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In mice, retinal direction selectivity is organized in a map that aligns to the body and gravitational axes of optic flow, and little is known about how this map develops. We find direction selectivity maps are largely present at eye opening and develop normally in the absence of visual experience. Remarkably, in mice lacking the beta2 subunit of neuronal nicotinic acetylcholine receptors (β2-nAChR-KO), which exhibit drastically reduced cholinergic retinal waves in the first postnatal week, selectivity to horizontal motion is absent while selectivity to vertical motion remains. We tested several possible mechanisms that could explain the loss of horizontal direction selectivity in β2-nAChR-KO mice (wave propagation bias, FRMD7 expression, starburst amacrine cell morphology), but all were found to be intact when compared with WT mice. This work establishes a role for retinal waves in the development of asymmetric circuitry that mediates retinal direction selectivity via an unknown mechanism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: In vitro maturation (IVM) is a technology that generates mature oocytes following culture of immature cumulus-oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for certain patient groups. Recently, a biphasic IVM system, capacitation (CAPA)-IVM, has shown improved clinical outcomes relative to standard IVM; however, it remains less efficient than IVF. This study assessed whether supplementation of CAPA-IVM culture media with the novel TGFβ superfamily proteins cumulin and super-GDF9 improves subsequent mouse embryo development., Methods: Immature mouse COCs were cultured by standard IVM or biphasic IVM ± cumulin or super-GDF9., Results: Both cumulin and super-GDF9 in standard IVM significantly improved day-6 blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; p = 0.006; n = 382-406 oocytes). Cumulin or super-GDF9 in CAPA-IVM did not alter embryo yield or blastocyst cell allocation in an unstimulated model. Moreover, cumulin did not alter these outcomes in a mild PMSG stimulation model. Cumulin in CAPA-IVM significantly increased cumulus cell expression of cumulus expansion genes (Ptgs2, Ptx3, Adamts1, Gfat2) and decreased Lhr expression relative to control. However, cumulin-induced mRNA expression of cumulus cell (Ptgs2, Ptx3) and oocyte genes (Gdf9, Bmp15, Oct4, Stella) in CAPA-IVM remained significantly lower than that of in vivo matured cells., Conclusion: Cumulin did not provide an additional beneficial effect in biphasic IVM in terms of blastocyst yield and cell allocation; however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The shape of a neuron facilitates its functionality within neural circuits. Dendrites integrate incoming signals from axons, receiving excitatory input onto small protrusions called dendritic spines. Therefore, understanding dendritic growth and development is fundamental for discerning neural function. We previously demonstrated that EphA7 receptor signaling during cortical development impacts dendrites in two ways: EphA7 restricts dendritic growth early and promotes dendritic spine formation later. Here, the molecular basis for this shift in EphA7 function is defined. Expression analyses reveal that EphA7 full-length (EphA7-FL) and truncated (EphA7-T1; lacking kinase domain) isoforms are dynamically expressed in the developing cortex. Peak expression of EphA7-FL overlaps with dendritic elaboration around birth, while highest expression of EphA7-T1 coincides with dendritic spine formation in early postnatal life. Overexpression studies in cultured neurons demonstrate that EphA7-FL inhibits both dendritic growth and spine formation, while EphA7-T1 increases spine density. Furthermore, signaling downstream of EphA7 shifts during development, such that in vivo inhibition of mTOR by rapamycin in EphA7-mutant neurons ameliorates dendritic branching, but not dendritic spine phenotypes. Finally, direct interaction between EphA7-FL and EphA7-T1 is demonstrated in cultured cells, which results in reduction of EphA7-FL phosphorylation. In cortex, both isoforms are colocalized to synaptic fractions and both transcripts are expressed together within individual neurons, supporting a model where EphA7-T1 modulates EphA7-FL repulsive signaling during development. Thus, the divergent functions of EphA7 during cortical dendrite development are explained by the presence of two variants of the receptor., Competing Interests: The authors have declared that no competing interests exist.

MYCBP2 is a ubiquitin (Ub) E3 ligase (E3) that is essential for neurodevelopment and regulates axon maintenance. MYCBP2 transfers Ub to nonlysine substrates via a newly discovered RING-Cys-Relay (RCR) mechanism, where Ub is relayed from an upstream cysteine to a downstream substrate esterification site. The molecular bases for E2-E3 Ub transfer and Ub relay are unknown. Whether these activities are linked to the neural phenotypes is also unclear. We describe the crystal structure of a covalently trapped E2~Ub:MYCBP2 transfer intermediate revealing key structural rearrangements upon E2-E3 Ub transfer and Ub relay. Our data suggest that transfer to the dynamic upstream cysteine, whilst mitigating lysine activity, requires a closed-like E2~Ub conjugate with tempered reactivity, and Ub relay is facilitated by a helix-coil transition. Furthermore, neurodevelopmental defects and delayed injury-induced degeneration in RCR-defective knock-in mice suggest its requirement, and that of substrate esterification activity, for normal neural development and programmed axon degeneration.

CRISPR/Cas9 machinery delivered as ribonucleoprotein (RNP) to the zygote has become a standard tool for the development of genetically modified mouse models. In recent years, a number of reports have demonstrated the effective delivery of CRISPR/Cas9 machinery via zygote electroporation as an alternative to the conventional delivery method of microinjection. In this study, we have performed side-by-side comparisons of the two RNP delivery methods across multiple gene loci and conclude that electroporation compares very favourably with conventional pronuclear microinjection, and report an improvement in mutagenesis efficiency when delivering CRISPR via electroporation for the generation of simple knock-in alleles using single-stranded oligodeoxynucleotide (ssODN) repair templates. In addition, we show that the efficiency of knock-in mutagenesis can be further increased by electroporation of embryos derived from Cas9-expressing donor females. The maternal supply of Cas9 to the zygote avoids the necessity to deliver the relatively large Cas9 protein, and high efficiency generation of both indel and knock-in allele can be achieved by electroporation of small single-guide RNAs and ssODN repair templates alone. Furthermore, electroporation, compared to microinjection, results in a higher rate of embryo survival and development. The method thus has the potential to reduce the number of animals used in the production of genetically modified mouse models.

Purpose: Human choroidal melanocytes become evident in the last trimester of development, but very little is known about them. To better understand normal and diseased choroidal melanocyte biology we examined their precursors, melanoblasts (MB), in mouse eyes during development, particularly their relation to the developing vasculature and immune cells., Methods: Naïve B6(Cg)-Tyrc-2J/J albino mice were used between embryonic (E) day 15.5 and postnatal (P) day 8, with adult controls. Whole eyes, posterior segments, or dissected choroidal wholemounts were stained with antibodies against tyrosinase-related protein 2, ionized calcium binding adaptor molecule-1 or isolectin B4, and examined by confocal microscopy. Immunoreactive cell numbers in the choroid were quantified with Imaris. One-way ANOVA with Tukey's post hoc test assessed statistical significance., Results: Small numbers of MB were present in the presumptive choroid at E15.5 and E18.5. The density significantly increased between E18.5 (381.4 ± 45.8 cells/mm2) and P0 (695.2 ± 87.1 cells/mm2; P = 0.032). In postnatal eyes MB increased in density and formed multiple layers beneath the choriocapillaris. MB in the periocular mesenchyme preceded the appearance of vascular structures at E15.5. Myeloid cells (Ionized calcium binding adaptor molecule-1-positive) were also present at high densities from this time, and attained adult-equivalent densities by P8 (556.4 ± 73.6 cells/mm2)., Conclusions: We demonstrate that choroidal MB and myeloid cells are both present at very early stages of mouse eye development (E15.5). Although MB and vascularization seemed to be unlinked early in choroidal development, they were closely associated at later stages. MB did not migrate into the choroid in waves, nor did they have a consistent relationship with nerves.

