RESUMEN
The glial environment is an important determinant of neuronal health in experimental models of neurodegeneration. Specifically, astrocytes have been shown, dependent on context, to be both injurious and protective. Human pluripotent stem cells offer a powerful new system to improve our understanding of the mechanisms underlying astrocyte-mediated neuroprotection. Here, we describe a human embryonic stem cell (HESC)-based system to assess the scope and mechanism of human astrocyte-mediated neuroprotection. We first report the generation of enriched and functional HESC-derived astrocytes, by combining BMP-mediated Smad and LIF-mediated JAK-STAT signalling. These astrocytes promote the protection of HESC-derived neurons against oxidative insults. Moreover, their neuroprotective capacity can be greatly enhanced by treatment with the nuclear factor-erythroid 2-related factor 2 (Nrf2)-activating triterpenoid 1[2-Cyano-3,12-dioxool-eana-1,9(11)-dien-28-oyl] trifluoroethylamide (CDDO(TFEA)). Activation of the transcription factor Nrf2 in human astrocytes by CDDO(TFEA) treatment induced expression of the glutamate-cysteine ligase (GCL) catalytic subunit, leading to enhanced GCL activity and glutathione production, and strong neuroprotection against H(2)O(2). This enhanced neuroprotection was found to be dependent on astrocytic GCL activity, unlike the basal neuroprotection afforded by untreated astrocytes. Direct treatment of HESC-derived neurons with CDDO(TFEA) elicited no induction of Nrf2 target genes, nor any neuroprotection. Thus, human astrocytes can mediate neuroprotection through glutathione-dependent and glutathione-independent mechanisms, and represent a therapeutic target for human disorders associated with neuronal oxidative stress.
Asunto(s)
Astrocitos/citología , Astrocitos/metabolismo , Células Madre Embrionarias/citología , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Astrocitos/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Inmunohistoquímica , Ratones , Ratones Mutantes , Factor 2 Relacionado con NF-E2/genética , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologíaRESUMEN
A major challenge in neurobiology is to understand mechanisms underlying human neuronal diversification. Motor neurons (MNs) represent a diverse collection of neuronal subtypes, displaying differential vulnerability in different human neurodegenerative diseases. The ability to manipulate cell subtype diversification is critical to establish accurate, clinically relevant in vitro disease models. Retinoid signalling contributes to caudal precursor specification and subsequent MN subtype diversification. Here we investigate the necessity for retinoic acid in motor neurogenesis from human embryonic stem cells. We show that activin/nodal signalling inhibition, followed by sonic hedgehog agonist treatment, is sufficient for MN precursor specification, which occurs even in the presence of retinoid pathway antagonists. Importantly, precursors mature into HB9/ChAT-expressing functional MNs. Furthermore, retinoid-independent motor neurogenesis results in a ground state biased to caudal, medial motor columnar identities from which a greater retinoid-dependent diversity of MNs, including those of lateral motor columns, can be selectively derived in vitro.
Asunto(s)
Células Madre Embrionarias/metabolismo , Neuronas Motoras/metabolismo , Neurogénesis , Tretinoina/metabolismo , Animales , Línea Celular , Células Madre Embrionarias/citología , Humanos , Ratones , Neuronas Motoras/citología , Transducción de SeñalRESUMEN
Under the current allocation system for liver transplantation (LTx), primary and retransplantation (ReTx) are treated identically. The aims of this study were (1) to compare the risk of death between ReTx and primary LTx candidates at a given MELD score and (2) to gauge the impact of the MELD-based allocation system on the waitlist outcome of ReTx candidates. Based on data of all waitlist registrants in the United States between 2000 and 2006, unique adult patients with chronic liver disease were identified and followed forward to determine mortality within six months of registration. There were a total of 45,943 patients waitlisted for primary LTx and 2081 registered for ReTx. In the MELD era (n = 30,175), MELD was significantly higher among ReTx candidates than primary LTx candidates (median, 21 vs. 15). Within a range of MELD scores where most transplantation took place, mortality was comparable between ReTx and primary candidates after adjusting for MELD. The probability for LTx increased significantly following implementation of the MELD-based allocation in both types of candidates. We conclude that by and large, primary and ReTx candidates fare equitably under the current MELD-based allocation system, which has contributed to a significant increase in the probability of LTx.
