Copyright (c) 2013 University of Massachusetts Medical School All rights reserved. http://escholarship.umassmed.edu/rivera Recent documents in Rivera Lab en-us Fri, 17 May 2013 11:21:19 PDT 3600 http://escholarship.umassmed.edu/rivera/22 http://escholarship.umassmed.edu/rivera/22 Wed, 02 Jan 2013 06:07:47 PST The formation of the anteroposterior axis in mice requires a Wnt3-dependent symmetry-breaking event that leads to the formation of the primitive streak and gastrulation. Wnt3 is expressed sequentially in two distinct areas of the mouse embryo before the appearance of the primitive streak; first in the posterior visceral endoderm and soon after in the adjacent posterior epiblast. Hence, although an axial requirement for Wnt3 is well established, its temporal and tissue specific requirements remain an open question. Here, we report the conditional inactivation of Wnt3 in the epiblast of developing mouse embryos. Contrary to previous studies, our data shows that embryos lacking Wnt3 specifically in the epiblast are able to initiate gastrulation and advance to late primitive streak stages but fail to thrive and are resorbed by E9.5. At the molecular level, we provide evidence that Wnt3 regulates its own expression and that of other primitive streak markers via activation of the canonical Wnt signaling pathway.

]]> Giovane G. Tortelote et al. Wnt3 Protein Gastrulation http://escholarship.umassmed.edu/rivera/21 http://escholarship.umassmed.edu/rivera/21 Thu, 04 Oct 2012 08:55:23 PDT Aurora A is a mitotic kinase essential for cell proliferation. In mice, ablation of Aurora A results in mitotic arrest and pre-implantation lethality, preventing studies at later stages of development. Here we report the effects of Aurora A ablation on embryo patterning at early post-implantation stages. Inactivation of Aurora A in the epiblast or visceral endoderm layers of the conceptus leads to apoptosis and inhibition of embryo growth, causing lethality and resorption at approximately E9.5. The effects on embryo patterning, however, depend on the tissue affected by the mutation. Embryos with an epiblast ablation of Aurora A properly establish the anteroposterior axis but fail to progress through gastrulation. In contrast, mutation of Aurora A in the visceral endoderm, leads to posteriorization of the conceptus or failure to elongate the anteroposterior axis. Injection of ES cells into Aurora A epiblast knockout blastocysts reconstitutes embryonic development to E9.5, indicating that the extra-embryonic tissues in these mutant embryos can sustain development to organogenesis stages. Our results reveal new ways to induce apoptosis and to ablate cells in a tissue-specific manner in vivo. Moreover, they show that epiblast-ablated embryos can be used to test the potency of stem cells.

]]> Yeonsoo Yoon et al. Protein-Serine-Threonine Kinases Body Patterning Embryo, Mammalian Mice http://escholarship.umassmed.edu/rivera/20 http://escholarship.umassmed.edu/rivera/20 Thu, 03 Feb 2011 12:58:16 PST Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.

]]> Paul Hasty et al. Animals Blotting, Southern Cloning, Molecular Embryonal Carcinoma Stem Cells *Genetic Vectors Hypoxanthine Phosphoribosyltransferase Mice *Mutagenesis, Insertional Neoplastic Stem Cells Recombination, Genetic Restriction Mapping http://escholarship.umassmed.edu/rivera/19 http://escholarship.umassmed.edu/rivera/19 Thu, 03 Feb 2011 12:58:15 PST Homologous recombination has been used to introduce site-specific mutations into murine embryonic stem (ES) cells with both insertion and replacement vectors. In this study, we compared the frequency of gene targeting with various lengths of homology and found a dramatic increase in targeting with an increase in homology from 1.3 to 6.8 kb. We examined in detail the relationship between the length of homology and the gene-targeting frequency for replacement vectors and found that a critical length of homology is needed for targeting. Adding greater lengths of homology to this critical length has less of an effect on the targeting frequency. We also analyzed the lengths of homology necessary on both arms of the vector for gene replacement events and found that 472 bp of homology is used as efficiently as 1.2 kb in the formation and resolution of crossover junctions.