The features of herpes simplex virus 1 (HSV-1) strain 129 (H129), including natural neurotropism and anterograde transneuronal trafficking, make it a potential tool for anterograde neural circuitry tracing. Recently anterograde polysynaptic and monosynaptic tracers were developed from H129 and have been applied for the identification of novel connections and functions of different neural circuitries. However, how H129 viral particles are transported in neurons, especially those of the central nervous system, remains unclear. In this study, we constructed recombinant H129 variants with mCherry-labeled capsids and/or green fluorescent protein (GFP)-labeled envelopes and infected the cortical neurons to study axonal transport of H129 viral particles. We found that different types of viral particles were unevenly distributed in the nucleus, cytoplasm of the cell body, and axon. Most H129 progeny particles were unenveloped capsids and were transported as capsids rather than virions in the axon. Notably, capsids acquired envelopes at axonal varicosities and terminals where the sites forming synapses are connected with other neurons. Moreover, viral capsids moved more frequently in the anterograde direction in axons, with an average velocity of 0.62 ± 0.18 μm/s and maximal velocity of 1.80 ± 0.15 μm/s. We also provided evidence that axonal transport of capsids requires the kinesin-1 molecular motor. These findings support that H129-derived tracers map the neural circuit anterogradely and possibly transsynaptically. These data will guide future modifications and improvements of H129-based anterograde viral tracers. IMPORTANCE Anterograde transneuronal tracers derived from herpes simplex virus 1 (HSV-1) strain 129 (H129) are important tools for mapping neural circuit anatomic and functional connections. It is, therefore, critical to elucidate the transport pattern of H129 within neurons and between neurons. We constructed recombinant H129 variants with genetically encoded fluorescence-labeled capsid protein and/or glycoprotein to visualize viral particle movement in neurons. Both electron microscopy and light microscopy data show that H129 capsids and envelopes move separately, and notably, capsids are enveloped at axonal varicosity and terminals, which are the sites forming synapses to connect with other neurons. Superresolution microscopy-based colocalization analysis and inhibition of H129 particle movement by inhibitors of molecular motors support that kinesin-1 contributes to the anterograde transport of capsids. These results shed light into the mechanisms for anterograde transport of H129-derived tracer in axons and transmission between neurons via synapses, explaining the anterograde labeling of neural circuits by H129-derived tracers., (Copyright © 2020 American Society for Microbiology.)

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Armadillo repeat and Armadillo-like helical domain containing proteins form a large family with diverse and fundamental functions in many eukaryotes. Herein we investigated the spatiotemporal expression pattern of Armadillo-like helical domain containing 4 (or Armh4) as an uncharacterized protein coding mouse gene, within the mouse embryo during the initial stages of heart morphogenesis. We found Armh4 is initially expressed in both first heart field as well as the second heart field progenitors and subsequently within predominantly their cardiomyocyte derivatives. Armh4 expression is initially cardiac-restricted in the developing embryo and is expressed in second heart field subpharyngeal mesoderm prior to cardiomyocyte differentiation, but Armh4 diminishes as the embryonic heart matures into the fetal heart. Armh4 is subsequently expressed in craniofacial structures and neural crest-derived dorsal root and trigeminal ganglia. Whereas lithium chloride-induced stimulation of Wnt/β-catenin signaling elevated Armh4 expression in both second heart field subpharyngeal mesodermal progenitors and outflow tract, right ventricle and atrial cardiomyocytes, neither a systemic loss of Islet-1 nor an absence of cardiac neural crest cells had any effect upon Armh4 expression. These results confirm that Wnt/β-catenin-responsive Armh4 is a useful specific biomarker of the FHF and SHF cardiomyocyte derivatives only., (Copyright © 2019 Elsevier B.V. All rights reserved.)

CENP-A is an essential histone H3 variant that epigenetically marks the centromeric region of chromosomes. Here we show that CENP-A nucleosomes form characteristic clusters during the G1 phase of the cell cycle. 2D and 3D super-resolution microscopy and segmentation analysis reveal that these clusters encompass a globular rosette-like structure, which evolves into a more compact structure in late G1. The rosette-like clusters contain numerous CENP-A molecules and form a large cellular structure of ∼250-300 nm diameter with remarkably similar shapes for each centromere. Co-localization analysis shows that HJURP, the CENP-A chaperone, is located in the center of the rosette and serves as a nucleation point. The discovery of an HJURP-mediated CENP-A nucleation in human cells and its structural description provide important insights into the mechanism of CENP-A deposition and the organization of CENP-A chromatin in the centromeric region.

Midkine (MDK) and Pleiotrophin (PTN) belong to a group of heparin-binding growth factors that has been shown to have pleiotropic functions in various biological processes during development and disease. Development of the vertebrate eye is a multistep process that involves coordinated interactions between neuronal and non-neuronal cells, but very little is known about the potential function of MDK and PTN in these processes. In this study, we demonstrate by section in situ hybridization, the spatiotemporal expression of MDK and PTN during ocular development in chick and mouse. We show that MDK and PTN are expressed in dynamic patterns that overlap in a few non-neuronal tissues in the anterior eye and in neuronal cell layers of the posterior eye. We show that the expression patterns of MDK and PTN are only conserved in a few tissues in chick and mouse but they overlap with the expression of some of their receptors LRP1, RPTPZ, ALK, NOTCH2, ITGβ1, SDC1, and SDC3. The dynamic expression patterns of MDK, PTN and their receptors suggest that they function together during the multistep process of ocular development and they may play important roles in cell proliferation, adhesion, and migration of neuronal and non-neuronal cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We have previously reported the deregulatory impact of ethanol on global DNA methylation of brain-derived neural stem cells (NSC). Here, we conducted a genome-wide RNA-seq analysis in differentiating NSC exposed to different modes of ethanol exposure. RNA-seq results showed distinct gene expression patterns and canonical pathways induced by ethanol exposure and withdrawal. Short-term ethanol exposure caused abnormal up-regulation of synaptic pathways, while continuous ethanol treatment profoundly affected brain cells' morphology. Ethanol withdrawal restored the gene expression profile of differentiating NSC without rescuing impaired expression of epigenetics factors. Ingenuity Pathway Analysis (IPA) analysis predicated that ethanol may impact synaptic functions via GABA receptor signalling pathway and affects neural system and brain morphology. We identified Sptbn2, Dcc, and Scn3a as candidate genes which may link alcohol-induced neuronal morphology to brain structural abnormalities, predicted by IPA analysis. Cross-examination of Scn3a and As3mt in differentiated NSC from two different mouse strains (BL6 and CD1) showed a consistent pattern of induction and reduction, respectively. Collectively, our study identifies genetic networks, which may contribute to alcohol-mediated cellular and brain structural dysmorphology, contributing to our knowledge of alcohol-mediated damage to central nervous system, paving the path for better understanding of FASD pathobiology.

