Developmental biology. The worm keeps turning.
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Animals↗
Biomedical subjects
B Bowerman
Publications and source records attributed to B Bowerman.
Wnt signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm.
A polarizing signal induces endoderm production by a 4-cell stage blastomere in C. elegans called EMS. We identified 16 mutations in five genes, mom-1 through mom-5, required for EMS to produce endoderm. mom-1, mom-2, and mom-3 are required in the signaling cell, P2, while mom-4 is required in EMS. P2 signaling downregulates an HMG domain protein, POP-1, in one EMS daughter. The sequence of mom-2 predicts that it encodes a member of the Wnt family of secreted glycoproteins, which in other systems activate HMG domain proteins. Defective mitotic spindle orientations in mom mutant embryos indicate that Wnt signaling influences cytoskeletal polarity in blastomeres throughout the early embryo.
Alleles↗
The maternal par genes and the segregation of cell fate specification activities in early Caenorhabditis elegans embryos.
After fertilization in C. elegans, activities encoded by the maternally expressed par genes appear to establish cellular and embryonic polarity. Loss-of-function mutations in the par genes disrupt anterior-posterior (a-p) asymmetries in early embryos and result in highly abnormal patterns of cell fate. Little is known about how the early asymmetry defects are related to the cell fate patterning defects in par mutant embryos, or about how the par gene products affect the localization and activities of developmental regulators known to specify the cell fate patterns made by individual blastomeres. Examples of such regulators of blastomere identity include the maternal proteins MEX-3 and GLP-1, expressed at high levels anteriorly, and SKN-1 and PAL-1, expressed at high levels posteriorly in early embryos. To better define par gene functions, we examined the expression patterns of MEX-3, PAL-1 and SKN-1, and we analyzed mex-3, pal-1, skn-1 and glp-1 activities in par mutant embryos. We have found that mutational inactivation of each par gene results in a unique phenotype, but in no case do we observe a complete loss of a-p asymmetry. We conclude that no one par gene is required for all a-p asymmetry and we suggest that, in some cases, the par genes act independently of each other to control cell fate patterning and polarity. Finally, we discuss the implications of our findings for understanding how the initial establishment of polarity in the zygote by the par gene products leads to the proper localization of more specifically acting regulators of blastomere identity.
Animals↗
MEX-3 is a KH domain protein that regulates blastomere identity in early C. elegans embryos.
After the first division of the C. elegans embryo, the posterior blastomere can produce numerous muscles while the anterior blastomere cannot. We show here that maternal-effect lethal mutations in the gene mex-3 cause descendants of the anterior blastomere to produce muscles by a pattern of development similar to that of a descendant of the wild-type posterior blastomere. mex-3 encodes a probable RNA-binding protein that is distributed unequally in early embryos and that is a component of germline-specific granules called P granules. We propose that MEX-3 contributes to anterior-posterior asymmetry by regulating one or more mRNAs involved in specifying the fate of the posterior blastomere.
Alleles↗
Time-dependent responses to glp-1-mediated inductions in early C. elegans embryos.
In an embryo of the nematode Caenorhabditis elegans, two blastomeres at the 4-cell stage, ABa and ABp, are born with equivalent developmental potential. Subsequently, interactions with the P2 blastomere at the 4-cell stage and the MS blastomere at the 12-cell stage generate differences in developmental fate among descendants of ABa and ABp. We have reproduced these inductions in vitro using embryonic blastomeres isolated in cell-culture medium. We show that during these inductions only the responding AB descendants require the activity of the glp-1 gene, which is similar in sequence to Drosophila Notch, supporting models in which GLP-1 protein acts as a receptor for both the P2 and MS signals. We also show that P2 signaling requires the activity of the apx-1 gene, similar in sequence to Drosophila Delta, and that MS signaling requires the putative transcription factor SKN-1. We present evidence that the primary factor determining the different responses to these two signals is the age of the AB descendants, not the identity of the signaling cell or ligand. Therefore, we suggest that time-dependent changes in factors within AB descendants are responsible for their different responses to inductive signals that use a common receptor.
Aging↗
Determinants of blastomere identity in the early C. elegans embryo.
Genetic and molecular studies of development in Caenorhabditis elegans have identified regulators that appear to control pattern formation in the cellularized nematode embryo. Two genes, skn-1 and pie-1, are required for specifying the different identities of two sister blastomeres in a 4-cell embryo, called P2 and EMS. The skn-1 gene encodes a DNA binding protein that may control blastomere development by regulating transcription in EMS and its descendants. ABa and ABp, the other two sisters in a 4-cell embryo, are influenced to develop differently by cell signaling events that require the two genes apx-1 and glp-1. In this review, I summarize evidence that some or all of these genes may encode embryonic determinants of blastomere identity.
Animals↗
Formation of a monomeric DNA binding domain by Skn-1 bZIP and homeodomain elements.