]]> Paul Hasty et al. Animals Blotting, Southern Cells, Cultured DNA DNA Transposable Elements Embryo, Mammalian Exons Genetic Vectors Hypoxanthine Phosphoribosyltransferase Mice *Mutagenesis, Site-Directed Recombination, Genetic Restriction Mapping *Sequence Homology, Nucleic Acid Stem Cells *Transfection http://escholarship.umassmed.edu/rivera/18 http://escholarship.umassmed.edu/rivera/18 Thu, 03 Feb 2011 12:58:12 PST Many experimental designs require the analysis of genomic DNA from a large number of samples. Although the polymerase chain reaction (PCR) can be used, the Southern blot is preferred for many assays because of its inherent reliability. The rapid acceptance of PCR, despite a significant rate of false positive/negative results, is partly due to the disadvantages of the sample preparation process for Southern blot analysis. We have devised a rapid protocol to extract high-molecular-weight genomic DNA from a large number of samples. It involves the use of a single 96-well tissue culture dish to carry out all the steps of the sample preparation. This, coupled with the use of a multichannel pipette, facilitates the simultaneous analysis of multiple samples. The procedure may be automated since no centrifugation, mixing, or transferring of the samples is necessary. The method has been used to screen embryonic stem cell clones for the presence of targeted mutations at the Hox-2.6 locus and to obtain data from human blood.

]]> Ramiro Ramirez-Solis et al. Animals Blotting, Southern Clone Cells DNA Genome Humans Mice Restriction Mapping Stem Cells http://escholarship.umassmed.edu/rivera/17 http://escholarship.umassmed.edu/rivera/17 Thu, 03 Feb 2011 12:58:10 PST We have analyzed the gene-targeting frequencies and recombination products generated by a series of vectors which target the hprt locus in embryonic stem cells and found the existence of alternative pathways that depend on the location of the double-strand break within the vector. A double-strand break in the targeting homology was found to increase the targeting frequency compared with a double-strand break at the edge of or outside the target homology; this finding agrees with the double-strand break repair model proposed for Saccharomyces cerevisiae. Although a double-strand break in the homology is important for efficient targeting, observations reported here suggest that the terminal ends are not always directly involved in the initial recombination event. Short terminal heterologous sequences which block the homologous ends of the vector may be incorporated into the target locus. A modification of the double-strand break repair model is described to account for this observation.

]]> Paul Hasty et al. Animals Base Sequence Cells, Cultured DNA DNA, Circular DNA, Superhelical Genetic Vectors Hypoxanthine Phosphoribosyltransferase Mice Molecular Sequence Data Oligodeoxyribonucleotides Polymerase Chain Reaction *Recombination, Genetic Sequence Homology, Nucleic Acid Transfection http://escholarship.umassmed.edu/rivera/16 http://escholarship.umassmed.edu/rivera/16 Thu, 03 Feb 2011 12:58:08 PST We analysed DNA through Southern blot and polymerase chain reaction using blood samples of northeastern Mexican families affected by hemophilia A. Our aim was to identify possible carriers of the mutated gene by indirect detection using the Bcl I polymorphism (RFLP) at intron 18 of the factor VIII gene. The sample studied consisted of 43 individuals within eight families with hereditary hemophilia A. Of 17 possible carrier women, three were positive, five were negative, and in the remaining nine, the lack of informativeness (heterozygosity for the polymorphism) of their mothers precluded reaching conclusions. The frequencies found for the Bcl I polymorphism were 63% for the 1.2 kb allelic fragment and 37% for the 0.9 kb allelic fragment. Heterozygote women were found in 48.2% of the families studied. Our results show that probably, the Bcl I RFLP is more useful for HA carrier diagnosis in our sample (northeastern Mexico).

