Grainyhead-like 2 interacts with noggin to regulate tissue fusion in mouse.

Defective tissue fusion during mammalian embryogenesis results in congenital anomalies, such as exencephaly, spina bifida and cleft lip and/or palate. The highly conserved transcription factor grainyhead-like 2 (Grhl2) is a crucial regulator of tissue fusion, with mouse models lacking GRHL2 function presenting with a fully penetrant open cranial neural tube, facial and abdominal clefting (abdominoschisis), and an open posterior neuropore. Here, we show that GRHL2 interacts with the soluble morphogen protein and bone morphogenetic protein (BMP) inhibitor noggin (NOG) to impact tissue fusion during development. The maxillary prominence epithelium in embryos lacking Grhl2 shows substantial morphological abnormalities and significant upregulation of NOG expression, together with aberrantly distributed pSMAD5-positive cells within the neural crest cell-derived maxillary prominence mesenchyme, indicative of disrupted BMP signalling. Reducing this elevated NOG expression (by generating Grhl2-/-;Nog+/- embryos) results in delayed embryonic lethality, partial tissue fusion rescue, and restoration of tissue form within the craniofacial epithelia. These data suggest that aberrant epithelial maintenance, partially regulated by noggin-mediated regulation of BMP-SMAD pathways, may underpin tissue fusion defects in Grhl2-/- mice.

Dysregulation of Grainyhead-like 3 expression causes widespread developmental defects.

BACKGROUND: The gene encoding the transcription factor, Grainyhead-like 3 (Grhl3), plays critical roles in mammalian development and homeostasis. Grhl3-null embryos exhibit thoraco-lumbo-sacral spina bifida and soft-tissue syndactyly. Additional studies reveal that these embryos also exhibit an epidermal proliferation/differentiation imbalance. This manifests as skin barrier defects resulting in peri-natal lethality and defective wound repair. Despite these extensive analyses of Grhl3 loss-of-function models, the consequences of gain-of-function of this gene have been difficult to achieve. RESULTS: In this study, we generated a novel mouse model that expresses Grhl3 from a transgene integrated in the Rosa26 locus on an endogenous Grhl3-null background. Expression of the transgene rescues both the neurulation and skin barrier defects of the knockout mice, allowing survival into adulthood. Despite this, the mice are not normal, exhibiting a range of phenotypes attributable to dysregulated Grhl3 expression. In mice homozygous for the transgene, we observe a severe Shaker-Waltzer phenotype associated with hearing impairment. Micro-CT scanning of the inner ear revealed profound structural alterations underlying these phenotypes. In addition, these mice exhibit other developmental anomalies including hair loss, digit defects, and epidermal dysmorphogenesis. CONCLUSION: Taken together, these findings indicate that diverse developmental processes display low tolerance to dysregulation of Grhl3.

Inhibition of retinoic acid signaling impairs cranial and spinal neural tube closure in mice lacking the Grainyhead-like 3 transcription factor.

Neural tube closure is a dynamic morphogenic event in early embryonic development. Perturbations of this process through either environmental or genetic factors induce the severe congenital malformations known collectively as neural tube defects (NTDs). Deficiencies in maternal folate intake have long been associated with NTDs, as have mutations in critical neurulation genes that include the Grainyhead-like 3 (Grhl3) gene. Mice lacking this gene exhibit fully penetrant thoraco-lumbo-sacral spina bifida and a low incidence of exencephaly. Previous studies have shown that exposure of pregnant mice carrying hypomorphic Grhl3 alleles to exogenous retinoic acid (RA) increases the incidence and severity of NTDs in their offspring. Here, we demonstrate that inhibition of RA signaling using a high affinity pan-RA receptor antagonist administered to pregnant mice at E7.5 induces fully penetrant exencephaly and more severe spina bifida in Grhl3-null mice. Later administration, although prior to neural tube closure has no effect. Similarly, blockade of RA in the context of reduced expression of Grhl2, a related gene known to induce NTDs, has no effect. Taken together, these findings provide new insights into the complexities of the interplay between RA signaling and Grhl3-induced neurulation.

Grainyhead-like transcription factors: guardians of the skin barrier.