Asunto(s)
Enfermedad Hepática en Estado Terminal/cirugía , Trasplante de Hígado/mortalidad , Obtención de Tejidos y Órganos/estadística & datos numéricos , Adulto , Femenino , Asignación de Recursos para la Atención de Salud , Humanos , Masculino , Persona de Mediana Edad , Sistema de Registros , Reoperación/mortalidad , Resultado del Tratamiento , Estados Unidos/epidemiología , Listas de Espera/mortalidadRESUMEN
BACKGROUND: The role of interleukin 2 (IL-2) receptor antibodies to avoid the nephrotoxic effects of calcineurin inhibitors in the early post-liver transplant (LT) period is not well defined. AIM: To examine the use of daclizumab induction in LT recipients with renal insufficiency. METHODS: Between 2002 and 2005, 62 patients (median pre-LT creatinine 2.4 mg/dL, IQR 1.9-3.7) received daclizumab induction with tacrolimus being administered when serum creatinine was <2.0 mg/dL. A concurrent comparison group (n = 221, 2002-2005) received tacrolimus-based immunosuppression without daclizumab (median pre-LT creatinine 1.1 mg/dL, IQR 0.9-1.4). A second historical comparison group (n = 103, 1995-2005) not receiving daclizumab was matched to the daclizumab patients by pre-LT serum creatinine (2.2 mg/dL, IQR 1.8-3.1). All patients received mycophenolate mofetil and steroids. RESULTS: Serum creatinine improved in the daclizumab group (-1.0 mg/dL, IQR -2.2 to -0.4) and worsened in the concurrent comparison group (+0.2 mg/dL, IQR 0-0.5) from pre-LT to 4 months. However, there was no difference when daclizumab group was compared with the historical comparison group matched on pre-LT creatinine (median change: -0.8 mg/dL vs. -0.7 mg/dL). Daclizumab induction was not associated with improvement in renal function at 4 months (P = 0.34) after adjusting for pre-LT creatinine, age, gender, hepatitis C status and simultaneous liver kidney transplantation. CONCLUSION: The incremental benefit offered by induction therapy with IL-2 receptor antibodies to preserve renal function is questionable.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Rechazo de Injerto/prevención & control , Inmunoglobulina G/uso terapéutico , Inmunosupresores/uso terapéutico , Hepatopatías/cirugía , Trasplante de Hígado/inmunología , Insuficiencia Renal/complicaciones , Adulto , Anticuerpos Monoclonales Humanizados , Estudios de Casos y Controles , Daclizumab , Femenino , Humanos , Hepatopatías/fisiopatología , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
Although mortality rates following liver transplantation (LT) are well described, there is a lack of detailed, prospective studies determining patterns of and risk factors for long-term mortality. We analyzed the multicenter, prospectively obtained The National Institute of Diabetes and Digestive and Kidney Diseases LT Database of 798 transplant recipients from 1990 to 1994 (follow-up 2003). Overall, 327 recipients died. Causes of death >1 year: 28% hepatic, 22% malignancy, 11% cardiovascular, 9% infection, 6% renal failure. Renal-related death increased dramatically over time. Risk factors for death >1 year (univariate): male gender, age/decade, pre-LT diabetes, post-LT diabetes, post-LT hypertension, post-LT renal insufficiency, retransplantation >1 year, pre-LT malignancy, alcoholic disease (ALD) and metabolic liver disease, with similar risks noted for death >5 years. Hepatitis C, retransplantation, post-LT diabetes, hypertension and renal insufficiency were significant risk factors for liver-related death. Cardiac deaths associated with age, male gender, ALD, cryptogenic disease, pre-LT hypertension and post-LT renal insufficiency. In summary, the leading causes of late deaths after transplant were graft failure, malignancy, cardiovascular disease and renal failure. Older age, diabetes and renal insufficiency identified patients at highest risk of poor survival overall. Diligent management of modifiable post-LT factors including diabetes, hypertension and renal insufficiency may impact long-term mortality.