]]> Jaime A. Rivera-Pérez et al. Alleles Blotting, Southern Factor VIII Female Genes Hemophilia A Heterozygote Detection Humans Incidence Introns Male Mexico Pedigree Polymerase Chain Reaction Polymorphism, Restriction Fragment Length http://escholarship.umassmed.edu/rivera/15 http://escholarship.umassmed.edu/rivera/15 Thu, 03 Feb 2011 12:58:07 PST To develop an animal model for cystic fibrosis (CF), targeted gene disruption in embryonic stem (ES) cells was used to generate a duplication of exon 3 (cftrm1Bay allele) of the mouse CF gene. ES cells containing this mutation were used to generate chimeric animals that transmitted the mutant allele through the germline. Homozygous mutant animals display a severe phenotype, with approximately 40% dying within 1 week from intestinal obstruction. RNAase protection analysis of the cftrm1Bay allele did not detect any normal mRNA (< 1-2% of wild-type) in mutant animals. Pathologic changes in the intestines from mutant mice included mucus accumulation in the crypts and intestinal lumen, dilatation of the bases of the crypts, enlargement of goblet cells, and the presence of concretions in the crypts or between the villi. Changes were also present in the mucosal glands of the pharynx and the minor sublingual glands, where dilatation of acini and accumulation of eosinophilic material were evident. Atrophy of acinar cells that may be secondary to nutritional deficiency and mild inflammation in the main pancreatic duct were present in the pancreas of mutant animals. No changes were noted in the lung, trachea, liver, or male reproductive tract of mutant animals, and mutant males were fertile. Homozygous mutant mice showed defects in cAMP-mediated ion transport both in ileum and in cultured fetal tracheal explants. Thus, an additional mouse model for CF has been generated that should prove useful for the understanding of the pathogenesis and the development of treatments for CF.

]]> Wanda K. O'Neal et al. Alleles Animals Base Sequence Chloride Channels Chlorides Cyclic AMP Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator *Disease Models, Animal Exocrine Glands Exons Female Fertility Fetal Diseases Intestinal Obstruction Intestine, Small Lung Male Membrane Proteins Mice Mice, Inbred C57BL Mice, Knockout Molecular Sequence Data Pancreas Phenotype Sodium http://escholarship.umassmed.edu/rivera/14 http://escholarship.umassmed.edu/rivera/14 Thu, 03 Feb 2011 12:58:05 PST Recombination of an insertion vector into its chromosomal homologue is a conservative event in that both the chromosomal and the vector sequences are preserved. However, gene conversion may accompany homologous recombination of an insertion vector. To examine gene conversion in more detail we have determined the targeting frequencies and the structure of the recombinant alleles generated with a series of vectors which target the hprt gene in embryonic stem cells. We demonstrate that gene conversion of the introduced mutation does not significantly limit homologous recombination and that gene conversion occurs without a sequence specific bias for five different mutations. The frequency of the loss of a vector mutation and the gain of a chromosomal sequence is inversely proportional to the distance between the vector mutation and the double-strand break. The loss of a chromosomal sequence and the gain of a vector mutation occurs at a low frequency.

]]> Paul Hasty et al. Alleles Animals Base Sequence Cells, Cultured *DNA Transposable Elements Electroporation *Genetic Vectors Molecular Sequence Data Mutation *Stem Cells http://escholarship.umassmed.edu/rivera/13 http://escholarship.umassmed.edu/rivera/13 Thu, 03 Feb 2011 12:58:03 PST Goosecoid (gsc) is an evolutionarily conserved homeobox gene expressed in the gastrula organizer region of a variety of vertebrate embryos, including zebrafish, Xenopus, chicken and mouse. To understand the role of gsc during mouse embryogenesis, we generated gsc-null mice by gene targeting in embryonic stem cells. Surprisingly, gsc-null embryos gastrulated and formed the primary body axes; gsc-null mice were born alive but died soon after birth with numerous craniofacial defects. In addition, rib fusions and sternum abnormalities were detected that varied depending upon the genetic background. Transplantation experiments suggest that the ovary does not provide gsc function to rescue gastrulation defects. These results demonstrate that gsc is not essential for organizer activity in the mouse but is required later during embryogenesis for craniofacial and rib cage development.

