Localization of neogenin protein during morphogenesis in the mouse embryo

Neogenin, a close relative of the axon guidance receptor DCC, has been shown to be a receptor for members of the Netrin and Repulsive Guidance Molecule families. Recent studies have begun to uncover a role for Neogenin in organogenesis. Here we examine the localization of Neogenin protein in the developing mouse embryo (embryonic day 14.5) when organogenesis is progressing rapidly. We observe that Neogenin protein is restricted to distinct tissue layers within a given organ. In some embryonic epithelia such as the gut and pancreas, Neogenin protein is predominantly polarized to the basal surfaces of the epithelial cells. In contrast, Neogenin is restricted to mesenchymal cells within the lung and kidney. Neogenin is also seen in differentiating skeletal muscle and condensing cartilage throughout the embryo, and in the trigeminal and dorsal root ganglia of the peripheral nervous system. This study supports the emerging role for Neogenin as a key receptor in the establishment of the morphological architecture in many developing organ systems.

Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure

A key factor in the use of assisted reproductive technologies (ART) for diverse species is the safety of procedures for long-term health. By using a mouse model, we have investigated the effect of in vitro culture and embryo transfer (ET) of superovulated embryos on postnatal growth and physiological activity compared with that of embryos developing in vivo. Embryo culture from two-cell to blastocyst stages in T6 medium either with or without a protein source reduced blastocyst trophectoderm and inner cell mass cell number compared with that of embryos developing in vivo. Embryo culture and ET had minimal effects on postnatal growth when compared with in vivo development with an equivalent litter size. However, embryo culture, and to a lesser extent ET, led to an enhanced systolic blood pressure at 21 weeks compared with in vivo development independent of litter size, maternal origin, or body weight. Moreover, activity of enzymatic regulators of cardiovascular and metabolic physiology, namely, serum angiotensin-converting enzyme and the gluconeogenesis controller, hepatic phosphoeno/pyruvate carboxykinase, were significantly elevated in response to embryo culture and/or ET in female offspring at 27 weeks, independent of maternal factors and postnatal growth. These animal data indicate that postnatal physiological criteria important in cardiovascular and metabolic health may be more sensitive to routine ART procedures than growth. © 2007 by The National Academy of Sciences of the USA.

In vitro fertilization, embryo development, and cell lineage segregation after pre- and/or postnatal exposure of female mice to ambient fine particulate matter

Objective: To evaluate effects of pre- and/or postnatal exposure to ambient fine particulate matter on fertilization, embryo development, and cell lineage segregation in preimplantation blastocysts using the IVF mouse model. Design: Animal model. Setting: Academic institution. Animal(S): Six-week-old, superovulated mice. Intervention(s): Pre- and postnatal exposure to filtered air (FA-FA), filtered-ambient air (FA-AA), or ambient air (AA-AA) in exposure chambers 24 hours a day for 9 weeks. Main Outcome Measure(S): Gestation length, litter size, sex ratio, ovarian response to superovulation, fertilization rate, embryo development, blastocyst and hatching rates, total cell count, and proportion of cell allocation to inner-cell mass (ICM) and trophectoderm (TE). Result(S): Gestation length, litter size and birth weight, live-birth index, and sex ratio were similar among exposure groups. Ovarian response was not affected by the exposure protocol. A multivariate effect for pre- and/or postnatal exposure to ambient fine particulate matter on IVF, embryo development, and blastocyst differential staining was found. Cell counts in ICM and ICM/TE ratios in blastocysts produced in the FA-FA protocol were significantly higher than in blastocysts produced in the FA-AA and AA-AA protocols. No difference in total cell count was observed among groups. Conclusion(S): Our study suggests that exposure to ambient fine particulate matter may negatively affect female reproductive health by disrupting the lineage specification at the blastocyst stage without interfering in early development of the mouse embryo. (Fertil Steril (R) 2009;92:1725-35. (C) 2009 by American Society for Reproductive Medicine.)