Maternally expressed Skn-1 protein is required for the correct specification of certain blastomere fates in early Caenorhabditis elegans embryos. Skn-1 contains a basic region similar to those of basic leucine zipper (bZIP) proteins but, paradoxically, it lacks a leucine zipper dimerization segment. Random sequence selection methods were used to show that Skn-1 binds to specific DNA sequences as a monomer. The Skn-1 basic region lies at the carboxyl terminus of an 85-amino acid domain that binds preferentially to a bZIP half-site and also recognizes adjacent 5' AT-rich sequences in the minor groove, apparently with an amino (NH2)-terminal "arm" related to those of homeodomain proteins. The intervening residues appear to stabilize interactions of these two subdomains with DNA. The Skn-1 DNA binding domain thus represents an alternative strategy for promoting binding of a basic region segment recognition helix to its cognate half-site. The results point to an underlying modularity in subdomains within established DNA binding domains.
Amino Acid Sequence↗
Two maternal genes, apx-1 and pie-1, are required to distinguish the fates of equivalent blastomeres in the early Caenorhabditis elegans embryo.
In a 4-cell Caenorhabditis elegans embryo, two sister blastomeres called ABa and ABp are born with equivalent developmental potential, but eventually produce distinct patterns of cell fate. The different fates of ABa and ABp are specified at least in part by inductive interactions with neighboring blastomeres. Previous studies indicate that, at the 4-cell stage, a signal from the posterior-most blastomere, P2, is required for ABp to produce at least one of its unique cell types. This P2/ABp interaction depends on glp-1, a putative receptor for intercellular interactions. To investigate this early induction further, we isolated mutants in which ABp developed abnormally. We describe the effects of recessive mutations in apx-1, a maternal gene that appears to be required for P2 to signal ABp. In embryos from mothers homozygous for mutations in apx-1 (apx-1 embryos), ABp fails to produce its characteristic cell types. Instead, ABp from apx-1 embryos develops more like its sister ABa: it produces ABa-like pharyngeal cells and it recapitulates ABa-like cell lineages. Because mutations in apx-1 affect the development of only the ABp blastomere, we suggest that the wild-type gene encodes a component of the P2/ABp signalling pathway. To explain the observation that ABp in apx-1 embryos adopts an ABa-like fate, we propose a model in which the P2 signal is required to break the initial equivalence of ABa and ABp. We performed two independent tests of this model. First, we examined ABp development in pie-1 mutant embryos, in which P2 adopts the identity of another blastomere. We find that, in pie-1 embryos, APp fails to produce its characteristic cell types and instead adopts a fate similar to that of ABa. We conclude that the changed identity of P2 in pie-1 embryos prevents the P2/ABp interaction. As a second test, we examined ABp development in wild-type embryos after physically removing P2. These operated embryos produce extra pharyngeal cells, consistent with out proposal that a signal from P2 breaks the initially equivalent developmental state of ABa and ABp. We discuss the possibility that apx-1 acts as a ligand in this glp-1-dependent signalling pathway.
Animals↗
The maternal gene skn-1 encodes a protein that is distributed unequally in early C. elegans embryos.
The autonomous or cell-intrinsic developmental properties of early embryonic blastomeres in nematodes are thought to result from the action of maternally provided determinants. After the first cleavage of the C. elegans embryo, only the posterior blastomere, P1, has a cell-intrinsic ability to produce pharyngeal cells. The product of the maternal gene skn-1 is required for P1 to produce pharyngeal cells. We show here that the Skn-1 protein is nuclear localized and that P1 appears to accumulate markedly higher levels of Skn-1 protein than its sister, the AB blastomere. We have examined the distribution of Skn-1 protein in embryos from mothers with maternal-effect mutations in the genes mex-1, par-1, and pie-1. These results suggest that mex-1(+) and par-1(+) activities are required for the unequal distribution of the Skn-1 protein and that pie-1(+) activity may function to regulate the activity of Skn-1 protein in the descendants of the posterior blastomere P1.
Animals↗
skn-1, a maternally expressed gene required to specify the fate of ventral blastomeres in the early C. elegans embryo.
By the 4-cell stage of C. elegans embryogenesis, a ventral blastomere, called EMS, is already committed to producing pharyngeal and intestinal cell types. Recessive, maternal-effect mutations in the gene skn-1 prevent EMS from producing both pharyngeal and intestinal cells. In skn-1 mutant embryos, EMS instead produces hypodermal cells and body wall muscle cells, much like its sister blastomere. Genetic analysis suggests that the skn-1 gene product is also required post-embryonically for development of the intestine. We have cloned and sequenced the skn-1 gene and describe sequence similarities to the basic regions of bZIP transcription factors. We propose that the maternally expressed skn-1 gene product acts to specify the fate of the EMS blastomere.
Amino Acid Sequence↗
Cell interactions involved in development of the bilaterally symmetrical intestinal valve cells during embryogenesis in Caenorhabditis elegans.
