RESUMO
The accumulation of senescent cells in the tumor microenvironment can drive tumorigenesis in a paracrine manner through the senescence-associated secretory phenotype (SASP). Using a new p16-FDR mouse line, we show that macrophages and endothelial cells are the predominant senescent cell types in murine KRAS-driven lung tumors. Through single cell transcriptomics, we identify a population of tumor-associated macrophages that express a unique array of pro-tumorigenic SASP factors and surface proteins and are also present in normal aged lungs. Genetic or senolytic ablation of senescent cells, or macrophage depletion, result in a significant decrease in tumor burden and increased survival in KRAS-driven lung cancer models. Moreover, we reveal the presence of macrophages with senescent features in human lung pre-malignant lesions, but not in adenocarcinomas. Taken together, our results have uncovered the important role of senescent macrophages in the initiation and progression of lung cancer, highlighting potential therapeutic avenues and cancer preventative strategies.
Assuntos
Senescência Celular , Neoplasias Pulmonares , Idoso , Animais , Humanos , Camundongos , Carcinogênese/genética , Carcinogênese/metabolismo , Senescência Celular/genética , Células Endoteliais , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Microambiente TumoralRESUMO
Ensuring the fitness of the pluripotent cells that will contribute to future development is important both for the integrity of the germline and for proper embryogenesis. Consequently, it is becoming increasingly apparent that pluripotent cells can compare their fitness levels and signal the elimination of those cells that are less fit than their neighbours. In mammals the nature of the pathways that communicate fitness remain largely unknown. Here we identify that in the early mouse embryo and upon exit from naive pluripotency, the confrontation of cells with different fitness levels leads to an inhibition of mTOR signalling in the less fit cell type, causing its elimination. We show that during this process, p53 acts upstream of mTOR and is required to repress its activity. Finally, we demonstrate that during normal development around 35% of cells are eliminated by this pathway, highlighting the importance of this mechanism for embryonic development.
Assuntos
Embrião de Mamíferos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Comunicação Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Serina-Treonina Quinases TOR/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/ß-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo-pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke's pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with ß-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH.
Assuntos
Sistema Hipotálamo-Hipofisário , Proteína 1 Semelhante ao Fator 7 de Transcrição/fisiologia , Animais , Estudos de Coortes , Humanos , Camundongos , Hipófise/anormalidades , Hipófise/metabolismo , Hipófise/fisiopatologia , Prosencéfalo/anormalidades , Prosencéfalo/metabolismoRESUMO
BACKGROUND: Tamoxifen (TAM) is an important cancer therapeutic and an experimental tool for effecting genetic recombination using the inducible Cre-Lox technique. Despite its widespread use in the clinic and laboratory, we know little about its effects on the nervous system. This is of significant concern because TAM, via unknown mechanisms, induces cognitive impairment in humans. A hallmark of cellular stress is induction of Activating Transcription Factor 3 (Atf3), and so to determine whether TAM induces cellular stress in the adult nervous system, we generated a knock-in mouse in which Atf3 promoter activity drives transcription of TAM-dependent Cre recombinase (Cre-ERT2); when crossed with tdtomato reporter mice, Atf3 induction results in robust and permanent genetic labeling of cells in which it is up-regulated even transiently. RESULTS: We found that granular neurons of the olfactory bulb and dentate gyrus, vascular cells and ependymal cells throughout the brain, and peripheral sensory neurons expressed tdtomato in response to TAM treatment. We also show that TAM induced Atf3 up-regulation through inhibition of cholesterol epoxide hydrolase (ChEH): reporter expression was mitigated by delivery in vitamin E-rich wheat germ oil (vitamin E depletes ChEH substrates), and was partially mimicked by a ChEH-specific inhibitor. CONCLUSIONS: This work demonstrates that TAM stresses cells of the adult central and peripheral nervous systems and highlights concerns about clinical and experimental use of TAM. We propose TAM administration in vitamin E-rich vehicles such as wheat germ oil as a simple remedy.