Asunto(s)
Trasplante de Hígado/efectos adversos , Trasplante de Hígado/mortalidad , Alcohólicos , Enfermedades Cardiovasculares/inducido químicamente , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/mortalidad , Diabetes Mellitus/inducido químicamente , Diabetes Mellitus/etiología , Diabetes Mellitus/mortalidad , Femenino , Estudios de Seguimiento , Hepatitis C/inducido químicamente , Hepatitis C/etiología , Hepatitis C/mortalidad , Humanos , Hipertensión/inducido químicamente , Hipertensión/etiología , Hipertensión/mortalidad , Riñón , Trasplante de Riñón/mortalidad , Hígado , Hepatopatías/etiología , Hepatopatías/mortalidad , Estudios Longitudinales , Masculino , Persona de Mediana Edad , National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) , Estudios Prospectivos , Insuficiencia Renal/inducido químicamente , Insuficiencia Renal/etiología , Insuficiencia Renal/mortalidad , Factores de Riesgo , Resultado del Tratamiento , Estados UnidosRESUMEN
Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing multi-color FISH-based technologies to score all 24 chromosomes in single cells within a three-day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20-50% of invasive CTB cells from uncomplicated pregnancies were found to be aneuploid, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate the feasibility of a full karyotype analysis of individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.
Asunto(s)
Blastocisto/citología , Aberraciones Cromosómicas/embriología , Hibridación Fluorescente in Situ , Cariotipificación , Trofoblastos/citología , Blastocisto/patología , Femenino , Fertilización In Vitro , Humanos , Intercambio Materno-Fetal , Metafase , Embarazo , Trisomía/genética , Trofoblastos/patologíaRESUMEN
Two independent studies have recently suggested similar models in which the embryonic and abembryonic parts of the mouse blastocyst become separated already by the first cleavage division. However, no lineage tracing studies carried out so far on early embryos provide the support for such a hypothesis. Thus, to re-examine the fate of blastomeres of the two-cell mouse embryo, we have undertaken lineage tracing studies using a non-perturbing method. We show that two-cell stage blastomeres have a strong tendency to develop into cells that comprise either the embryonic or the abembryonic parts of the blastocyst. Moreover, the two-cell stage blastomere that is first to divide will preferentially contribute its progeny to the embryonic part. Nevertheless, we find that the blastocyst embryonic-abembryonic axis is not perfectly orthogonal to the first cleavage plane, but often shows some angular displacement from it. Consequently, there is a boundary zone adjacent to the interior margin of the blastocoel that is populated by cells derived from both earlier and later dividing blastomeres. The majority of cells that inhabit this boundary region are, however, derived from the later dividing two-cell stage blastomere that contributes predominantly to the abembryonic part of the blastocyst. Thus, at the two-cell stage it is already possible to predict which cell will contribute a greater proportion of its progeny to the abembryonic part of the blastocyst (region including the blastocyst cavity) and which to the embryonic part (region containing the inner cell mass) that will give rise to the embryo proper.
Asunto(s)
Blastómeros/citología , Blastómeros/fisiología , Animales , Blastocisto/citología , Blastocisto/fisiología , Linaje de la Célula , Femenino , Ratones , Ratones Endogámicos , Biología Molecular/métodosRESUMEN
Numerical chromosome aberrations are incompatible with normal human development. Our laboratories develop hybridization-based screening tools that generate a maximum of cytogenetic information for each polar body or blastomere analyzed. The methods are developed considering that the abnormality might require preparation of case-specific probes and that only one or two cells will be available for diagnosis, most of which might be in the interphase stage. Furthermore, assay efficiencies have to be high, since there is typically not enough time to repeat an experiment or reconfirm a result prior to fertilization or embryo transfer. Structural alterations are delineated with breakpoint-spanning probes. When screening for numerical abnormalities, we apply a Spectral Imaging-based approach to simultaneously score as many as ten different chromosome types in individual interphase cells. Finally, DNA micro-arrays are under development to score all of the human chromosomes in a single experiment and to increase the resolution with which micro-deletions can be delineated.
Asunto(s)
Aberraciones Cromosómicas , Trastornos de los Cromosomas/diagnóstico , Hibridación Fluorescente in Situ/métodos , Interfase/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Diagnóstico Preimplantación/métodos , Blastómeros , Sondas de ADN , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Cariotipificación , Tamizaje Masivo , EmbarazoRESUMEN
Chromosome abnormalities are common causes of congenital malformations and spontaneous abortions. They include structural abnormalities, polyploidy, trisomy, and mosaicism. In in vitro fertilization (IVF) programs, preimplantation genetic diagnosis (PGD) of oocytes and embryos has become the technique of choice to select against abnormal embryos before embryo transfer. For diagnosis of structural abnormalities, we developed case-specific breakpoint-spanning DNA probes. Screening of an in-house yeast artificial chromosome (YAC) library is facilitated by information from publicly available databases and published articles. Most numerical chromosome abnormalities, on the other hand, are detrimental to early embryonic development and increase with maternal age. We therefore developed a multichromosome screening technique based on spectral imaging to simultaneously detect and score as many as 10 different chromosome types. The probe set was chosen to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions. Detecting structural and numerical abnormalities in single interphase cells using spectral imaging is a powerful technique for multilocus genetic screening.