]]> Jaime A. Rivera-Pérez et al. Animals DNA-Binding Proteins Facial Bones Female Fetal Death Gastrula Gene Expression Gene Targeting *Genes, Homeobox Goosecoid Protein *Homeodomain Proteins Mice Mice, Mutant Strains Morphogenesis Phenotype *Repressor Proteins Ribs Skull *Transcription Factors http://escholarship.umassmed.edu/rivera/12 http://escholarship.umassmed.edu/rivera/12 Thu, 03 Feb 2011 12:58:01 PST Maki Wakamiya et al. Amino Acid Sequence Animals Chickens Crosses, Genetic *Embryonic and Fetal Development Female *Gene Expression Regulation, Developmental Goosecoid Protein Homeodomain Proteins Humans In Situ Hybridization Male Mice Mice, Inbred C57BL Mice, Inbred Strains Mice, Knockout Molecular Sequence Data Polymerase Chain Reaction *Repressor Proteins Sequence Alignment Sequence Homology, Amino Acid *Transcription Factors http://escholarship.umassmed.edu/rivera/11 http://escholarship.umassmed.edu/rivera/11 Thu, 03 Feb 2011 12:58:00 PST The homeobox gene goosecoid (gsc) and the winged-helix gene Hepatic Nuclear Factor-3beta (HNF-3beta) are co-expressed in all three germ layers in the anterior primitive streak and at the rostral end of mouse embryos during gastrulation. In this paper, we have tested the possibility of functional synergism or redundancy between these two genes during embryogenesis by generating double-mutant mice for gsc and HNF-3beta. Double-mutant embryos of genotype gsc(-/-);HNF-3beta(+/-) show a new phenotype as early as embryonic days 8.75. Loss of Sonic hedgehog (Shh) and HNF-3beta expression was observed in the notochord and ventral neural tube of these embryos. These results indicate that gsc and HNF-3beta interact to regulate Shh expression and consequently dorsal-ventral patterning in the neural tube. In the forebrain of the mutant embryos, severe growth defects and absence of optic vesicles could involve loss of expression of fibroblast growth factor-8, in addition to Shh. Our results also suggest that interaction between gsc and HNF-3beta regulates other signalling molecules required for proper development of the foregut, branchial arches and heart.

]]> Stefania Filosa et al. Animals Body Patterning DNA-Binding Proteins Digestive System Fibroblast Growth Factor 8 *Fibroblast Growth Factors Gene Expression Regulation, Developmental Genes, Homeobox Gestational Age Goosecoid Protein Growth Substances Hedgehog Proteins Hepatocyte Nuclear Factor 3-beta Heterozygote Homeodomain Proteins In Situ Hybridization Mice Mice, Inbred C57BL Mice, Inbred Strains Mutation Nervous System Notochord Nuclear Proteins Prosencephalon Proteins RNA, Messenger *Repressor Proteins *Trans-Activators *Transcription Factors http://escholarship.umassmed.edu/rivera/10 http://escholarship.umassmed.edu/rivera/10 Thu, 03 Feb 2011 12:57:58 PST Gscl encodes a Goosecoid-related homeodomain protein that is expressed during mouse embryogenesis. In situ hybridization and immunohistochemistry studies show that Gscl is expressed in the pons region of the developing central nervous system and primordial germ cells. Gscl expression is also detected in a subset of adult tissues, including brain, eye, thymus, thyroid region, stomach, bladder and testis. Gscl is located within a region of the mouse genome that is syntenic with the region commonly deleted in DiGeorge and velocardiofacial syndrome (DGS/VCFS) patients. DGS/VCFS patients have craniofacial abnormalities, cardiac outflow defects and hypoplasia of the parathyroid gland and thymus due to haploinsufficiency of a gene or genes located within the deleted region. Thus, the genomic location of Gscl and its expression in a subset of the tissues affected in DGS/VCFS patients suggest that Gscl may contribute to the pathogenesis of DGS/VCFS. To determine the role of Gscl during mouse embryogenesis and in DGS/VCFS, we have deleted Gscl by gene targeting in mouse embryonic stem cells. Both Gscl heterozygous and Gscl null mice were normal and fertile, suggesting that Gscl is not a major factor in DGS/VCFS. Interestingly, expression of the adjacent Es2 gene in the pons region of Gscl null fetuses was absent, suggesting that mutations within the DGS/VCFS region can influence expression of adjacent genes. In addition, embryos that lacked both Gscl and the related Gsc gene appeared normal. These studies represent the first functional analysis of a DGS/VCFS candidate gene in vivo. These Gscl null mice will be an important genetic resource for crosses with other mouse models of the DGS/VCFS.