There has been selective pressure to maintain a skin barrier since terrestrial animals evolved 360 million years ago. These animals acquired an unique integumentary system with a keratinized, stratified, squamous epithelium surface barrier. The barrier protects against dehydration and entry of microbes and toxins. The skin barrier centres on the stratum corneum layer of the epidermis and consists of cornified envelopes cemented by the intercorneocyte lipid matrix. Multiple components of the barrier undergo cross-linking by transglutaminase (TGM) enzymes, while keratins provide additional mechanical strength. Cellular tight junctions also are crucial for barrier integrity. The grainyhead-like (GRHL) transcription factors regulate the formation and maintenance of the integument in diverse species. GRHL3 is essential for formation of the skin barrier during embryonic development, whereas GRHL1 maintains the skin barrier postnatally. This is achieved by transactivation of Tgm1 and Tgm5, respectively. In addition to its barrier function, GRHL3 plays key roles in wound repair and as an epidermal tumour suppressor. In its former role, GRHL3 activates the planar cell polarity signalling pathway to mediate wound healing by providing directional migration cues. In squamous epithelium, GRHL3 regulates the balance between proliferation and differentiation, and its loss induces squamous cell carcinoma (SCC). In the skin, this is mediated through increased expression of MIR21, which reduces the expression levels of GRHL3 and its direct target, PTEN, leading to activation of the PI3K-AKT signalling pathway. These data position the GRHL family as master regulators of epidermal homeostasis across a vast gulf of evolutionary history.

Il y a eu une pression de sélection pour maintenir la barrière cutanée depuis l'évolution des animaux terrestres pendant 360 millions d'années. Ces animaux ont acquis un système tégumentaire unique avec un épithélium squameux, stratifié, kératinisé comme barrière de surface. La barrière protège contre la déshydratation et l'entée de microbes et de toxines. La barrière cutanée est centrée sur la couche du stratum corneum de l'épiderme et consiste en des enveloppes cimentées par une matrice lipidique intercornéocytaire. Les composants multiples de la barrière subissent des remaniements par les enzymes transglutaminases (TGM) tandis que la kératine fournit un soutien mécanique supplémentaire. Les jonctions serrées cellulaires jouent aussi un rôle crucial pour l'intégrité de barrière. Les facteurs de transcriptions GRHL (grainyhead-like) régulent la formation et le maintien du tégument dans différentes espèces. GRHL3 est essentielle pour la formation de la barrière cutanée au cours du développement embryonnaire tandis que GRHL1 maintient la barrière cutanée après la naissance. Ceci est permis respectivement par transactivation de Tgm1 et Tgm5. En plus de cette fonction barrière, GRHL3 joue un rôle clé dans la cicatrisation et en tant que suppresseur de tumeur épidermique. Dans ses rôles principaux, GRHL3 active la voie de signal de polarité cellulaire plane pour soutenir la cicatrisation en fournissant des repaires directionnels de migration. Dans les épithéliums squameux, GRHL3 régule la balance entre prolifération et différentiation, et sa perte induit le carcinome épidermoïde (SCC). Dans la peau ceci est médié par une augmentation de l'expression de MIR21, qui réduit le niveau d'expression de GRHL3 et sa cible directe, PTEN, menant à l'activation de la voie de signal PI3K-AKT. Ces données positionnent la famille GRHL comme régulatrice majeure de l'homéostasie épidermique à travers le vaste gouffre de l'histoire de l'évolution.

Ha habido una presión selectiva para mantener una barrera cutánea desde que los animales terrestres evolucionaron hace 360 ​​millones de años. Estos animales adquirieron un sistema tegumentario único con una barrera superficial de epitelio escamoso estratificado queratinizado. La barrera protege contra la deshidratación y la entrada de microbios y toxinas. La barrera cutánea se centra en la capa de estrato córneo de la epidermis y consta de membranas cornificadas cementadas por una matriz lipídica intercorneocitaria. Múltiples componentes de la barrera se unen por la actividad de enzimas transglutaminasas (TGM), mientras que las queratinas proporcionan resistencia mecánica adicional. Las uniones celulares estrechas también son cruciales para la integridad de la barrera. Los factores de transcripción similares a grainyhead (cabeza granulada) (GRHL) regulan la formación y mantenimiento del tegumento en diversas especies. GRHL3 es esencial para la formación de la barrera cutánea durante el desarrollo embrionario, mientras que GRHL1 mantiene la barrera cutánea postnatal. Esto se logra mediante la transactivación de Tgm1 y Tgm5, respectivamente. Además de su función de barrera, GRHL3 juega un papel clave en la reparación de heridas y como supresor de tumores epidérmicos. En su función de cicatrización, GRHL3 activa la vía de señalización de la polaridad celular plana para mediar en la cicatrización de heridas proporcionando señales de migración direccional. En el epitelio escamoso, GRHL3 regula el equilibrio entre la proliferación y la diferenciación, y su pérdida induce el carcinoma de células escamosas (SCC). En la piel, esto está mediado por una mayor expresión de MIR21, que reduce los niveles de expresión de GRHL3 y su sustrato directo, PTEN, lo que lleva a la activación de la vía de señal intracelular PI3K-AKT. Estos datos colocan la familia de factores de transcripción GRHL como reguladores críticos de la homeostasis epidérmica a través de una extensa historia evolutiva.