DNA polymerase activity in trophoblast giant cells in the mouse /

In the developing mouse embryo, the diploid trophectoderm is known to undergo a diploid to giant cell transformation. These cells arise by a process of endoreduplication, characterized by replication of the entire genome without subsequent mitosis or cell division, leading to polyploidy and the formation of giant nuclei. Studies of 13.5 day rat trophoblast derived from the parietal yolk sac have indicated a relatively low rate of DNA polymerase a activity, the noinnal eukaryotic replicase, in comparison to that of DNA polymerase g. These results have suggested that endoreduplication in trophoblast giant cells may not employ the normal replicase enzyme, DNA polymerase a. In order to determine whether a 'switch' from DNA polymerase to DNA polymerase is a necessary concomitant of the diploid to giant cell transformation, two distinct populations of trophoblast giant cells, the primary giant cell derived from the mural trophectoderm and the secondary giant cell derived from the polar trophoectoderm were used. These two populations of trophoblast giant cells can be obtained from the tissue outgrowths of 3.5da blastocysts and the extraembryonic ectoderm (EX) and ectoplacental cone (EPC) of 7.5 day embryos respectively. Tissue outgrowths were treated with aphidicolin, a specific reversible inhibitor of eukaryotic DNA polymerase a, on various days after explantation. The effect of aphidicolin treatment was assessed both qualitatively, using autoradiography and quantitatively by scintillation counting and Feulgen staining. 3 DNA synthesis was measured in control and treated cultures after a Hthymidine pulse. Scintillation counts of the embryo proper revealed that DNA synthesis was consistently inhibited by greater than 907. in the presence of aphidicolin. Inhibition of DNA synthesis in the EX and EPC varied between 81-957. and 82-987. respectively, indicating that most DNA synthesis was mediated by DNA polymerase a, but that a small but significant amount of residual synthesis was indicated. A qualitative approach was then applied to determine whether the apparent residual DNA synthesis was restricted to a subpopulation of giant cells or whether all giant cells displayed a low level of DNA synthesis. Autoradiographs of the ICM of blastocysts and the embryo proper of 7.5da embryos, which acted as diploid control population, was completely inhibited regardless of duration in explant culture. In contrast, primary trophoblast giant cells derived from blastocysts and secondary giant cells derived from the EX and EPC were observed to possess some heavily labelled cells after aphidicolin treatment. These results suggest that although DNA polymerase a is the primary replicating enzyme responsible for endoreduplication in mouse trophoblast giant cells, some nonactivity is also observed. A DNA polymerase assay employing tissue lysates of outgrown 7.5da embryo, EX and EPC tissues was used to attempt to confirm the presence of higher nonactivity in tissues possessing trophoblast giant cells. Employing a series of inhibitors of DNA polymerases, it would appear that DNA polymerase a is the major polymerase active in all tissues of the 7.5da mouse embryo. The nature of the putative residual DNA synthetic activity could not be unequivically determined in this study. Therefore, these results suggest that both primary and secondary trophoblast giant cells possess and use DNA polymerase a in endoreduplicative DNA synthesis. It would appear that the high levels of DNA polymerase g activity reported in trophoblast tissue derived from the 13.5 da rat yolk sac was not a general feature of all endoreduplication.

Assessment of chromatin activity in mouse and human tissues

Pancreatic deoxyribonuclease preferentially digests active genes during all phases of the cell cycle including mitosis. Recently, a DNAse I-directed in ~ nick translation technique has been used to demonstrate differences in the DNAse I sensitivity of euchromatic and heterochromatic regions of mitotic chromosomes. This ill ~ technique has been used in this study to ask whether facultative heterochromatin of the inactive X chromosome can be distinguished from the active X chromosome in mouse and human tissues. In addition to this, in ~ nick translation has been used to distinguish constitutive heterochromatin in mouse and human mitotic chromosomes. Based on relative levels of DNAse I sensitivity, the inactive X chromosome could not be distinguished from the active X chromosome in either mouse or human tissues but regions of constitutive heterochromatin could be distinguished by their relative DNAse I insensitivity. The use of !D situ nick translation was also applied to tissue sections of 7.5 day mouse embryos to ask whether differing levels of DNAse I sensitivity could be detected between different tissue types. Differences in DNAse I sensitivities were detected in three tissues examined; embryonic ectoderm, an embryo-derived tissue, and two extraembryonic tissues, extraembryonic ectoderm and ectoplacental cone. Embryonic ectoderm and extraembryonic ectoderm nuclei possessed comparable levels of DNAse I sensitivity while ectoplacental cone was significantly less DNAse I sensitive. This suggests that tissue-specific mechanisms such as chromatin structure may be involved in the regulation of gene activity in certain tissue types. This may also shed some light on possible tissue specific mechanisms regulating X chromosome activity in the developing mouse embryo.