]]> Maki Wakamiya et al. Abnormalities, Multiple Animals Brain DiGeorge Syndrome Female Fertility Gene Deletion Gene Expression Regulation, Developmental Goosecoid Protein Homeodomain Proteins In Situ Hybridization Male Mice Mice, Inbred C57BL Mice, Inbred Strains Mice, Knockout Mutagenesis, Site-Directed Nuclear Proteins Pregnancy Proteins RNA Probes *Repressor Proteins Survival Syndrome *Transcription Factors http://escholarship.umassmed.edu/rivera/9 http://escholarship.umassmed.edu/rivera/9 Thu, 03 Feb 2011 12:57:56 PST Mice homozygous for a targeted deletion of the homeobox gene Goosecoid (Gsc) have multiple craniofacial defects. To understand the mechanisms responsible for these defects, the behavior of Gsc-null cells was examined in morula aggregation chimeras. In these chimeras, Gsc-null cells were marked with beta-galactosidase (beta-gal) activity using the ROSA26 lacZ allele. In addition, mice with a lacZ gene that had been introduced into the Gsc locus were used as a guide to visualize the location of Gsc-expressing cells. In Gsc-null<->wild-type chimeras, tissues that would normally not express Gsc were composed of both Gsc-null and wild-type cells that were well mixed, reflecting the overall genotypic composition of the chimeras. However, craniofacial tissues that would normally express Gsc were essentially devoid of Gsc-null cells. Furthermore, the nasal capsules and mandibles of the chimeras had defects similar to Gsc-null mice that varied in severity depending upon the proportion of Gsc-null cells. These results combined with the analysis of Gsc-null mice suggest that Gsc functions cell autonomously in mesenchyme-derived tissues of the head. A developmental analysis of the tympanic ring bone, a bone that is always absent in Gsc-null mice because of defects at the cell condensation stage, showed that Gsc-null cells had the capacity to form the tympanic ring condensation in the presence of wild-type cells. However, analysis of the tympanic ring bones of 18.5 d.p.c. chimeras suggests that Gsc-null cells were not maintained. The participation of Gsc-null cells in the tympanic ring condensation of chimeras may be an epigenetic phenomenon that results in a local environment in which more precursor cells are present. Thus, the skeletal defects observed in Gsc-null mice may reflect a regional reduction of precursor cells during embryonic development.