Tem havido uma pressão seletiva para manter a barreira cutânea desde a evolução dos animais terrestres há 360 milhões de anos. Estes animais adquiriram um sistema tegumentar único com uma barreira de superfície escamosa, estratificada e queratinizada. A barreira protege contra a desidratação e entrada de micróbios e toxinas. A barreira cutânea é centrada na camada do estrato córneo da epiderme e consiste em envelopes cornificados revestidos pela matriz lipídica intercorneocítica. Vários componentes da barreira sofrem ligação cruzada por enzimas transglutaminase (TGM), enquanto as queratinas fornecem resistência mecânica adicional. As junções celulares também são cruciais para a integridade da barreira. Os fatores de transcrição do tipo grainyhead (GRHL) regulam a formação e manutenção do tegumento em diversas espécies. GRHL3 é essencial para a formação da barreira cutânea durante o desenvolvimento embrionário, enquanto GRHL1 mantém a barreira cutânea pós-natal. Isso é obtido pela transativação de Tgm1 e Tgm5, respectivamente. Além de sua função de barreira, GRHL3 desempenha papéis importantes no reparo de feridas e como supressor de tumor epidérmico. Em sua função anterior, GRHL3 ativa a via de sinalização de polaridade celular planar para mediar a cicatrização de feridas, fornecendo pistas de migração direcional. No epitélio escamoso, o GRHL3 regula o equilíbrio entre a proliferação e a diferenciação, e sua perda induz o carcinoma de células escamosas (CCE). Na pele, isso é mediado pelo aumento da expressão de MIR21, que reduz os níveis de expressão de GRHL3 e seu alvo direto, PTEN, levando à ativação da via de sinalização PI3K-AKT. Esses dados posicionam a família GRHL como reguladores mestres da homeostase epidérmica em um vasto abismo da história evolutiva.

Delineating the roles of Grhl2 in craniofacial development through tissue-specific conditional deletion and epistasis approaches in mouse.

BACKGROUND: The highly conserved Grainyhead-like (Grhl) family of transcription factors play critical roles in the development of the neural tube and craniofacial skeleton. In particular, deletion of family member Grainyhead-like 2 (Grhl2) leads to mid-gestational embryonic lethality, maxillary clefting, abdominoschisis, and both cranial and caudal neural tube closure defects. These highly pleiotropic and systemic defects suggest that Grhl2 plays numerous critical developmental roles to ensure correct morphogenesis and patterning. RESULTS: Here, using four separate Cre-lox conditional deletion models, as well as one genetic epistasis approach (Grhl2+/- ;Edn1+/- double heterozygous mice) we have investigated tissue-specific roles of Grhl2 in embryonic development, with a particular focus on the craniofacial skeleton. We find that loss of Grhl2 in the pharyngeal epithelium (using the ShhCre driver) leads to low-penetrance micrognathia, whereas deletion of Grhl2 within the ectoderm of the pharynx (NestinCre ) leads to small, albeit significant, differences in the proximal-distal elongation of both the maxilla and mandible. Loss of Grhl2 in endoderm (Sox17-2aiCre ) resulted in noticeable lung defects and a single instance of secondary palatal clefting, although formation of other endoderm-derived organs such as the stomach, bladder and intestines was not affected. Lastly, deletion of Grhl2 in cells of the neural crest (Wnt1Cre ) did not lead to any discernible defects in craniofacial development, and similarly, our epistasis approach did not detect any phenotypic consequences of loss of a single allele of both Grhl2 and Edn1. CONCLUSION: Taken together, our study identifies a pharyngeal-epithelium intrinsic, non-cell-autonomous role for Grhl2 in the patterning and formation of the craniofacial skeleton, as well as an endoderm-specific role for Grhl2 in the formation and establishment of the mammalian lung.

Inactivation of Zeb1 in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure.

Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like (Grhl1-3) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, Grhl2, is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. Grhl2-null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of Grhl2-null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors Ovol1 and Ovol2, which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of Zeb1 expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in Zeb1-/- mice is evident, with rescue of both palatal and facial fusion seen in Grhl2-/-;Zeb1-/- embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.

An intronic mutation in Chd7 creates a cryptic splice site, causing aberrant splicing in a mouse model of CHARGE syndrome.