Life-long regeneration of healthy muscle by cell transplantation is an ideal therapy for patients with degenerative muscle diseases. Yet, obtaining muscle stem cells from patients is very limited due to their exhaustion in disease condition. Thus, development of a method to obtain healthy myogenic stem cells is required. Here, we showed that the four transcription factors, Six1, Eya1, Esrrb, and Pax3, converts fibroblasts into induced myogenic stem cells (iMSCs). The iMSCs showed effective differentiation into multinucleated myotubes and also higher proliferation capacity than muscle derived stem cells both in vitro and in vivo. The iMSCs do not lose their proliferation capacity though the passaging number is increased. We further isolated CD106-negative and α7-integrin-positive iMSCs (sort-iMSCs) showing higher myogenic differentiation capacity than iMSCs. Moreover, genome-wide transcriptomic analysis of iMSCs and sort-iMSCs, followed by network analysis, revealed the genes and signaling pathways associated with enhanced proliferation and differentiation capacity of iMSCs and sort-iMSCs, respectively. The stably expandable iMSCs provide a new source for drug screening and muscle regenerative therapy for muscle wasting disease.

Nucleated circulating red blood cells (RBCs) of developing zebrafish, chick and mouse embryos can actively proliferate. While marrow- or organ-mediated erythropoiesis has been widely studied, transforming in vivo processes of circulating RBCs are under little scrutiny. We employed confocal, stereo- and electron microscopy to document the maturation of intravascular RBCs . In zebrafish embryos (32-72 h post-fertilization), RBC splitting in the caudal vein plexus follows a four-step program: (i) nuclear division with continued cytoplasmic connection between somata; (ii) dumbbell-shaped RBCs tangle at transluminal vascular pillars; (iii) elongation; and (iv) disruption of soma-to-soma connection. Dividing RBCs of chick embryos, however, retain the nucleus in one of their somata. Here, RBC splitting acts to pinch off portions of cytoplasm, organelles and ribosomes. Dumbbell-shaped primitive RBCs re-appeared as circulation constituents in mouse embryos. The splitting of circulating RBCs thus represents a biologically relevant mechanism of RBC division and maturation during early vertebrate ontogeny., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Mutation in a growing spectrum of genes is known to either cause or contribute to primary or secondary microcephaly. In primary microcephaly the genetic determinants frequently involve mutations that contribute to or modulate the microtubule cytoskeleton by causing perturbations of neuronal proliferation and migration. Here we describe four patients from two unrelated families each with an infantile neurodegenerative disorder characterized by loss of developmental milestones at 9–24 months of age followed by seizures, dystonia and acquired microcephaly. The patients harboured homozygous missense mutations (A475T and A586V) in TBCD, a gene encoding one of five tubulin-specific chaperones (termed TBCA-E) that function in concert as a nanomachine required for the de novo assembly of the α/β tubulin heterodimer. The latter is the subunit from which microtubule polymers are assembled. We found a reduced intracellular abundance of TBCD in patient fibroblasts to about 10% (in the case of A475T) or 40% (in the case of A586V) compared to age-matched wild type controls. Functional analyses of the mutant proteins revealed a partially compromised ability to participate in the heterodimer assembly pathway. We show via in utero shRNA-mediated suppression that a balanced supply of tbcd is critical for cortical cell proliferation and radial migration in the developing mouse brain. We conclude that TBCD is a novel functional contributor to the mammalian cerebral cortex development, and that the pathological mechanism resulting from the mutations we describe is likely to involve compromised interactions with one or more TBCD-interacting effectors that influence the dynamics and behaviour of the neuronal cytoskeleton., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Notch signaling plays a crucial role in controling the proliferation and differentiation of stem and progenitor cells during embryogenesis or organogenesis, but its regulation is incompletely understood. BLOS2, encoded by the Bloc1s2 gene, is a shared subunit of two lysosomal trafficking complexes, biogenesis of lysosome-related organelles complex-1 (BLOC-1) and BLOC-1-related complex (BORC). Bloc1s2 -/- mice were embryonic lethal and exhibited defects in cortical development and hematopoiesis. Loss of BLOS2 resulted in elevated Notch signaling, which consequently increased the proliferation of neural progenitor cells and inhibited neuronal differentiation in cortices. Likewise, ablation of bloc1s2 in zebrafish or mice led to increased hematopoietic stem and progenitor cell production in the aorta-gonad-mesonephros region. BLOS2 physically interacted with Notch1 in endo-lysosomal trafficking of Notch1. Our findings suggest that BLOS2 is a novel negative player in regulating Notch signaling through lysosomal trafficking to control multiple stem and progenitor cell homeostasis in vertebrates., Competing Interests: The authors declare that no competing interests exist.

Accurate mouse sexing is vital when conducting research examining sexual dimorphisms. Late fetal and newborn mouse pups are more immature than many previously described sexing methods allow. This study compares the sexing accuracy of a newly described internal gonad sexing method to a recently described peritoneal pigmentation sexing method in embryonic day 20 C57BL/6J mouse pups, using Sry genotyping to confirm the sex. The internal gonad sexing method was found to be highly accurate, while the peritoneal pigmentation method was slightly less accurate. Therefore, while Sry genotyping remains the gold standard, immediate and less expensive sexing methods can be performed accurately as early as the late fetal period in C57BL/6J mice.

Interspecific hybridization between closely related mammalian species, including various species of the genus Mus, is commonly associated with abnormal growth of the placenta and hybrid foetuses, a phenomenon known as hybrid placental dysplasia (HPD). The role of HPD in speciation is anticipated but still poorly understood. Here, we studied placental and foetal growth in F1 crosses between four inbred mouse strains derived from two house mouse subspecies, Mus musculus musculus and Mus musculus domesticus. These subspecies are in the early stage of speciation and still hybridize in nature. In accordance with the maternal-foetal genomic conflict hypothesis, we found different parental influences on placental and foetal development, with placental weight most affected by the father's body weight and foetal weight by the mother's body weight. After removing the effects of parents' body weight, we did not find any significant differences in foetal or placental weights between intra-subspecific and inter-subspecific F1 crosses. Nevertheless, we found that the variability in placental weight in inter-subspecific crosses is linked to the X chromosome, similarly as for HPD in interspecific mouse crosses. Our results suggest that maternal-foetal genomic conflict occurs in the house mouse system, but has not yet diverged sufficiently to cause abnormalities in placental and foetal growth in inter-subspecific crosses. HPD is thus unlikely to contribute to speciation in the house mouse system. However, we cannot rule out that it might have contributed to other speciation events in the genus Mus, where differences in the levels of polyandry exist between the species., (© 2015 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2015 European Society For Evolutionary Biology.)