]]> Jaime A. Rivera-Pérez et al. Animals Chimera Craniofacial Abnormalities Ear, Middle Facial Bones Gene Expression Regulation, Developmental *Genes, Homeobox Goosecoid Protein Homeodomain Proteins Mandible Mesoderm Mice Mice, Inbred C57BL Mice, Knockout Nasal Bone *Repressor Proteins Skull *Transcription Factors beta-Galactosidase http://escholarship.umassmed.edu/rivera/8 http://escholarship.umassmed.edu/rivera/8 Thu, 03 Feb 2011 12:57:55 PST The anterior midline tissue (AML) of the late gastrula mouse embryo comprises the axial mesendoderm and the ventral neuroectoderm of the prospective forebrain, midbrain and rostral hindbrain. In this study, we have investigated the morphogenetic role of defined segments of the AML by testing their inductive and patterning activity and by assessing the impact of their ablation on the patterning of the neural tube at the early-somite-stage. Both rostral and caudal segments of the AML were found to induce neural gene activity in the host tissue; however, the de novo gene activity did not show any regional characteristic that might be correlated with the segmental origin of the AML. Removal of the rostral AML that contains the prechordal plate resulted in a truncation of the head accompanied by the loss of several forebrain markers. However, the remaining tissues reconstituted Gsc and Shh activity and expressed the ventral forebrain marker Nkx2.1. Furthermore, analysis of Gsc-deficient embryos reveals that the morphogenetic function of the rostral AML requires Gsc activity. Removal of the caudal AML led to a complete loss of midline molecular markers anterior to the 4th somite. In addition, Nkx2.1 expression was not detected in the ventral neural tube. The maintenance and function of the rostral AML therefore require inductive signals emanating from the caudal AML. Our results point to a role for AML in the refinement of the anteroposterior patterning and morphogenesis of the brain.

]]> Anne Camus et al. Animals Body Patterning Ectoderm Embryonic Induction Embryonic and Fetal Development Gene Expression Regulation, Developmental Goosecoid Protein Hedgehog Proteins Homeodomain Proteins In Situ Hybridization Lac Operon Mesencephalon Mesoderm Mice Morphogenesis Mutation Nerve Tissue Proteins Nuclear Proteins Prosencephalon Proteins *Repressor Proteins Tissue Transplantation *Trans-Activators Transcription Factors http://escholarship.umassmed.edu/rivera/7 http://escholarship.umassmed.edu/rivera/7 Thu, 03 Feb 2011 12:57:54 PST Several lines of evidence suggest that the extraembryonic endoderm of vertebrate embryos plays an important role in the development of rostral neural structures. In mice, neural inductive signals are thought to reside in an area of visceral endoderm that expresses the Hex gene. Here, we have conducted a morphological and lineage analysis of visceral endoderm cells spanning pre- and postprimitive streak stages. Our results show that Hex-expressing cells have a tall, columnar epithelial morphology, which distinguishes them from other visceral endoderm cells. This region of visceral endoderm thickening (VET) is found overlying first the distal and then one side of the epiblast at stages between 5.5 and 5.75 days post coitum (d.p.c.). In addition, we show that the epiblast has an anteroposterior-compressed appearance that is aligned with the position of the VET. Intracellular labeling of VET/Hex-expressing cells reveals an anterior and anterolateral shift from their distal epiblast position. VET/Hex-expressing cells are first localized to the anterior side of the epiblast by 5.75 d.p.c. and form a crescent on the anterior half of the embryo at the onset of gastrulation. Subsequently, VET descendants are distributed along the embryonic/extraembryonic boundary by headfold stages at 7.5 d.p.c. The morphological characteristics and position of VET/Hex-expressing cells distinguishes the future anteroposterior axis of the embryo and provide landmarks to stage mouse embryos at preprimitive streak stages. Moreover, the morphological characteristics of pregastrulation mouse embryos together with the stereotyped shift in the position of visceral endoderm cells reveal similarities among amniote embryos that suggest an evolutionary conservation of the mechanisms that pattern the rostral neurectoderm at pregastrula stages.

]]> Jaime A. Rivera-Pérez et al. Animals Blastocyst Endoderm Gastrula Homeodomain Proteins Mice Transcription Factors http://escholarship.umassmed.edu/rivera/6 http://escholarship.umassmed.edu/rivera/6 Thu, 03 Feb 2011 12:57:52 PST Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4(K1361R)). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4(K1361R) embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4(K1361R) embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4(K1361R) fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.