The characterization of mouse carboxypeptidase N small subunit gene structure and presence in developing embryos

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits and two large subunits that protect the protein from degradation. CPN cleaves carboxy-terminal arginines and lysines from peptides found in the bloodstream such as complement anaphylatoxins, kinins, and creatine kinase MM. In this study, the mouse CPN small subunit (CPN1) coding region, gene structure, and chromosomal location were characterized and the expression of CPN1 was investigated in mouse embryos at different stages of development. The CPN1 gene, which was approximately 29 kb in length, contained nine exons and localized to mouse chromosome 19D2. The fifth and sixth exons of CPN1 encoded the amino acids necessary for substrate binding and catalytic activity. CPN1 RNA was expressed predominately in adult liver and contained a 1371 bp open reading frame encoding 457 amino acids. In the mouse embryo, CPN1 RNA was observed at 8.5 days post coitus (dpc), while its protein was detected at 10.5 dpc. In situ hybridization of the fetal liver detected CPN1 RNA in erythroid progenitor cells at 10.5, 13.5, and 16.5 dpc and in hepatocytes at 16.5 dpc. This was compared to the expression of the complement component C3, the parent molecule of complement anaphylatoxin C3a. Consistently throughout the experiments, CPN1 message and protein preceded the expression of C3. To obtain a better understanding of the biological significance of CPN1 in vivo, studies were initiated to produce a genetically engineered mouse in which the CPN1 gene was ablated. To facilitate this project a targeting vector was constructed by removing the functionally important fifth and sixth exons of the CPN1 gene. Collectively, these studies have: (1) provided important detailed information regarding the structure and organization of the murine CPN1 gene, (2) yielded insights into the developmental expression of mouse CPN1 in relationship to C3 expression, and (3) set the stage for the generation of a CPN1 “knock-out” mouse, which can be used to determine the biological significance of CPN1 in both normal and diseased conditions. ^

Targeted Deletion Of Functionally Validated Enhancers Defines Their Role in Mouse Limb Development