In mice, a minimum number of healthy embryos is required to trigger and maintain pregnancy. Therefore, when recovering mouse embryos from a limited litter, one useful technique is to transfer carrier ICR embryos along with the embryos of interest, a technique referred to as cotransfer. In this study, we examined suitable mouse strains for cotransfer with C57BL/6J (B6) embryos in regards to the maintenance of pregnancy, number of pups born, intrauterine growth, and postnatal growth. Because the coat color of B6 is black, we compared two white coat-colored strains, SJL/J and ICR. Cotransfer of SJL/J and ICR embryos had similar effects on maintenance of pregnancy, number of pups born, and intrauterine growth. However, the postnatal growth of B6 mouse pups cotransferred and grown with SJL/J pups was better than for B6 mouse pups cotransferred and grown with ICR pups, suggesting competition among littermates. These results demonstrate that cotransfer of SJL/J embryos will be useful not only as carrier embryos with B6-background embryos but also as a model system to examine littermate competition.

c-Abl is a proto-oncogene that is essential for mouse development and tissue homeostasis. Misregulation of c-Abl, as seen in the constitutively active BCR-ABL, is the leading cause of human chronic myeloid leukemia. However, how the Abl proteins execute their functions still remains largely unknown. Here, we report an important role for c-Abl in replicative senescence and immortalization by regulating the expression of two tumor suppressors that induce cellular senescence, p53 and p16(INK4a). Using primary mouse embryonic fibroblasts (MEFs), we show that c-Abl (-/-) cells were more resistant to immortalization than wildtype cells using a standard 3T3 or 3T9 protocol. We could only immortalize three out of nine c-Abl (-/-) MEF cultures even when we increased the number of starting cells. This resistance was attributed to premature senescence and reduced survival in senescent c-Abl (-/-) cells due to an increase in p16(INK4a) and p53 expression. Deleting p53 allows c-Abl (-/-) p53 (-/-) MEFs to bypass senescence to be spontaneously immortalized. Cell immortalization, but not senescence, was generally accompanied by mutations in p53 in both wildtype and c-Abl (-/-) MEFs, although the spectrum is different from that of human tumors. The role for c-Abl in regulating cell senescence and immortalization might explain some of the developmental defects in c-Abl (-/-) mice and how BCR-ABL transforms cells.

Common anaesthetic and analgesic agents used during pregnancy in mice have been observed to cause fetal growth restriction. We investigated the impact of therapeutic doses of three anaesthetics (ketamine/xylazine, isoflurane, and tribromoethanol) and two analgesics (buprenorphine and meloxicam) on fetal and placental growth. Pregnant mice were treated with one of these agents at fertilization (E0), attachment (E4), beginning of organogenesis (E6), end of organogenesis (E12), or during the logarithmic growth phase (E15), or they were placed into an untreated control group. At term (E18), fetal and placental growth were evaluated, morphological analyses were performed, and skeletal measurements were conducted. Fetal growth was reduced significantly (P < 0.01) by ketamine/xylazine treatment at E0, E4, E12, or E15, by isoflurane administered at E0 or E6, and by tribromoethanol administered at E6 or E12. Two-day treatment with buprenorphine beginning at E4 or E6, or with meloxicam at E0 also significantly reduced fetal growth (P < 0.01). Neither placental growth nor litter size was significantly affected by any of these agents. The occurrence of microphthalmia was nearly eight-fold higher (P < 0.05) in response to buprenorphine administration at E6 compared with controls. The length of the humerus was reduced at most gestation times in response to each of these agents and was correlated (P < 0.01) with fetal weight for ketamine/xylazine, tribromoethanol, and meloxicam. These data reveal patterns of acceptable and detrimental anaesthetic and analgesic use during fetal development and have refined our capability to provide recommendations for the use of these agents during pregnancy in the mouse.

Micro X-ray computed tomography (micro-CT) is widely used in preclinical studies of small animals. However, due to the low soft tissue contrast, segmentation of soft tissues in the micro-CT image is a challenging problem. To gain a better understanding of the macroscopic anatomy of the mouse embryo, 3 fixation methods and 3 metal stainings were examined for micro-CT using C57BL/6J mouse embryos in the present study. The examination demonstrated that 1% acetic acid/95% ethanol fixative together with zinc staining provided a high contrast micro-CT image, enabling the segmentation of soft tissues. Then, using this condition, the macroscopic embryo structure of the nude mouse was examined, revealing lack of a thymus. It appears that micro-CT with the fixation and staining condition devised in the present study could be a powerful tool in detecting the effects of various mutations at embryonic stages.

Eccrine sweat glands in the mouse are found only on the footpads and, when mature, resemble human eccrine glands. Eccrine gland anlagen were first apparent at 16.5 days postconception (DPC) in mouse embryos as small accumulations of cells in the mesenchymal tissue beneath the developing epidermis resembling hair follicle placodes. These cells extended into the dermis where significant cell organization, duct development, and evidence of the acrosyringium were observed in 6- to 7-postpartum day (PPD) mice. Mouse-specific keratin 1 (K1) and 10 (K10) expression was confined to the strata spinosum and granulosum. In 16.5 and 18.5 DPC embryos, K14 and K17 were both expressed in the stratum basale and diffusely in the gland anlagen. K5 expression closely mimicked K17 throughout gland development. K6 expression was not observed in the developing glands of the embryo but was apparent in the luminal cell layer of the duct by 6 to 7 PPD. By 21 PPD, the gland apertures appeared as depressions in the surface surrounded by cornified squames, and the footpad surface lacked the organized ridge and crease system seen in human fingers. These data serve as a valuable reference for investigators who use genetically engineered mice for skin research.

The C57BL/6 mouse strain is used widely for producing transgenic and knockout strains. Sperm motility is extremely low after a freeze-thaw process. Although intracytoplasmic sperm injection (ICSI) can be used to produce embryos from sperm with low or even no motility, its success rate is poor in the C57BL/6 strain. In particular, the survival of C57BL/6 oocytes after ICSI is extremely low compared with that of hybrid strains. We found that the survival percentages of C57BL/6J oocytes (63% and 64%) were lower than those of B6D2F1 oocytes (80% and 80%) when B6D2F1 and C57BL/6J sperm were injected, respectively. For C57BL/6J mice, 87%, 72%, 64%, 56%, and 59% of oocytes survived after ICSI in media containing 61.62, 71.62, 81.62, 91.62, and 101.62 mM NaCl, respectively. In addition, 64%, 81%, and 79% of oocytes survived after ICSI in media with 4.83, 14.83, and 24.83 mM KCl, respectively. Our results suggest that the survival of C57BL/6J oocytes after ICSI is improved by using Na(+)-deficient and K(+)-rich media.

The objective of this study was to investigate the capability of bank vole (Myodes glareolus) embryonic cells to sustain their pluripotent character during in vitro culture, and to determine the optimal conditions for derivation of embryonic stem (ES) cells. We compared the presence of specific pluripotency (Oct4, Ssea1) and differentiation markers (Gata4 - primitive endoderm marker; Cdx2 - trophectoderm marker) in blastocysts and inner cell mass (ICM) outgrowths obtained from blastocysts of bank vole, and two mouse hybrids F1(C57Bl/6xCBA/H) and F1(C57Bl/6x129/Sv), which differ in the permissiveness of giving rise to ES cells. We found that, in contrast to mouse, the expression of pluripotency markers in the cells of bank vole ICM outgrowths is progressively downregulated and rapidly lost by the 4th day of culture. This correlates with the appearance of cells expressing Gata4 and Cdx2, indicating differentiation towards primitive endoderm and derivatives of trophectoderm, respectively. We have also shown that heterologous cytokine leukaemia inhibitory factor (LIF) in conjunction with either homologous or heterologous feeder layer is unable to delay differentiation and preserve pluripotency of bank vole embryonic cells. Thus, the conditions optimised for mouse do not support the maintenance of bank vole embryonic cells in the undifferentiated state and do not allow for the isolation of the ES cells. Instead, combination of fibroblast growth factor 2 and activin A allows retention of Oct4 expression in bank vole blastocyst outgrowths during 4-day culture, indicating that signaling pathways operating in human, rather than mouse ES cells, might be involved in the process of self-renewal of bank vole embryonic cells.

