subsequently, in the left heart and gut of mouse, chick and Xenopus embryos. Misexpression of Shh and Nodal induces Pitx2 expression, whereas inhibition of activin signalling blocks it. Misexpression of Pitx2 alters the relative position of organs and the direction of body rotation in chick and Xenopus embryos. Changes in Pitx2 expression are evident in mouse mutants with laterality defects. Thus, Pitx2 seems to serve as a critical downstream transcription target that mediates left-right asymmetry in vertebrates.

Activin Receptors, Type II↗
PubMed1998

Smad2 signaling in extraembryonic tissues determines anterior-posterior polarity of the early mouse embryo.

Smad proteins transmit TGFbeta signals from the cell surface to the nucleus. Here we analyze Smad2 mutant embryos created using ES cell technology. Smad2 function is not required for mesoderm production per se, but, rather unexpectedly, in the absence of Smad2 the entire epiblast adopts a mesodermal fate giving rise to a normal yolk sac and fetal blood cells. In contrast, Smad2 mutants entirely lack tissues of the embryonic germ layers. Smad2 signals serve to restrict the site of primitive streak formation and establish anterior-posterior identity within the epiblast. Chimera experiments demonstrate these essential activities are contributed by the extraembryonic tissues. Thus, the extraembryonic tissues play critical roles in establishing the body plan during early mouse development.

Animals↗
PubMed1998

Mice lacking Bmp6 function.

Bmp6, a member of the 60A subgroup of bone morphogenetic proteins (BMPs), is expressed in diverse sites in the developing mouse embryo from preimplantation stages onwards. To evaluate roles for Bmp6 signaling in vivo, gene targeting was used to generate a null mutation at the Bmp6 locus. The resulting Bmp6 mutant mice are viable and fertile, and show no overt defects in tissues known to express Bmp6 mRNA. The skeletal elements of newborn and adult mutants are indistinguishable from wild-type. However, careful examination of skeletogenesis in late gestation embryos reveals a consistent delay in ossification strictly confined to the developing sternum. In situ hybridization studies in the developing long bones and sternum show that other BMP family members are expressed in overlapping domains. In particular we find that Bmp2 and Bmp6 are coexpressed in hypertrophic cartilage, suggesting that Bmp2 may functionally compensate in Bmp6 null mice. The defects in sternum development in Bmp6 null mice are likely to be associated with a transient early expression of Bmp6 in the sternal bands, prior to ossification. These sternal defects are slightly exacerbated in Bmp5/6 double mutant animals.

Animals↗
PubMed1998

Anterior patterning in mouse.

The anteroposterior axis of the vertebrate embryo becomes explicit during gastrulation, the process that converts a relatively featureless embryonic precursor population into new tissues assembled into a recognisable body pattern. Vertebrate embryos arrive at gastrulation in very different states in terms of their size, cell number and reliance on factors inherited from the unfertilized egg. However, they emerge from gastrulation looking very similar, and there is now extensive molecular genetic evidence to indicate that the bare essentials of the gastrulation process have been well conserved during evolution. Here, we review recent findings in the mouse that suggest that anterior identity is, in fact, established before gastrulation starts. They suggest that it is first manifest in extraembryonic tissue and that this tissue is essential for the embryo to develop normal anterior structures, such as the forebrain. We also argue that this precocious anterior pattern could have a counterpart in other non-mammalian vertebrates.

Animals↗