Assuntos
Colesterol/metabolismo , Sistema Nervoso/citologia , Neurônios/fisiologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologia , Regulação para Cima/efeitos dos fármacos , Fator 3 Ativador da Transcrição/genética , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Relação Dose-Resposta a Droga , Epóxido Hidrolases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Óleos de Plantas/farmacologia , Regiões Promotoras Genéticas , Vitamina E/farmacologiaRESUMO
Sox2(+) adult mouse pituitary cells can self-renew and terminally differentiate in vitro, but their physiological role in vivo and possible contribution to oncogenesis remain largely unknown. Using genetic lineage tracing, we show here that the Sox2(+) cell compartment of both the embryonic and adult pituitary contains stem/progenitor cells that are able to differentiate into all hormone-producing lineages and contribute to organ homeostasis during postnatal life. In addition, we show that targeted expression of oncogenic ß-catenin in Sox2(+) cells gives rise to pituitary tumors, but, unexpectedly, the tumor mass is not derived from the Sox2(+) mutation-sustaining cells, suggesting a paracrine role of Sox2(+) cells in pituitary oncogenesis. Our data therefore provide in vivo evidence of a role for Sox2(+) stem/progenitor cells in long-term physiological maintenance of the adult pituitary, and highlight an unexpected non-cell-autonomous role for these cells in the induction of pituitary tumors.
Assuntos
Homeostase , Hipófise/citologia , Neoplasias Hipofisárias/patologia , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos Transgênicos , Mutação , Hipófise/metabolismo , Neoplasias Hipofisárias/metabolismo , Fatores de Transcrição SOXB1/genéticaRESUMO
Sex-determining region Y (SRY) box 2 (SOX2) haploinsufficiency causes a form of hypopituitarism in humans that is characterized by gonadotrophin deficiency known as hypogonadotrophic hypogonadism. Here, we conditionally deleted Sox2 in mice to investigate the pathogenesis of hypogonadotrophic hypogonadism. First, we found that absence of SOX2 in the developing Rathke pouch of conditional embryos led to severe anterior lobe hypoplasia with drastically reduced expression of the pituitary-specific transcription factor POU class 1 homeobox 1 (POU1F1) as well as severe disruption of somatotroph and thyrotroph differentiation. In contrast, corticotrophs, rostral-tip POU1F1-independent thyrotrophs, and, interestingly, lactotrophs and gonadotrophs were less affected. Second, we identified a requirement for SOX2 in normal proliferation of periluminal progenitors; in its absence, insufficient precursors were available to produce all cell lineages of the anterior pituitary. Differentiated cells derived from precursors exiting cell cycle at early stages, including corticotrophs, rostral-tip thyrotrophs, and gonadotrophs, were generated, while hormone-producing cells originating from late-born precursors, such as somatotrophs and POU1F1-dependent thyrotrophs, were severely reduced. Finally, we found that 2 previously characterized patients with SOX2 haploinsufficiency and associated hypogonadotrophic hypogonadism had a measurable response to gonadotropin-releasing hormone (GnRH) stimulation, suggesting that it is not the absence of gonadotroph differentiation, but rather the deficient hypothalamic stimulation of gonadotrophs, that underlies typical hypogonadotrophic hypogonadism.