The wings apart-like (Wapl) protein is required to hold sister chromatids together in mitotic heterochromatin in Drosophila melanogaster. It is localized on the synaptonemal complex (SC), a meiosis-specific structure connecting one pair of sister chromatids to the homologous pair in mouse pachytene spermatocytes. The human Wapl is a cohesin-binding protein that facilitates cohesin's timely release from chromosome arms during prophase. The objective of the present study was to determine the subcellular localization of the mouse Wapl on female meiotic chromosomes at pachynema. The pachytene oocytes were isolated from foetal ovaries at 18.5 dpc and double immunostained with anti-synaptonemal complex protein 2 (SYCP2) and anti-Wapl. In the pachytene oocytes examined, mouse Wapl was colocalized with SYCP2 on the SC. Our results further implicated that Wapl might play a crucial role in meiotic chromosome remodelling at early meiosis.

Genetically modified mouse strains derived from embryonic stem (ES) cells are powerful tools for gene function analysis. ES cells from the C57BL/6 mouse strain are not widely used to generate mouse models despite the advantage of a defined genetic background. We assessed genetic variation in six such ES cell lines with 275 SSLP markers. Compared to C57BL/6, Bruce4 differed at 34 SSLP markers and had significant heterozygosity on three chromosomes. BL/6#3 and Dale1 ES cell lines differed at only 3 SSLP makers. The C2 and WB6d ES cell lines differed at 6 SSLP markers. It is important to compare the efficiency of producing mouse models with available C57BL/6 ES cells relative to standard 129 mouse strain ES cells. We assessed genetic stability (the tendency of cells to become aneuploid) in 110 gene-targeted ES cell clones from the most widely used C57BL/6 ES cell line, Bruce4, and 710 targeted 129 ES cell clones. Bruce4 clones were more likely to be aneuploid and unsuitable for ES cell-mouse chimera production. Despite their tendency to aneuploidy and consequent inefficiency, use of Bruce4 ES cells can be valuable for models requiring behavioral studies and other mouse models that benefit from a defined C57BL/6 background.

Brain functions are the products of dynamic interactions between multiple genes and environments. Accordingly, there are large differences among mouse strains at the behavioral and neurobiological levels. Therefore, it is crucial to manipulate genes on the same and homogenous genetic background and then to analyze and compare the phenotypes of various genetically modified mice. Furthermore, a conditional gene targeting to restrict the gene knockout to specific cells and time is a powerful tool to investigate the molecular basis of higher brain functions such as learning and memory. We have developed a system employing Cre-progesterone receptor fusion recombinase for temporal regulation of gene targeting and Flp/frt recombination system for elimination of marker genes. Importantly, both the recombinase lines and target mice have been produced with embryonic stem cells derived from the C57BL/6 strain suitable for brain function analysis. Thus, we have established an inducible and neuron-specific gene targeting system on the pure C57BL/6 genetic background.

Embryonic stem (ES) cells can differentiate spontaneously into various lineages in vitro. However, spontaneous commitment of ES cells to the adipocyte lineage is rare. In the present study, bone morphogenic protein-4 (BMP-4) is described as a factor inducing adipocyte differentiation from ES cells at a high rate. For this reason, ES-cell-derived embryoid bodies (EBs) in suspension cultures were exposed to different doses of BMP-4 for 5 days before they were plated onto gelatin-coated tissue culture plates. Moreover, the effect of serum-containing and serum-free media in three different combinations was assessed. Plated EBs, stained with Sudan Black and processed for transmission and scanning electron microscopy, were observed daily for adipocyte formation. Treatment with BMP-4 resulted in the appearance of adipocyte clusters in EBs' outgrowth, depending on the doses applied. Early in differentiation, many small fat droplets were observed in adipocytes, while later on they coalesced and formed a few large fat droplets. Adipocyte clusters had a fibrillar and vascular stroma, and each adipocyte was surrounded with a reticular external lamina. Furthermore, the appearance and development of adipocytes and their changes following 2-3 weeks of starvation mimicked live adipose tissue. In fact, understanding the biological activity of growth and differentiation factors is needed to regulate and direct stem cell differentiation to specific cell types in vitro.

Rationale: Cyp2a5, the mouse homologue of human CYP2A6, encodes for the enzyme responsible for the primary metabolism of nicotine. Variation in human CYP2A6 activity can alter the amount smoked such as number of cigarettes smoked per day and smoking intensity. Different mouse strains self-administer different amounts of oral nicotine and quantitative trait loci analyses in mice suggested that Cyp2a5 may be involved in differential nicotine consumption behaviors., Objectives: The goal of this study was to examine whether in vivo nicotine consumption levels were associated with CYP2A5 protein levels and in vitro nicotine metabolism in mice., Methods: F2 mice propagated from high (C57Bl/6) and low (St/bJ) nicotine consuming mice were analyzed for CYP2A5 hepatic protein levels and in vitro nicotine metabolizing activity., Results: We found that F2 male high-nicotine (n=8; 25.1+/-1.2 microg nicotine/day) consumers had more CYP2A5 protein, compared to low (n=11; 3.8+/-1.4 microg nicotine/day) consumers (10.2+/-1.0 vs 6.5+/-1.3 CYP2A5 units). High consumers also metabolized nicotine faster than the low consumers (6 microM: 0.18+/-0.04 vs 0.14+/-0.07; 30 microM: 0.36+/- 0.06 vs 0.26+/-0.13; 60 microM: 0.49+/-0.05 vs 0.32+/-0.17 nmol/min/mg). In contrast, female high- (25.1+/-2.1 microg nicotine/day) and low-nicotine (4.7+/-1.4 microg nicotine/day) consumers did not show pronounced differences in nicotine metabolism or CYP2A4/5 protein levels; this is consistent with other studies of sex differences in response to nicotine., Conclusions: These data suggested that among male F2 mice, increased nicotine self-administration is associated with increased rates of nicotine metabolism, most likely, as a result of greater CYP2A5 protein levels.

Aneuploidy results from nondisjunction of chromosomes in meiosis and is the leading cause of developmental disabilities and mental retardation in humans. Therefore, understanding aspects of chromosome segregation in a genetic model is of value. Mice heterozygous for a (2.8) Robertsonian translocation were intercrossed with chromosomally normal mice and Chromosome 2 was genotyped for number and parental origin in 836 individuals at 8.5 dpc. The frequency of nondisjunction of this Robertsonian chromosome is 1.58%. Trisomy of Chromosome 2 with two maternally derived chromosomes is the most developmentally successful aneuploid karyotype at 8.5 dpc. Trisomy of Chromosome 2 with two paternally derived chromosomes is developmentally delayed and less frequent than the converse. Individuals with maternal or paternal uniparental disomy of Chromosome 2 were not detected at 8.5 dpc. Nondisjunction events were distributed randomly across litters, i.e., no evidence for clustering was found. Transmission ratio distortion is frequently observed in Robertsonian chromosomes and a bias against the transmission of the (2.8) Chromosome was detected. Interestingly, this was observed for female and male transmitting parents.