Asunto(s)
Aberraciones Cromosómicas/diagnóstico , Procesamiento de Imagen Asistido por Computador/métodos , Interfase/genética , Microscopía Fluorescente/métodos , Diagnóstico Preimplantación/métodos , Rotura Cromosómica , Trastornos de los Cromosomas , Sondas de ADN , Femenino , Humanos , EmbarazoAsunto(s)
Tipificación del Cuerpo , Polaridad Celular , Interacciones Espermatozoide-Óvulo , Espermatozoides/fisiología , Animales , División Celular , Embrión de Mamíferos/citología , Embrión no Mamífero/citología , Desarrollo Embrionario , Femenino , Humanos , Masculino , Ratones , Embarazo , Xenopus laevisRESUMEN
We have established mouse embryonic stem (ES) cell lines from blastocysts derived by transfer of nuclei of fetal neuronal cells. These neuronal cell-derived embryonic cell lines had properties that characterize them as ES cells, including typical cell markers and alkaline phosphatase activity. Moreover, the cells had a normal karyotype and were pluripotent, as they were capable of differentiating into all three germ layers. Although they were derived from neuronal donor nuclei, the cells no longer expressed neuronal markers; however, they were capable of differentiating into cells with neuronal characteristics. These results suggest that the clone-derived cells have fully acquired an ES cell character. Thus, ES cells can be derived from embryos resulting from nuclear transfer, which results in reprogramming of the genetic information and acquisition of pluripotency. ES cells established from somatic cell-derived blastocysts could be useful not only as research tools for studying reprogramming but also as models for cell-based transplantation therapy.
Asunto(s)
Blastocisto/citología , Línea Celular , Neuronas/citología , Células Madre , Fosfatasa Alcalina/metabolismo , Animales , Diferenciación Celular , Línea Celular/citología , Línea Celular/metabolismo , Células Clonales , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Neuronas/metabolismo , Técnicas de Transferencia Nuclear , Células Madre/citología , Células Madre/metabolismoRESUMEN
The homeobox gene goosecoid, originally identified in Xenopus, is expressed in the organizer or its equivalent during gastrulation in the frog, chick, zebrafish and mouse. To investigate the role of goosecoid in mouse development, we have generated embryonic stem cells that stably overexpress the murine homolog of goosecoid. These cells show a repression of the gastrulation-associated gene Brachyury. Interestingly, repression of Brachyury is conserved between Xenopus and mouse despite the lack of conservation of the Brachyury promoter. Further characterization of the goosecoid-overexpressing ES cells revealed that they maintain the expression of stage-specific embryonic antigen-1, and teratomas derived from goosecoid-overexpressing cells show the presence of cell types derived from all three germ layers. Some highly chimeric mice derived from goosecoid-overexpressing cells displayed skull defects. These observations suggest that goosecoid may play a role in specification of anterior mesendodermal fates and specifically in mouse craniofacial development.
Asunto(s)
Embrión de Mamíferos/metabolismo , Cara/embriología , Proteínas Fetales , Proteínas de Homeodominio/metabolismo , Proteínas Represoras , Cráneo/embriología , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción , Animales , Secuencia de Bases , Línea Celular , Linaje de la Célula , Genes Reporteros , Proteína Goosecoide , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Células Madre/metabolismo , Transcripción Genética , TransfecciónRESUMEN
During neurogenesis of the mammalian neocortex, neural progenitor cells divide to generate daughter cells that either become neurons or remain as progenitor cells. The mouse numb (m-numb) gene encodes a membrane-associated protein that is asymmetrically localized to the apical cell membrane of dividing cortical progenitor cells and may be segregated to only the apical daughter cell that has been suggested to remain as a progenitor cell. To examine m-numb function during neural development, we generated a loss-of-function mutant allele of m-numb. Mice homozygous for this mutation exhibit severe defects in cranial neural tube closure and precocious neuron production in the forebrain and die around embryonic day 11.5 (E11. 5). These findings suggest that m-numb is an essential gene that plays a role in promoting progenitor cell fate during cortical neurogenesis.