Assuntos
Hipogonadismo/genética , Sistema Hipotálamo-Hipofisário/fisiologia , Fatores de Transcrição SOXB1/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hormônio Liberador de Gonadotropina/uso terapêutico , Heterozigoto , Proteínas de Homeodomínio/genética , Humanos , Hipogonadismo/tratamento farmacológico , Hipogonadismo/fisiopatologia , Camundongos , Camundongos Knockout , Organogênese/genética , Organogênese/fisiologia , Adeno-Hipófise/anormalidades , Adeno-Hipófise/embriologia , Adeno-Hipófise/metabolismo , Adeno-Hipófise/patologia , Proteínas Repressoras/genética , Fatores de Transcrição SOXB1/deficiência , Fatores de Transcrição SOXB1/genética , Somatotrofos/patologia , Tireotrofos/patologia , Fator de Transcrição Pit-1/deficiênciaRESUMO
CONTEXT: Fibroblast growth factor (FGF) 8 is important for GnRH neuronal development with human mutations resulting in Kallmann syndrome. Murine data suggest a role for Fgf8 in hypothalamo-pituitary development; however, its role in the etiology of wider hypothalamo-pituitary dysfunction in humans is unknown. OBJECTIVE: The objective of this study was to screen for FGF8 mutations in patients with septo-optic dysplasia (n = 374) or holoprosencephaly (HPE)/midline clefts (n = 47). METHODS: FGF8 was analyzed by PCR and direct sequencing. Ethnically matched controls were then screened for mutated alleles (n = 480-686). Localization of Fgf8/FGF8 expression was analyzed by in situ hybridization in developing murine and human embryos. Finally, Fgf8 hypomorphic mice (Fgf8(loxPNeo/-)) were analyzed for the presence of forebrain and hypothalamo-pituitary defects. RESULTS: A homozygous p.R189H mutation was identified in a female patient of consanguineous parentage with semilobar HPE, diabetes insipidus, and TSH and ACTH insufficiency. Second, a heterozygous p.Q216E mutation was identified in a female patient with an absent corpus callosum, hypoplastic optic nerves, and Moebius syndrome. FGF8 was expressed in the ventral diencephalon and anterior commissural plate but not in Rathke's pouch, strongly suggesting early onset hypothalamic and corpus callosal defects in these patients. This was consolidated by significantly reduced vasopressin and oxytocin staining neurons in the hypothalamus of Fgf8 hypomorphic mice compared with controls along with variable hypothalamo-pituitary defects and HPE. CONCLUSION: We implicate FGF8 in the etiology of recessive HPE and potentially septo-optic dysplasia/Moebius syndrome for the first time to our knowledge. Furthermore, FGF8 is important for the development of the ventral diencephalon, hypothalamus, and pituitary.
Assuntos
Anormalidades Craniofaciais/genética , Fator 8 de Crescimento de Fibroblasto/genética , Holoprosencefalia/genética , Doenças Hipotalâmicas/genética , Sistema Hipotálamo-Hipofisário/fisiopatologia , Mutação/fisiologia , Doenças da Hipófise/genética , Agenesia do Corpo Caloso/complicações , Agenesia do Corpo Caloso/genética , Arginina Vasopressina/metabolismo , Análise Mutacional de DNA , Feminino , Fator 8 de Crescimento de Fibroblasto/fisiologia , Hormônio do Crescimento Humano/sangue , Humanos , Hidrocortisona/sangue , Imuno-Histoquímica , Hibridização In Situ , Lactente , Imageamento por Ressonância Magnética , Hipófise/crescimento & desenvolvimento , Hipófise/fisiologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/biossíntese , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Displasia Septo-Óptica/genética , Tireotropina/sangueRESUMO
Wingless (Wnt)/ß-catenin signaling plays an essential role during normal development, is a critical regulator of stem cells, and has been associated with cancer in many tissues. Here we demonstrate that genetic expression of a degradation-resistant mutant form of ß-catenin in early Rathke's pouch (RP) progenitors leads to pituitary hyperplasia and severe disruption of the pituitary-specific transcription factor 1-lineage differentiation resulting in extreme growth retardation and hypopituitarism. Mutant mice mostly die perinatally, but those that survive weaning develop lethal pituitary tumors, which closely resemble human adamantinomatous craniopharyngioma, an epithelial tumor associated with mutations in the human ß-catenin gene. The tumorigenic effect of mutant ß-catenin is observed only when expressed in undifferentiated RP progenitors, but tumors do not form when committed or differentiated cells are targeted to express this protein. Analysis of affected pituitaries indicates that expression of mutant ß-catenin leads to a significant increase in the total numbers of pituitary progenitor/stem cells as well as in their proliferation potential. Our findings provide insights into the role of the Wnt pathway in normal pituitary development and demonstrate a causative role for mutated ß-catenin in an undifferentiated RP progenitor in the genesis of murine and human craniopharyngioma.