]]> Amy N. Abell et al. Animals Apoptosis Base Sequence Body Patterning Bone Development DNA Female Gene Expression Regulation, Developmental Gene Expression Regulation, Enzymologic Gene Targeting Humans MAP Kinase Kinase Kinase 4 MAP Kinase Signaling System Male Mice Mice, Inbred C57BL Mice, Mutant Strains Neural Tube Defects Phenotype Phosphorylation Pregnancy Rhombencephalon p38 Mitogen-Activated Protein Kinases http://escholarship.umassmed.edu/rivera/5 http://escholarship.umassmed.edu/rivera/5 Thu, 03 Feb 2011 12:57:51 PST The prevalent model for the generation of axial polarity in mouse embryos proposes that a radial to a linear transition in the expression of primitive streak markers precedes the formation of the primitive streak on one side of the epiblast. This model contrasts with the models of mesoderm formation in other vertebrates as it suggests that the primitive streak is initially established in a radial pattern rather than a localized region of the epiblast. Here, we examine the proposed correlation between the expression of Brachyury and Wnt3, two genes reported as expressed radially in the proximal epiblast, with the movements of proximal anterior epiblast cells at stages leading to the formation of the primitive streak. Our results reveal that neither Brachyury nor Wnt3 forms a ring of expression in the proximal epiblast as previously thought. In embryos dissected between 5.5 and 6.5 dpc, Brachyury is first expressed in the distal extra-embryonic ectoderm and subsequently on one side of the epiblast. Wnt3 expression is evident first in the posterior visceral endoderm of 5.5 dpc embryos and later in the posterior epiblast. Lineage analysis shows that the movements of the proximal epiblast do not restrict Brachyury expression to the posterior epiblast. Our data suggest a model whereby the localized expression of these genes in the posterior epiblast, and hence the formation of the primitive streak, is the result of local cell-cell interactions in the future posterior portion of the egg cylinder rather than regionalization of a radial pattern of expression in proximal epiblast cells.

]]> Jaime A. Rivera-Pérez et al. Animals Body Patterning Cell Communication Cell Movement Ectoderm Endoderm Female Fetal Proteins Gastrula Gene Expression Regulation, Developmental Male Mice Models, Biological T-Box Domain Proteins Wnt Proteins http://escholarship.umassmed.edu/rivera/4 http://escholarship.umassmed.edu/rivera/4 Thu, 03 Feb 2011 12:57:49 PST Crucial aspects of axial development in mice occur at early postimplantation stages from the time of implantation to the appearance of the primitive streak. However, this period of development is notoriously refractory to experimental approaches due to the small size of the conceptus and to the presence of the parietal yolk sac, a protective tripartite membrane that surrounds the developing egg cylinder. Here, we describe a method that combines enzymatic digestion and mechanical manipulation to remove the parietal yolk sac of conceptuses at stages between 5.5 and 6.5 days post coitum. This method, which is compatible with whole-mount in situ hybridization and immunostaining techniques, offers a significant improvement over conventional dissection techniques, and it will greatly facilitate research in early mammalian development.

]]> Jaime A. Rivera-Pérez et al. Animals Collagenases Embryonic Development Endopeptidases Enzymes Fluorescent Antibody Technique Hyaluronoglucosaminidase In Situ Hybridization Mice Microdissection Yolk Sac http://escholarship.umassmed.edu/rivera/3 http://escholarship.umassmed.edu/rivera/3 Thu, 28 Oct 2010 10:22:56 PDT The definitive axes of the mouse embryo can be unequivocally identified in embryos dissected at 5.5 days of gestation. However, how and when are these axes established remains an open question. At pre-implantation stages, different approaches have been aimed at determining if events occurring in the zygote influence the geometrical arrangement of the blastocyst. An intense debate has focused on whether the mouse embryo is a pre-patterned or a regulative structure. At post-implantation stages, the efforts have been concentrated in understanding how extra-embryonic tissues affect the formation of the primitive streak, the caudal marker of the anteroposterior axis. Here I summarize the last 10 years of research in this field.

]]> Jaime A. Rivera-Pérez Animals Blastocyst Body Patterning Cell Polarity Embryo Implantation Embryo, Mammalian Embryonic Development Endoderm Female Gastrula Mice Models, Biological Morphogenesis Morula Pregnancy Primitive Streak Zygote