Complement regulatory proteins prevent excessive complement system activation and deposition, which can lead to host tissue damage, including fetal loss during pregnancy. To further understand the regulation of complement during development, we examined the expression of the complement protein, C3, and the active subunit of carboxypeptidase N (CPN1), the complement anaphylatoxin regulator. RNA and protein analyses indicated that CPN1 expression occurred as early as 8.5 days post coitus (dpc) and continued through birth. At 10.5 and 13.5 dpc, in situ hybridization revealed CPN1 RNA in erythroid progenitor cells. At 16.5 dpc, expression of CPN1 was also detected in hepatocytes. In comparison to CPN1, C3 RNA expression occurred later (after 13.5 dpc). Moreover, C3 expression was limited to the liver erythroid progenitor cells at 16.5 dpc. These results demonstrated that mouse embryos contain RNA and protein for both C3 and CPN1, and CPN1 expression precedes that of C3 by several days., (Copyright 2003 Elsevier Ltd.)

According to observations in various cell lines, elimination of the cyclin-dependent kinase inhibitor p27(KIP1) during the late G1 phase of the cell cycle is required for progression to the S phase. Eyes from C57BL/6 mice at embryonic days 13, 14, and 18, and at 4 weeks of age, were analyzed by a bromodeoxyuridine cell proliferation assay and by immunocytochemistry using anti-p27(KIP1) antibody. On embryonic days 14 and 18, p27(KIP1) was detected in the ciliary body. This protein also was detected in the nuclei of the many cells of the retinal pigment epithelium on embryonic day 18, and was present in all such cells at 4 weeks of age. When p27(KIP1)-/- knockout and control mice were injected with bromodeoxyuridine between postnatal days 7 and 10 and analyzed on day 11, positive cells were abundant in the retinal pigment epithelium and the ciliary body of p27(KIP1)-/- mice, whereas few cells were positive in control mice. By fluorescent nuclear staining in whole mounts of retinal pigment epithelium at 12 weeks of age, more nuclei were present in p27(KIP1)-/- than in the wild-type mice. These results suggest that p27(KIP1) was involved in regulation of proliferation in the RPE and the ciliary body.

Herpesvirus entry mediator (HVEM) is a member of the tumor necrosis factor (TNF) receptor family used as a cellular receptor by virion glycoprotein D (gD) of herpes simplex virus (HSV). Both human and mouse forms of HVEM can mediate entry of HSV-1 but have no entry activity for pseudorabies virus (PRV). To assess the antiviral potential of HVEM in vivo, three transgenic mouse lines expressing a soluble form of HVEM (HVEMIg) consisting of an extracellular domain of murine HVEM and the Fc portion of human IgG1 were generated. All of the transgenic mouse lines showed marked resistance to HSV-1 infection when the mice were challenged intraperitoneally with HSV-1, but not to PRV infection. The present results demonstrate that HVEMIg is able to exert a significant antiviral effect against HSV-1 infection in vivo.

ETnII elements are mobile members of the repetitive early transposon family of mouse long terminal repeat (LTR) retroelements and have caused a number of mutations by inserting into genes. ETnII sequences lack retroviral genes, but the recent discovery of related MusD retroviral elements with regions similar to gag, pro, and pol suggests that MusD provides the proteins necessary for ETnII transposition in trans. For this study, we analyzed all ETnII elements in the draft sequence of the C57BL/6J genome and classified them into three subtypes (alpha, beta, and gamma) based on structural differences. We then used database searches and quantitative real-time PCR to determine the copy number and expression of ETnII and MusD elements in various mouse strains. In 7.5-day-old embryos of a mouse strain in which two mutations due to ETnII-beta insertions have been identified (SELH/Bc), we detected a three- to sixfold higher level of ETnII-beta and MusD transcripts than in control strains (C57BL/6J and LM/Bc). The increased ETnII transcription level can in part be attributed to a higher number of ETnII-beta elements, but 70% of the MusD transcripts appear to have been derived from one or a few MusD elements that are not detectable in C57BL/6J mice. This element belongs to a young MusD subgroup with intact open reading frames and identical LTRs, suggesting that the overexpressed element(s) in SELH/Bc mice might provide the proteins for the retrotransposition of ETnII and MusD elements. We also show that ETnII is expressed up to 30-fold more than MusD, which could explain why only ETnII, but not MusD, elements have been positively identified as new insertions.

A Robertsonian translocation results in a metacentric chromosome produced by the fusion of two acrocentric chromosomes. Rb heterozygous mice frequently generate aneuploid gametes and embryos, providing a good model for studying meiotic nondisjunction. We intercrossed mice heterozygous for a (7.18) Robertsonian translocation and performed molecular genotyping of 1812 embryos from 364 litters with known parental origin, strain, and age. Nondisjunction events were scored and factors influencing the frequency of nondisjunction involving chromosomes 7 and 18 were examined. We concluded the following: 1. The frequency of nondisjunction among 1784 embryos (3568 meioses) was 15.9%. 2. Nondisjunction events were distributed nonrandomly among progeny. This was inferred from the distribution of the frequency of trisomics and uniparental disomics (UPDs) among all litters. 3. There was no evidence to show an effect of maternal or paternal age on the frequency of nondisjunction. 4. Strain background did not play an appreciable role in nondisjunction frequency. 5. The frequency of nondisjunction for chromosome 18 was significantly higher than that for chromosome 7 in males. 6. The frequency of nondisjunction for chromosome 7 was significantly higher in females than in males. These results show that molecular genotyping provides a valuable tool for understanding factors influencing meiotic nondisjunction in mammals.

The innervation of hair follicles offers an intriguing, yet hardly studied model for the dissection of the stepwise innervation during cutaneous morphogenesis. We have used immunofluorescence and a panel of neuronal markers to characterize the developmental choreography of C57BL/6 mouse backskin innervation. The development of murine skin innervation occurs in successive waves. The first cutaneous nerve fibers appeared before any morphological evidence of hair follicle development at embryonic day 15 (E15). Stage 1 and 2 developing hair follicles were already associated with nerve fibers at E16. These fibers approached a location where later in development the follicular (neural) network A (FNA) is located on fully developed pelage hair follicles. Prior to birth (E18), some nerve fibers had penetrated the epidermis, and an additional set of perifollicular nerve fibers arranged itself around the isthmus and bulge region of stage 5 hair follicles, to develop into the follicular (neural) network B (FNB). By the day of birth (P1), the neuropeptides substance P and calcitonin gene-related peptide became detectable in subcutaneous and dermal nerve fibers first. Newly formed hair follicles on E18 and P1 displayed the same innervation pattern seen in the first wave of hair follicle development. Just prior to epidermal penetration of hair shafts (P5), peptide histidine methionine-IR nerve fibers became detectable and epidermal innervation peaked; such innervation decreased after penetration (P7- P17). Last, tyrosine hydroxylase-IR and neuropeptide Y-IR became readily detectable. This sequence of developing innervation consistently correlates with hair follicle development, indicating a close interdependence of neuronal and epithelial morphogenesis., (Copyright 2002 Wiley-Liss, Inc.)