Asunto(s)
Corteza Cerebral/embriología , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Neuronas/fisiología , Animales , Pérdida del Embrión , Femenino , Ratones , Mutación , Defectos del Tubo Neural/etiología , EmbarazoRESUMEN
Balanced reciprocal translocations are known to interfere with homolog pairing in meiosis. Many individuals carrying such chromosomal abnormalities suffer from reduced fertility or spontaneous abortions and seek help in the form of assisted reproductive technology. Although most translocations are relatively easy to detect in metaphase cells, the majority of embryonic cells biopsied in the course of in vitro fertilization (IVF) procedures are in interphase. These nuclei are, thus, unsuitable for analysis by chromosome banding or painting using fluorescence in situ hybridization (FISH). Our assay, based on FISH detection of breakpoint-spanning DNA probes, identifies translocations in interphase nuclei by microscopic inspection of hybridization domains. Probes are selected that span the breakpoint regions on normal homologs. The probes should hybridize to several hundred kilobases of DNA flanking the breakpoint. The two breakpoint-spanning DNA probes for the translocation chromosomes are labeled in separate colors (e.g., red and green). The translocation event producing two fused red/green hybridization domains can then be detected in interphase cell nuclei using a fluorescence microscope. We applied this scheme to analyze somatic and germ cells from 21 translocation patients, each with distinct breakpoints. Here, we summarize our experience and provide a description of strategies, cost estimates, as well as typical time frames.
Asunto(s)
Sondas de ADN , Interfase , Translocación Genética , Blastómeros , Femenino , Genotipo , Humanos , Hibridación Fluorescente in Situ , MasculinoRESUMEN
Numerical chromosome aberrations are detrimental to early embryonic, fetal and perinatal development of mammals. When fetuses carrying a chromosomal imbalance survive to term, an aberrant gene dosage typically leads to stillbirth or causes a severely altered phenotype. Aneuploidy of any of the 24 chromosomes will negatively impact on human development, and a preimplantation and prenatal genetic diagnosis test should thus score as many chromosomes as possible. Since cells available for analysis are likely to be in interphase, we set out to develop a rapid enumeration procedure based on hybridization of chromosome-specific probes and spectral imaging detection. The probe set was chosen to allow the simultaneous enumeration of ten chromosome types and was expected to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions, i.e., human chromosomes 9, 13, 14, 15, 16, 18, 21, 22, X, and Y. Cell fixation protocols were optimized to achieve the desired detection sensitivity and reproducibility. We were able to resolve and identify ten separate chromosomal signals in interphase nuclei from different types of cells, including lymphocytes, uncultured amniocytes, and blastomeres. In summary, this study demonstrates the strength of spectral imaging, allowing us to construct partial spectral imaging karyotypes for individual interphase cells by assessing the number of each of the target chromosome types.
Asunto(s)
Interfase/genética , Cariotipificación/métodos , Análisis Espectral , Blastómeros/citología , Células Cultivadas , Sondas de ADN , Humanos , Linfocitos/citología , Masculino , Metafase , Microscopía FluorescenteRESUMEN
The autosomal beta1 integrin knockout mouse mutation was selected as a model to experimentally determine preimplantation diagnosis test reliability for autosomal gene deletions and duplications. In experiment 1, which analyzed 198 individually disaggregated single blastomeres, the observed test frequencies matched the mathematically predicted frequencies calculated from the independently derived values of 90% normal allele amplification, 92% mutant allele amplification, 4% alternate allele contamination, and 4% failure to transfer amplifiable target DNA into the PCR reaction mix. This experiment correctly predicted a normal embryonic phenotype in 143 (99.3%) of the 144 phenotypically normal autosomal recessive results. Experiment 2 compared single biopsied blastomere test results to test results on the remaining embryonic cells cultured 1 week until trophoblast outgrowth. Single biopsied blastomere analysis correctly predicted a normal autosomal recessive phenotype in 87 (98%) of the 89 embryos that would have been selected for implantation. Experiment 3 compared the PCR results of two biopsied blastomeres tested independently to the PCR result from the remaining cultured blastomeres to improve test reliability. Given that embryos would have been implanted only when two normal results were obtained, 17 of 17 phenotypically normal embryos would have been implanted from among the 44 embryos tested. These experiment 3 results are consistent with the mathematical prediction that about 99.9% of embryos implanted with two unaffected biopsied blastomere results would have had a phenotypically normal genotype.