Assuntos
Hipófise/citologia , Hipófise/metabolismo , Neoplasias Hipofisárias/etiologia , Neoplasias Hipofisárias/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , Animais , Diferenciação Celular , Craniofaringioma/etiologia , Craniofaringioma/genética , Craniofaringioma/metabolismo , Craniofaringioma/patologia , Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Mutantes , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Hipófise/crescimento & desenvolvimento , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , beta Catenina/genética , beta Catenina/metabolismoRESUMO
A homozygous substitution of the highly conserved isoleucine at position 26 by threonine (I26T) in the transcriptional repressor HESX1 has been associated with anterior pituitary hypoplasia in a human patient, with no forebrain or eye defects. Two individuals carrying a homozygous substitution of the conserved arginine at position 160 by cysteine (R160C) manifest septo-optic dysplasia (SOD), a condition characterised by pituitary abnormalities associated with midline telencephalic structure defects and optic nerve hypoplasia. We have generated two knock-in mouse models containing either the I26T or R160C substitution in the genomic locus. Hesx1(I26T/I26T) embryos show pituitary defects comparable with Hesx1(-/-) mouse mutants, with frequent occurrence of ocular abnormalities, although the telencephalon develops normally. Hesx1(R160C/R160C) mutants display forebrain and pituitary defects that are identical to those observed in Hesx1(-/-) null mice. We also show that the expression pattern of HESX1 during early human development is very similar to that described in the mouse, suggesting that the function of HESX1 is conserved between the two species. Together, these results suggest that the I26T mutation yields a hypomorphic allele, whereas R160C produces a null allele and, consequently, a more severe phenotype in both mice and humans.
Assuntos
Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Hipopituitarismo/genética , Mutação , Proteínas Repressoras/genética , Displasia Septo-Óptica/genética , Animais , Padronização Corporal , Camundongos , Prosencéfalo/anormalidades , Prosencéfalo/embriologiaRESUMO
Hesx1 has been shown to be essential for normal pituitary development. The homeobox gene Six3 is expressed in the developing pituitary gland during mouse development but its function in this tissue has been precluded by the fact that in the Six3-deficient embryos the pituitary gland is not induced. To gain insights into the function of Six3 during pituitary development we have generated Six3+/- ;Hesx1Cre/+ double heterozygous mice. Strikingly, these mice show marked dwarfism, which is first detectable around weaning, and die by the 5th-6th week of age. Thyroid and gonad development is also impaired in these animals. Analysis of Six3+/- ;Hesx1Cre/+ compound embryos indicates that hypopituitarism is the likely cause of these defects since pituitary development is severely impaired in these mutants. Similar to the Hesx1-deficient embryos, Rathke's pouch is initially expanded in Six3+/- ;Hesx1Cre/+ compound embryos due to an increase in cell proliferation. Subsequently, the anterior pituitary gland appears bifurcated, dysmorphic and occasionally ectopically misplaced in the nasopharyngeal cavity, but cell differentiation is unaffected. Our research has revealed a role for Six3 in normal pituitary development, which has likely been conserved during evolution as SIX3 is also expressed in the pituitary gland of the human embryo.
Assuntos
Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Adeno-Hipófise/embriologia , Animais , Diferenciação Celular , Proliferação de Células , Nanismo Hipofisário/genética , Proteínas do Olho/genética , Genes Homeobox , Genótipo , Gônadas/anormalidades , Gônadas/citologia , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas do Tecido Nervoso/genética , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Proteínas Repressoras , Glândula Tireoide/anormalidades , Glândula Tireoide/citologia , Proteína Homeobox SIX3RESUMO
The homeobox gene Hesx1 is an essential repressor that is required within the anterior neural plate for normal forebrain development in mouse and humans. Combining genetic cell labelling and marker analyses, we demonstrate that the absence of Hesx1 leads to a posterior transformation of the anterior forebrain (AFB) during mouse development. Our data suggest that the mechanism underlying this transformation is the ectopic activation of Wnt/beta-catenin signalling within the Hesx1 expression domain in the AFB. When ectopically expressed in the developing mouse embryo, Hesx1 alone cannot alter the normal fate of posterior neural tissue. However, conditional expression of Hesx1 within the AFB can rescue the forebrain defects observed in the Hesx1 mutants. The results presented here provide new insights into the function of Hesx1 in forebrain formation.