Deviations from Mendelian 1:1 transmission ratio have been observed in mice and humans. With few exceptions, the mechanism leading to transmission-ratio distortion (TRD) remains obscure. We proposed that a genomic imprinting mechanism plays a key role in the genesis of grandparental origin-dependent TRD (Naumova et al. 2001). To further test this hypothesis, we analyzed the transmission of grandparental alleles at three imprinted regions of the mouse genome known to contain genes required for embryo development. We found and replicated moderate (58%: 42%) TRD in favor of grandmaternal alleles in the imprinted region of maternal distal Chromosome (Chr) 12 among female offspring. Comparison of transmission ratios at the distorted region of Chr 12 among 3-week-old mice with those in embryos suggests that the distortion in favor of grandmaternal alleles is owing to postimplantation embryo loss. The absence of grandparental origin-dependent TRD for maternal Chr 6 and 7 implies that the relationship between TRD and imprinting is complex. Most likely, multiple conditions are required for TRD to occur.

The early phase of granule cell migration in the developing chick cerebellum occurs within ribbons of cells moving through parasagittally arrayed gaps between Purkinje cell clusters. These parasagittal arrays of migrating granule cells, termed "granule cell raphes," also have been reported in rabbit and cat, but recent publications variously report that granule cell raphes are absent or present in rodents. By using Nissl counterstaining and Pax6 immunohistochemistry, we confirm that granule cells do migrate in raphes in the developing mouse cerebellum, and also in the primate cerebellum during a period of development that coincides with Purkinje cell compartmentation. In mouse and primate cerebellum, as in chick cerebellum, granule cell migratory streams occur at the borders of Purkinje cell clusters. GFAP immunostaining of Bergmann glial fibers shows no parasagittally localized pattern of distribution, indicating that the formation of granule cell ribbons is not prepatterned by heterogeneous distribution of radial glia. The conservation of the ribboned pattern of granule cell migration from bird to primate and the timing of this event suggest a possible role for granule cell raphes in parasagittal compartmentation of Purkinje cells. A potential mechanism for such an interaction is discussed., (Copyright 2001 Wiley-Liss, Inc.)

Dystrophin glycoprotein complex (DGC) assembly and function require mediation by dystrophin in skeletal muscle. The existence of such complexes and the correlation with DMD phenotypes are not yet established in the central nervous system. Here we have studied the expression of DMD gene mRNAs and proteins in retina from C57BL/6 and mdx(3Cv) mouse strains. Then we have comparatively investigated the localization of dystrophin and dystrophin-associated proteins (DAPs) in both strains to analyze the repercussion of the mdx(3Cv) mutation on the retinal distributions of alpha/beta-dystroglycan, alpha1-syntrophin, alpha-dystrobrevin, and delta/gamma-sarcoglycan. Results showed that DMD gene product deficiency affects the expression of dystroglycan assembly exclusively at the outer plexiform layer without an apparent effect on the other DAPs. We conclude that the localization of members of the DGC could be independent of the presence of the DMD gene products and/or utrophin., (Copyright 2001 Academic Press.)

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a basic-helix-loop-helix/Per- ARNT-Sim (bHLH/PAS) transcription factor that is involved in multiple signaling pathways. This study focuses on the tissue distribution and subcellular localization of ARNT during embryological development of the mouse and chicken. Two different species were chosen to determine the consistency of the ARNT staining pattern. Immunohistochemical techniques were used to stain sections of embryos over three developmental time points for each species. Mouse tissues evaluated from embryonic day 10.5, 12.5, and 15, exhibited predominant nuclear staining with little change in expression patterns over time. Chicken tissues evaluated from embryonic day 2, 4, and 10 also showed predominant nuclear staining within all cells and little change in expression over developmental time, as well as, low levels of cytoplasmic ARNT staining in some cells. Importantly, in all tissues, the level of ARNT staining within the nuclear compartment was greater than staining observed in the cytoplasm. Thus, the overall conclusions from these studies are that i) the predominant subcellular localization of ARNT protein is nuclear, and ii) that mouse and chicken appear to maintain ARNT protein expression in many cell types over developmental time. These data support vertebrate ARNT as a nuclear transcription factor and a model in which dimerization partners require nuclear localization for interaction., (Copyright 2000 Wiley-Liss, Inc.)

Nociceptin/orphanin FQ (N/OFQ) and its receptor share similarities to opioids and their receptors in terms of the molecular structure and signaling pathway, but the two systems exhibit different actions in vivo. To understand the mechanism of N/OFQ-system actions, we examined, by in situ hybridization analysis, the distribution of preproN/OFQ and N/OFQ receptor mRNAs in the developing and adult mouse central nervous systems (CNS). In most neural regions, preproN/OFQ mRNA was mainly expressed in a small population of middle-sized neurons. These neurons were scattered between large projection-type neurons or within the neuropil, suggestive of interneurons. In some other nuclei (lateral septum, bed nucleus of the stria terminalis, reticular thalamic nucleus, inferior colliculus, and rostral periolivery nucleus), preproN/OFQ mRNA was expressed in a number of large projection-type neurons. By contrast, N/OFQ receptor mRNA was evenly expressed in most neurons of the adult CNS. Considering the inhibitory actions of N/OFQ, the distinct cellular expression pattern of the N/OFQ system suggests that the release of N/OFQ from interneurons may lower neuronal and synaptic activities of neighboring neurons, leading to integration or modulation of local circuits. Furthermore, the cellular expression pattern, distinct from that of the opioid system, may provide a possible molecular/cellular basis for the different in vivo actions of N/OFQ and opioids. In embryonic stages, both preproN/OFQ and N/OFQ receptor mRNAs were highly and widely expressed in the mantle zone, suggesting the possible importance of N/OFQ signaling in CNS development.

Embigin and basigin are highly glycosylated transmembrane glycoproteins with two immunoglobulin domains and form a subgroup in the immunoglobulin superfamily. Previous studies have demonstrated the functional significance of these molecules. In the present study, in situ hybridization analysis of their expression was performed during mouse embryogenesis. Embigin was strongly expressed in the endoderm during early postimplantation embryogenesis, and in the somite stage in the gut and visceral endoderm. Embryonic ectoderm and its derivative tissues weakly to moderately expressed this molecule. From day 10 to 15 of gestation, no embigin signal was detected. Basigin was more broadly expressed. During the organogenesis period, basigin was expressed in various epithelial tissues, brain ventricles, the spinal cord and dorsal root ganglion. The modes of expression of these two proteins throughout the egg cylinder stage correlated with the expression of the carbohydrate markers that they carry; embigin with Dolichos biflorus agglutinin binding sites and basigin with LeX antigen and more closely with fucosyltransferase IV, which forms the antigenic epitope. These findings imply that proteins with specific carbohydrate epitopes play roles in early postimpantation embryogenesis.

It is found out that the acceleration of preimplantation morphogenesis is observed in mouse chimeras between single two-cell-stage blastomeres of BALB/c and [BALB/c x C57B1/6] x C57B1/6 (F2) embryos. The paired aggregation of blastomeres of two-cell stage made F2-BALB/c chimeras to start cavitation 8 +/- 2 hours earlier than F2<-->F2 and BALB/c-BALB/c control embryos. We have demonstrated for the first time the 'chimeric' heterosis on preimplantation stages. Possible approaches to the future studies of this phenomenon are discussed.