Asunto(s)
Aneuploidia , Integrina beta1/genética , Modelos Genéticos , Mutación , Diagnóstico Prenatal/métodos , Animales , Secuencia de Bases , Blastómeros/citología , Cartilla de ADN/genética , Desarrollo Embrionario/genética , Femenino , Enfermedades Genéticas Congénitas/diagnóstico , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/prevención & control , Genotipo , Humanos , Masculino , Ratones , Ratones Noqueados , Fenotipo , EmbarazoRESUMEN
In most species, the polarity of an embryo underlies the future body plan and is determined from that of the zygote. However, mammals are thought to be an exception to this; in the mouse, polarity is generally thought to develop significantly later, only after implantation. It has not been possible, however, to relate the polarity of the preimplantation mouse embryo to that of the later conceptus due to the lack of markers that endure long enough to follow lineages through implantation. To test whether early developmental events could provide cues that predict the axes of the postimplantation embryo, we have used the strategy of injecting mRNA encoding an enduring marker to trace the progeny of inner cell mass cells into the postimplantation visceral endoderm. This tissue, although it has an extraembryonic fate, plays a role in axis determination in adjacent embryonic tissue. We found that visceral endoderm cells that originated near the polar body (a marker of the blastocyst axis of symmetry) generally became distal as the egg cylinder formed, while those that originated opposite the polar body tended to become proximal. It follows that, in normal development, bilateral symmetry of the mouse blastocyst anticipates the polarity of the later conceptus. Moreover, our results show that transformation of the blastocyst axis of symmetry into the axes of the postimplantation conceptus involves asymmetric visceral endoderm cell movement. Therefore, even if the definitive axes of the mouse embryo become irreversibly established only after implantation, this polarity can be traced back to events before implantation.
Asunto(s)
Tipificación del Cuerpo , Desarrollo Embrionario/fisiología , Desarrollo Embrionario y Fetal , Animales , Blastocisto , Linaje de la Célula , Polaridad Celular , Endodermo , Femenino , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Óvulo , EmbarazoRESUMEN
We report the generation and analysis of mice homozygous for a targeted deletion of the Dlx5 homeobox gene. Dlx5 mutant mice have multiple defects in craniofacial structures, including their ears, noses, mandibles and calvaria, and die shortly after birth. A subset (28%) exhibit exencephaly. Ectodermal expression of Dlx5 is required for the development of olfactory and otic placode-derived epithelia and surrounding capsules. The nasal capsules are hypoplastic (e.g. lacking turbinates) and, in most cases, the right side is more severely affected than the left. Dorsal otic vesicle derivatives (e. g. semicircular canals and endolymphatic duct) and the surrounding capsule, are more severely affected than ventral (cochlear) structures. Dlx5 is also required in mandibular arch ectomesenchyme, as the proximal mandibular arch skeleton is dysmorphic. Dlx5 may control craniofacial development in part through the regulation of the goosecoid homeobox gene. goosecoid expression is greatly reduced in Dlx5 mutants, and both goosecoid and Dlx5 mutants share a number of similar craniofacial malformations. Dlx5 may perform a general role in skeletal differentiation, as exemplified by hypomineralization within the calvaria. The distinct focal defects within the branchial arches of the Dlx1, Dlx2 and Dlx5 mutants, along with the nested expression of their RNAs, support a model in which these genes have both redundant and unique functions in the regulation of regional patterning of the craniofacial ectomesenchyme.
Asunto(s)
Región Branquial/embriología , Huesos Faciales/embriología , Genes Homeobox , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Cráneo/embriología , Animales , Animales Recién Nacidos , Secuencia de Bases , Región Branquial/anomalías , Anomalías Craneofaciales/genética , Cartilla de ADN/genética , Oído Interno/anomalías , Oído Interno/embriología , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Ratones , Ratones Noqueados , FenotipoRESUMEN
We modified the Comet assay to enable the quantification of DNA strand breakage in individual cells of extremely small tissue samples. This modification was used to analyze cells isolated from the ectoplacental cone and egg cylinder of mouse embryos at embryonic day 7.5. We detected more naturally occurring DNA strand breaks and a higher number of apoptotic cells in the ectoplacental cone compared with the egg cylinder.