Transcriptional enhancers are genomic DNA sequences that contain clustered transcription factor (TF) binding sites. When combinations of TFs bind to enhancer sequences they act together with basal transcriptional machinery to regulate the timing, location and quantity of gene transcription. Elucidating the genetic mechanisms responsible for differential gene expression, including the role of enhancers, during embryological and postnatal development is essential to an understanding of evolutionary processes and disease etiology. Numerous methods are in use to identify and characterize enhancers. Several high-throughput methods generate large datasets of enhancer sequences with putative roles in embryonic development. However, few enhancers have been deleted from the genome to determine their roles in the development of specific structures, such as the limb. Manipulation of enhancers at their endogenous loci, such as the deletion of such elements, leads to a better understanding of the regulatory interactions, rules and complexities that contribute to faithful and variant gene transcription – the molecular genetic substrate of evolution and disease. To understand the endogenous roles of two distinct enhancers known to be active in the mouse embryo limb bud we deleted them from the mouse genome. I hypothesized that deletion of these enhancers would lead to aberrant limb development. The enhancers were selected because of their association with p300, a protein associated with active transcription, and because the human enhancer sequences drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. To confirm that the orthologous mouse enhancers, mouse 280 and 1442 (M280 and M1442, respectively), regulate expression in the developing limb we generated stable transgenic lines, and examined lacZ expression. In M280-lacZ mice, expression was detected in E11.5 fore- and hindlimbs in a region that corresponds to digits II-IV. M1442-lacZ mice exhibited lacZ expression in posterior and anterior margins of the fore- and hindlimbs that overlapped with digits I and V and several wrist bones. We generated mice lacking the M280 and M1442 enhancers by gene targeting. Intercrosses between M280 -/+ and M1442 -/+, respectively, generated M280 and M1442 null mice, which are born at expected Mendelian ratios and manifest no gross limb malformations. Quantitative real-time PCR of mutant E11.5 limb buds indicated that significant changes in transcriptional output of enhancer-proximal genes accompanied the deletion of both M280 and M1442. In neonatal null mice we observed that all limb bones are present in their expected positions, an observation also confirmed by histology of E18.5 distal limbs. Fine-scale measurement of E18.5 digit bone lengths found no differences between mutant and control embryos. Furthermore, when the developmental progression of cartilaginous elements was analyzed in M280 and M1442 embryos from E13.5-E15.5, transient development defects were not detected. These results demonstrate that M280 and M1442 are not required for mouse limb development. Though M280 is not required for embryonic limb development it is required for the development and/or maintenance of body size – adult M280 mice are significantly smaller than control littermates. These studies highlight the importance of experiments that manipulate enhancers in situ to understand their contribution to development.

Structure of the imprinted mouse Snrpn gene and establishment of its parental-specific methylation pattern

The mouse Snrpn gene encodes the Smn protein, which is involved in RNA splicing. The gene maps to a region in the central part of chromosome 7 that is syntenic to the Prader–Willi/Angelman syndromes (PWS-AS) region on human chromosome 15q11-q13. The mouse gene, like its human counterpart, is imprinted and paternally expressed, primarily in brain and heart. We provide here a detailed description of the structural features and differential methylation pattern of the gene. We have identified a maternally methylated region at the 5′ end (DMR1), which correlates inversely with the Snrpn paternal expression. We also describe a region at the 3′ end of the gene (DMR2) that is preferentially methylated on the paternal allele. Analysis of Snrpn mRNA levels in a methylase-deficient mouse embryo revealed that maternal methylation of DMR1 may play a role in silencing the maternal allele. Yet both regions, DMR1 and DMR2, inherit the parental-specific methylation profile from the gametes. This methylation pattern is erased in 12.5-days postcoitum (dpc) primordial germ cells and reestablished during gametogenesis. DMR1 is remethylated during oogenesis, whereas DMR2 is remethylated during spermatogenesis. Once established, these methylation patterns are transmitted to the embryo and maintained, protected from methylation changes during embryogenesis and cell differentiation. Transfections of DMR1 and DMR2 into embryonic stem cells and injection into pronuclei of fertilized eggs reveal that embryonic cells lack the capacity to establish anew the differential methylation pattern of Snrpn. That all PWS patients lack DMR1, together with the overall high resemblance of the mouse gene to the human SNRPN, offers an excellent experimental tool to study the regional control of this imprinted chromosomal domain.

Neural crest-directed gene transfer demonstrates Wnt1 role in melanocyte expansion and differentiation during mouse development

Wnt1 signaling has been implicated as one factor involved in neural crest-derived melanocyte (NC-M) development. Mice deficient for both Wnt1 and Wnt3a have a marked deficiency in trunk neural crest derivatives including NC-Ms. We have used cell lineage-directed gene targeting of Wnt signaling genes to examine the effects of Wnt signaling in mouse neural crest development. Gene expression was directed to cell lineages by infection with subgroup A avian leukosis virus vectors in lines of transgenic mice that express the retrovirus receptor tv-a. Transgenic mice with tva in either nestin-expressing neural precursor cells (line Ntva) or dopachrome tautomerase (DCT)-expressing melanoblasts (line DCTtva) were analyzed. We overstimulated Wnt signaling in two ways: directed gene transfer of Wnt1 to Ntva+ cells and transfer of β-catenin to DCTtva+ NC-M precursor cells. In both methods, NC-M expansion and differentiation were effected. Significant increases were observed in the number of NC-Ms [melanin+ and tyrosinase-related protein 1 (TYRP1)+ cells], the differentiation of melanin− TYRP1+ cells to melanin+ TYRP1+ NC-Ms, and the intensity of pigmentation per NC-M. These data are consistent with Wnt1 signaling being involved in both expansion and differentiation of migrating NC-Ms in the developing mouse embryo. The use of lineage-directed gene targeting will allow the dissection of signaling molecules involved in NC development and is adaptable to other mammalian developmental systems.