A study was made to determine if mouse zygotes can be effectively vitrified in 7 M ethylene glycol in modified Dulbecco's phosphate buffered saline (PB1) and to find out if the development of vitrified-warmed zygotes in vitro can be improved by renewing the culture medium. The results showed that without medium change, vitrification reduced the development of zygotes to the expanded blastocyst stage (p < 0.01). With medium change, the development rate of vitrified-warmed zygotes exposed in 7 M ethylene glycol for 1 or 2 min was similar to that of unvitrified zygotes. However, prolonged exposure (5 min) markedly reduced the development rates of vitrified-warmed zygotes to the expanded blastocyst stage (p < 0.05). When the zygotes were vitrified in 7 M ethylene glycol and diluted at 18 degrees C to 22 degrees C, a slower efflux of ethylene glycol from the cell might have occurred, leading to a toxic effect of ethylene glycol in culture. The development rates of vitrified embryos cultured with medium change at 24 hr did not significantly differ from the untreated control (89.0% vs 96.5%). In conclusion, this study showed that mouse zygotes can be vitrified in 7 M ethylene glycol in PB1 and that changing the culture medium can improve the in vitro development rates of vitrified-warmed zygotes to the expanded blastocyst stage.

We examined the relationships between litter size, embryonic growth, days of gestation, onset and duration of morphological stages and development of the first arch skeleton in three inbred strains of mice--C57BL/6, CBA/J and C3H/He. Detailed embryonic staging was based on craniofacial development between 11 and 18 days of gestation. Considerable intra- and interlitter variation of morphological stages of embryonic development exists in all three inbred strains. The relationship of morphological stages to days of gestation reveals that each stage has a different duration, being shortest at Theiler's stage 18 and longest at stage 21 in all three inbred strains. Embryos of CBA/J mice tend to reach each stage later than do embryos of the other two strains, i.e., morphological development is slowest in CBA/J. The greatest length, a measurement of embryonic growth, increases at a constant rate during gestation in all three strains. In C57BL/6 and CBA/J, more embryos tend to be implanted in the right horn of the uterus than in the left, whereas in C3H/He an even number of embryos tends to be implanted in both horns. Timing of the development of Meckel's cartilage differs between the three inbred strains: both condensation and onset of matrix deposition begin one stage earlier in C57BL/6 than in CBA/J and C3H/He. On the other hand, alkaline phosphatase, one of the earliest markers for bone development, is expressed at the same time in all three inbred strains. Differences in timing of skeletal development between the strains may be attributed in part to the genealogical closeness OF CBA/J and C3H/He mice.

The enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is a key enzyme in the biosynthesis of steroid hormones. To date, this laboratory has isolated and characterized five distinct 3beta-HSD complementary DNAs (cDNAs) in the mouse (3beta-HSD I through V). These different forms are expressed in a tissue- and developmentally-specific manner and fall into two functionally distinct enzymes. 3beta-HSD I and III, and most likely II, function as dehydrogenase/isomerases, whereas 3beta-HSD IV and V function as 3-ketosteroid reductases. This study describes the isolation, characterization, and tissue-specific expression of a sixth member of this gene family, 3beta-HSD VI. This new isoform functions as an NAD+-dependent dehydrogenase/isomerase exhibiting very low Michaelis-Menten constant (Km) values for pregnenolone (approximately 0.035 microM) and dehydroepiandrosterone (approximately 0.12 microM). 3beta-HSD VI is the earliest isoform to be expressed during embryogenesis in cells of embryonic origin at 7 and 9.5 days postcoitum (pc), and is the major isoform expressed in uterine tissue at the time of implantation (4.5 days pc) and continues to be expressed in uterine tissue at 6.5, 7.5, and 9.5 days pc. 3beta-HSD VI is expressed in giant trophoblasts at 9.5 days pc and is expressed in the placenta through day 15.5 pc. In the adult mouse, 3beta-HSD VI appears to be the only isoform expressed in the skin and also is expressed in the testis, but to a lesser extent than 3beta-HSD I. Mouse 3beta-HSD VI cDNA is orthologous to human 3beta-HSD I cDNA. Human type I 3beta-HSD has been shown to be the only isoform expressed in the placenta and skin. The demonstration that mouse 3beta-HSD VI functions as a dehydrogenase/isomerase and is the predominant isoform expressed during the first half of pregnancy in uterine tissue and in embryonic cells suggests that this isoform may be involved in local production of progesterone, which is needed for successful implantation of the blastocyst and/or maintenance of early pregnancy.

The effects of different concentrations of etoposide and cycloheximide (ETO-CHXM), used for chemical enucleation of mouse oocytes, on polar body extrusion and chromatin expulsion were tested. The developmental ability of blastomeres of late 2-cell stage embryos fused to chemically enucleated oocytes of different ages or cytoplasts from different sources was also examined in vitro. Metaphase I oocytes cultured in different concentrations of ETO-CHXM (10-50 micrograms/ml/each) extruded polar bodies at rates similar to those cultured without ETO-CHXM (58.5-65.9% and 64.6%, respectively). However, low percent of the oocytes (1.7-6.2%) expressed signs of meiotic perturbation, which was manifested by blebbing of the cytoplasmic membrane and extrusion of two or more polar body-like fragments. Twenty-three percent of the chemically enucleated oocytes cultured in ETO-CHXM-free medium spontaneously fused to their polar bodies. The rates of total chromatin expulsion were similar when ETO-CHXM concentrations were 36 and 50 micrograms/ml (93.5 and 98%, respectively). The results also showed that the cleavage rates of reconstituted embryos were significantly (P < 0.001) affected by the age of the chemically enucleated oocytes. Cytoplasts of bisected oocytes that matured in vivo supported the development of 31.7% of the reconstituted embryos to the blastocyst stage. However, both cytoplasts of chemically enucleated oocytes and in vitro matured oocytes did not support the development to the blastocyst stage. A high percentage (85.5%) of the reconstituted embryos with chemically enucleated recipients displayed abnormality of the metaphase plate. These results suggest that concentrations of etoposide between 36 and 50 micrograms/ml are optimum for enucleation of mouse oocytes. Furthermore, increasing the age or reducing the cytoplasmic volume of the chemically enucleated oocytes did not improve the development of the reconstituted embryos to the blastocyst stage.

The majority of embryo aggregation (EA) mouse chimeras between non-obese diabetic (NOD) mice and C57BL/6 (B6) mice show clear signs of insulitis frequently accompanied by beta-cell destruction. Less than 5% of these chimeras, however, spontaneously progress to autoimmune diabetes, an incidence far lower than observed in NOD mice. The resistance in chimeras can be accounted for by the target organ chimerism and/or the immune system chimerism. To investigate the mechanism(s) controlling diabetes resistance in these mice, we studied a total of 92 NOD<-->B6 EA chimeras that showed overt lymphoid chimerism and treated 34 chimeras with cyclophosphamide (CY), a compound known to precipitate an acute form of insulin-dependent diabetes mellitus (IDDM) in pre-diabetic NOD mice, by interfering with regulatory mechanisms. We found that CY-treated EA chimeras displayed an increase in the NOD:B6 lymphocyte ratio and 32% of them developed diabetes that could be adoptively transferred to irradiated NOD or NOD-rag-2-/- mice. These findings suggest that lymphocyte chimerism rather than beta-cell chimerism accounts for diabetes resistance in NOD<-->B6 EA chimeras and that the susceptibility to CY-induced diabetes may be related to the proportion of NOD versus B6 lymphoid cells.