Assuntos
Proteínas de Homeodomínio/fisiologia , Prosencéfalo/fisiologia , Animais , Proteína Axina , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Proteína Homeobox SIX3RESUMO
Planar-cell-polarity (PCP) signalling is necessary for initiation of neural tube closure in higher vertebrates. In mice with PCP gene mutations, a broad embryonic midline prevents the onset of neurulation through wide spacing of the neural folds. In order to evaluate the role of convergent extension in this defect, we vitally labelled the midline of loop-tail (Lp) embryos mutant for the PCP gene Vangl2. Injection of DiI into the node, and electroporation of a GFP expression vector into the midline neural plate, revealed defective convergent extension in both axial mesoderm and neuroepithelium, before the onset of neurulation. Chimeras containing both wild-type and Lp-mutant cells exhibited mainly wild-type cells in the midline neural plate and notochordal plate, consistent with a cell-autonomous disturbance of convergent extension. Inhibitor studies in whole-embryo culture demonstrated a requirement for signalling via RhoA-Rho kinase, but not jun N-terminal kinase, in convergent extension and the onset of neural tube closure. These findings identify a cell-autonomous defect of convergent extension, requiring PCP signalling via RhoA-Rho kinase, during the development of severe neural tube defects in the mouse.
Assuntos
Polaridade Celular/fisiologia , Sistema Nervoso Central/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Defeitos do Tubo Neural/genética , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Padronização Corporal , Sistema Nervoso Central/embriologia , Desenvolvimento Embrionário , Camundongos , MutaçãoRESUMO
We generated a ROSA26-eGFP-DTA mouse line by introducing an eGFP-DTA (enhanced green fluorescent protein -- diphtheria toxin fragment A) cassette into the ROSA26 locus by homologous recombination in ES cells. This mouse expresses eGFP ubiquitously, but DTA expression is prevented by the presence of eGFP, a Neo cassette, and a strong transcriptional stop sequence. Mice carrying this construct are normal and fertile, indicating the absence of DTA expression. However, upon Cre-mediated excision of the floxed region DTA expression is activated, resulting in the specific ablation of Cre-expressing cells. As an example of this approach, we ablated Nkx2.5 and Wnt1-expressing cells by using the Nkx2.5-Cre and Wnt1-Cre mouse lines, respectively. We observed loss of the precise tissues in which Nkx2.5 and Wnt1 are expressed. Apart from being a general GFP reporter, the ROSA26-GFP-DTA mouse line should provide a useful resource for genetic ablation of specific groups of cells.
Assuntos
Toxina Diftérica/genética , Regulação da Expressão Gênica no Desenvolvimento , Fragmentos de Peptídeos/genética , Animais , Encéfalo/embriologia , Morte Celular , Toxina Diftérica/metabolismo , Embrião de Mamíferos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Coração/embriologia , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização Genética , Hibridização In Situ , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
The relation between the role of the organizer at the gastrula stage and the activity of earlier signals in the specification, maintenance, and regionalization of the developing brain anlage is still controversial. Mouse embryos homozygous for null mutation in the cripto gene die at about 9.0 days postcoitum (d.p.c.) and fail to gastrulate and to form the node (the primary organizer). Here, we study the presence and the distribution of anterior neural plate molecular domains in cripto null mutants. We demonstrate that, in cripto(-/-) embryos, the main prosencephalic and mesencephalic regions are present and that they assume the correct topological organization. The identity of the anterior neural domains is maintained in mutant embryos at 8.5 d.p.c., as well as in mutant explants dissected at 8.5 d.p.c. and cultured in vitro for 24 h. Our data imply the existence of a stable neural regionalization of anterior character inside the cripto(-/-) embryos, despite the failure in both the gastrulation process and node formation. These results suggest that, in mouse embryos, the specification of the anterior neural identities can be maintained without an absolute requirement for the embryonic mesoderm and the node.