We describe two different cell interactions that appear to be required for the proper development of a pair of bilaterally symmetrical cells in Caenorhabditis elegans called the intestinal valve cells. Previous experiments have shown that at the beginning of the 4-cell stage of embryogenesis, two sister blastomeres called ABa and ABp are equivalent in development potential. We show that cell interactions between ABp and a neighboring 4-cell-stage blastomere called P2 distinguish the fates of ABa and ABp by inducing descendants of ABp to produce the intestinal valve cells, a cell type not made by ABa. A second cell interaction appears to occur later in embryogenesis when two bilaterally symmetrical descendants of ABp, which both have the potential to produce valve cells, contact each other; production of the valve cells subsequently becomes limited to only one of the two descendants. This second interaction does not occur properly if the two symmetrical descendants of ABp are prevented from contacting each other. Thus the development of the intestinal valve cells appears to require both an early cell interaction that establishes a bilaterally symmetrical pattern of cell fate and a later interaction that breaks the symmetrical cell fate pattern by restricting to only one of two cells the ability to produce a pair of valve cells.
Animals↗
Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein.
An essential step in the life cycle of a retrovirus is the integration of a DNA copy of the viral genome into a host cell chromosome. We have analyzed the structure of the initial covalent product of an in vitro retroviral integration reaction and determined the structure of the ends of the unintegrated linear viral DNA molecules present in vivo in cells infected with murine leukemia virus (MLV). Our results lead to the following conclusions: (i) Circularization of viral DNA plays no role in integration. The direct precursor to the integrated MLV provirus is a linear molecule. (ii) The initial step in the integration reaction is probably a cleavage that removes the terminal 2 bases from each 3' end of the viral DNA. This cleavage depends on a virally encoded protein, IN, that has previously been shown genetically to be required for integration. (iii) The resulting viral 3' ends are joined to target DNA to form the initial recombination intermediate.
Cell Transformation, Viral↗
A nucleoprotein complex mediates the integration of retroviral DNA.
The integration of viral DNA into the host genome is an essential step in the retrovirus life cycle. To understand this process better, we have examined the native state of viral DNA in cells acutely infected by murine leukemia virus (MLV), using both a physical assay for viral DNA and a functional assay for integration activity (Brown et al. 1987). The viral DNA and integration activity copurify during velocity sedimentation, gel filtration, and density equilibrium centrifugation, indicating that viral DNA is in a large (approximately 160S) nucleoprotein complex that includes all functions required for integration activity in vitro. Analysis by immunoprecipitation shows that the viral capsid protein is part of the active nucleoprotein complex, but recognition of the complex by only a subset of anti-capsid sera implies that the protein is constrained conformationally. The viral DNA within this structure is accessible to nucleases; the effects of nucleases on the integrity of the complex suggest that the integration-competent particle is derived from and similar to the core of extracellular virions.
Animals↗
Correct integration of retroviral DNA in vitro.
We have developed a cell-free system for studying the integration of retroviral DNA. In our assay, amber mutations in a bacteriophage lambda genome that serves as the target for integration are suppressed by integration of an MLV derivative that carries the E. coli supF gene. The structure of the reaction products is that expected from an authentic MLV integration reaction. Linear viral DNA from the cytoplasm of infected cells serves as a precursor, though not necessarily the immediate precursor, to the provirus integrated in vitro. The viral DNA in the infected cell appears to be tightly associated with the enzymatic machinery required for its integration. Supercoiling, chromatin structure, transcription, and replication are not required of the target DNA. Since no high-energy cofactor is necessary, the DNA breakage and joining steps in the integration reaction are probably coupled.
Animals↗
Inactive renin in peritoneal fluid--an independent system or a transport form of circulating renin?
Active and inactive renin have been studied in peritoneal fluid and plasma of six patients with ascites and four patients on peritoneal dialysis (using concentrated dialysate). Acid-pepsin and trypsin activation gave similar results. In ascitic patients mean (+/- s.e.) plasma active renin was very high (0.31 +/- 0.99 pmol angiotensin l/l/s) compared with normal subjects (0.31 +/- 0.08 pmol ANG l/l/s). Plasma inactive renin (1.27 +/- 0.30 pmol ANG l/l/s) was similar to normal subjects (1.31 +/- 0.31 pmol ANG l/l/s) while the mean percentage of total renin which was inactive was 29.7 and 76.5. In ascitic fluid, active renin was very low (0.36 +/- 0.09 pmol ANG l/l/s) but inactive renin (2.15 +/- 0.28 pmol ANG l/l/s) was higher than plasma. Percentage of total renin inactive was 85.8. Peritoneal dialysate showed even smaller amounts of active renin but similar inactive renin with 95.4 per cent inactive (versus 73.8 in their plasma). Peritoneal fluid inactive renin was similar to that in plasma by affigel chromatography and gel filtration and showed, after activation, similar pH optima and Michaelis constants to plasma and peritoneal active renin.
Ascitic Fluid↗