Efficient in vivo manipulation of mouse genomic sequences at the zygote stage.

We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. To assess the ability of this recombinase to excise loxP-flanked DNA sequences at early stages of development, we bred EIIa-cre transgenic mice to two different mouse lines carrying loxP-flanked target sequences: (i) a strain with a single gene-targeted neomycin resistance gene flanked by 1oxP sites and (ii) a transgenic line carrying multiple transgene copies with internal loxP sites. Mating either of these loxP-carrying mouse lines to EIIa-cre mice resulted in first generation progeny in which the loxP-flanked sequences had been efficiently deleted from all tissues tested, including the germ cells. Interbreeding of these first generation progeny resulted in efficient germ-line transmission of the deletion to subsequent generations. These results demonstrate a method by which loxP-flanked DNA sequences can be efficiently deleted in the early mouse embryo. Potential applications of this approach are discussed, including reduction of multicopy transgene loci to produce single-copy transgenic lines and introduction of a variety of subtle mutations into the line.

GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors.

The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) of the glucocorticoid receptor. In the yeast two-hybrid system and in vitro, GRIP1 interacted with the HBDs of the glucocorticoid, estrogen, and androgen receptors in a hormone-regulated manner. When fused to the DNA binding domain of a heterologous protein, the GRIP1 fragment activated a reporter gene containing a suitable enhancer site in yeast cells and in mammalian cells, indicating that GRIP1 contains a transcriptional activation domain. Overexpression of the GRIP1 fragment in mammalian cells interfered with hormone-regulated expression of mouse mammary tumor virus-chloramphenicol acetyltransferase gene and constitutive expression of cytomegalovirus-beta-galactosidase reporter gene, but not constitutive expression from a tRNA gene promoter. This selective squelching activity suggests that GRIM can interact with an essential component of the RNA polymerase II transcription machinery. Finally, while a steroid receptor HBD fused with a GAL4 DNA binding domain did not, by itself, activate transcription of a reporter gene in yeast, coexpression of this fusion protein with GRIP1 strongly activated the reporter gene. Thus, in yeast, GRIP1 can serve as a coactivator, potentiating the transactivation functions in steroid receptor HBDs, possibly by acting as a bridge between HBDs of the receptors and the basal transcription machinery.

Immunolocalization of estrogen receptor protein in the mouse blastocyst during normal and delayed implantation.

We previously showed that estrogen receptor (ER) mRNA is present in preimplantation mouse embryos. The apparent synthesis of ER mRNA by the blastocyst at the time of implantation when estrogen is required was of special interest. A demonstration of the presence of ER protein would support the idea that estrogen can act directly on the embryo. The mouse embryo at the blastocyst stage is differentiated into two cell types, the trophectoderm and the inner cell mass. To determine whether ER mRNA is translated into ER protein and its cell-specific distribution, immunocytochemical analyses were performed in mouse blastocysts. ER protein was detected in all cell types of the normal, dormant, or activated blastocyst. To trace the fate of ER in these cell types, immunocytochemistry was performed in implanting blastocysts and early egg cylinder stage embryos developed in culture. Again, ER was detected in all cells of the implanting blastocyst. At the early egg cylinder stage, continued expression of ER was observed in cells derived from the inner cell mass or the trophoblast. In trophoblast giant cells, ER was concentrated in small regions of the nucleus, possibly the nucleoli, which was similar to that observed in dormant and activated blastocysts. The embryonic expression of ER at such early stages in a broad array of cells suggests that ER may have a general role during early development.

Serotonin regulates mouse cranial neural crest migration.

Serotonergic agents (uptake inhibitors, receptor ligands) cause significant craniofacial malformations in cultured mouse embryos suggesting that 5-hydroxytryptamine (serotonin) (5-HT) may be an important regulator of craniofacial development. To determine whether serotonergic regulation of cell migration might underly some of these effects, cranial neural crest (NC) explants from embryonic day 9 (E9) (plug day = E1) mouse embryos or dissociated mandibular mesenchyme cells (derived from NC) from E12 embryos were placed in a modified Boyden chamber to measure effects of serotonergic agents on cell migration. A dose-dependent effect of 5-HT on the migration of highly motile cranial NC cells was demonstrated, such that low concentrations of 5-HT stimulated migration, whereas this effect was progressively lost as the dose of 5-HT was increased. In contrast, most concentrations of 5-HT inhibited migration of less motile, mandibular mesenchyme cells. To investigate the possible involvement of specific 5-HT receptors in the stimulation of NC migration, several 5-HT subtype-selective antagonists were used to block the effects of the most stimulatory dose of 5-HT (0.01 microM). Only NAN-190 (a 5-HT1A antagonist) inhibited the effect of 5-HT, suggesting involvement of this receptor. Further evidence was obtained by using immunohistochemistry with 5-HT receptor antibodies, which revealed expression of the 5-HT1A receptor but not other subtypes by migrating NC cells in both embryos and cranial NC explants. These results suggest that by activating appropriate receptors 5-HT may regulate migration of cranial NC cells and their mesenchymal derivatives in the mouse embryo.

Elimination of paternal mitochondrial DNA in intraspecific crosses during early mouse embryogenesis.

To examine whether mtDNA is uni- or biparentally transmitted in mice, we developed an assay that can detect sperm mtDNA in a single mouse embryo. In intraspecific hybrids of Mus musculus, paternal mtDNA was detected only through the early pronucleus stage, and its disappearance co-incided with loss of membrane potential in sperm-derived mitochondria. By contrast, in interspecific hybrids between M. musculus and Mus spretus, paternal mtDNA was detected throughout development from pronucleus stage to neonates. We propose that oocyte cytoplasm has a species-specific mechanism that recognizes and eliminates sperm mitochondria and mtDNA. This mechanism must recognize nuclearly encoded proteins in the sperm midpiece, and not the mtDNA or the proteins it encodes, because sperm mitochondria from the congenic strain B6.mtspr, which carries M. spretus mtDNA on background of M. musculus (B6) nuclear genes, were eliminated early by B6 oocytes as in intraspecific crosses. We conclude that cytoplasmic genomes are transmitted uniparentally in intraspecific crosses in mammals as in Chlamydomonas and that leakage of parental mtDNA is limited to interspecific crosses, which rarely occur in nature.

Germ cells enter meiosis in a rostro-caudal wave during development of the mouse ovary

Germ cells in the mouse embryo remain undifferentiated until about 13.5 days post-coitum (dpc), when male germ cells enter mitotic arrest and female germ cells enter meiosis. The molecular signals and transcriptional control mechanisms governing the differential fate of germ cells in males and females remain largely unknown. In order to gain insights into the behavior of germ cells around this period and into likely mechanisms controlling entry into meiosis, we have studied by wholemount in situ hybridization the expression pattern of two germ cell-specific markers, Oct4 and Sycp3, during mouse fetal gonad development. We observed a dynamic wave of expression of both genes in developing ovaries, with Oct4 expression being extinguished in a rostro-caudal wave and Sycp3 being upregulated in a corresponding wave, during the period 13.5-15.5 dpc. These results indicate that entry into meiosis proceeds in a rostro-caudal progression, in turn suggesting that somatically derived signals may contribute to the control of germ cell entry into meiosis in developing ovaries. (C) 2004 Wiley-Liss, Inc.