Alternate splicing is a critical regulator of gene expression in eukaryotes, however genetic mutations can cause erroneous splicing and disease. Most recorded splicing disorders are caused by mutations of splice donor/acceptor sites, however intronic mutations can affect splicing. Clinical exome analyses largely ignore intronic sequence, limiting the detection of mutations to within coding regions. We describe 'Trooper', a novel mouse model of CHARGE syndrome harbouring a pathogenic point mutation in Chd7. The mutation is 18 nucleotides upstream of exon 10 and creates a cryptic acceptor site, causing exon skipping and partial intron retention. This mutation, though detectable in exome sequence, was initially dismissed by computational filtering due to its intronic location. The Trooper strain exhibited many of the previously described CHARGE-like anomalies of CHD7 deficient mouse lines; including hearing impairment, vestibular hypoplasia and growth retardation. However, more common features such as facial asymmetry and circling were rarely observed. Recognition of these characteristic features prompted manual reexamination of Chd7 sequence and subsequent validation of the intronic mutation, highlighting the importance of phenotyping alongside exome analyses. The Trooper mouse serves as a valuable model of atypical CHARGE syndrome and reveals a molecular mechanism that may underpin milder clinical presentation of the syndrome.

Stage-dependent therapeutic efficacy in PI3K/mTOR-driven squamous cell carcinoma of the skin.

Cutaneous squamous cell carcinoma (SCC) is a recurrent cancer that is prevalent in predisposed subjects such as immunosuppressed patients and patients being treated for other malignancies. Model systems to trial therapies at different stages of SCC development are lacking, therefore precluding efficient therapeutic interventions. Here, we have disrupted the expression of the tumor suppressor GRHL3 to induce loss of PTEN and activation of the PI3K/mTOR signaling pathway in mice and human skin, promoting aggressive SCC development. We then examined the potential for targeting PI3K/mTOR and an oncogenic driver miR-21, alone and in combination, for the prevention and treatment of SCC during the initiation, promotion/progression and establishment stages. Treatment with PI3K/mTOR inhibitors completely prevented tumor initiation, and these inhibitors significantly delayed the course of papilloma progression to malignancy. However, established SCC did not undergo any growth regression, indicating that this therapy is ineffective in established cancers. Mechanistically, the resistant SCCs displayed increased miR-21 expression in mice and humans where antagonists of miR-21 rescued expression levels of GRHL3/PTEN, but the combination of miR-21 antagonism with PI3K/mTOR inhibition resulted in acquired SCC resistance in part via c-MYC and OCT-4 upregulation. In conclusion, our data provide molecular evidence for the efficacy of targeting oncogenic drivers of SCC during the initiation and promotion stages and indicate that combination therapy may induce an aggressive phenotype when applied in the establishment stage.

Mice Haploinsufficient for Ets1 and Fli1 Display Middle Ear Abnormalities and Model Aspects of Jacobsen Syndrome.

E26 transformation-specific 1 (ETS1) and friend leukemia integration 1 (FLI1) are members of the ETS family of transcription factors, of which there are 28 in humans. Both genes are hemizygous in Jacobsen syndrome, an 11q contiguous gene deletion disorder involving thrombocytopenia, facial dysmorphism, growth and mental retardation, malformation of the heart and other organs, and hearing impairment associated with recurrent ear infections. To determine whether any of these defects are because of hemizygosity for ETS1 and FLI1, we characterized the phenotype of mice heterozygous for mutant alleles of Ets1 and Fli1. Fli1(+/-) mice displayed mild thrombocytopenia, as did Ets1(+/-)Fli1(+/-) animals. Fli1(+/-) and Ets1(+/-)Fli1(+/-) mice also displayed craniofacial abnormalities, including a small middle ear cavity, short nasal bone, and malformed interface between the nasal bone process and cartilaginous nasal septum. They exhibited hearing impairment, otitis media, fusions of ossicles to the middle ear wall, and deformed stapes. Hearing impairment was more penetrant and stapes malformations were more severe in Ets1(+/-)Fli1(+/-) mice than in Fli1(+/-) mice, indicating partial functional redundancy of these transcription factors during auditory development. Our findings indicate that the short nose, otitis media, and hearing impairment in Jacobsen syndrome are likely because of hemizygosity for ETS1 and FLI1.

CHD7 deficiency in "Looper", a new mouse model of CHARGE syndrome, results in ossicle malformation, otosclerosis and hearing impairment.

CHARGE syndrome is a rare human disorder caused by mutations in the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). Characteristics of CHARGE are varied and include developmental ear and hearing anomalies. Here we report a novel mouse model of CHD7 dysfunction, termed Looper. The Looper strain harbours a nonsense mutation (c.5690C>A, p.S1897X) within the Chd7 gene. Looper mice exhibit many of the clinical features of the human syndrome, consistent with previously reported CHARGE models, including growth retardation, facial asymmetry, vestibular defects, eye anomalies, hyperactivity, ossicle malformation, hearing loss and vestibular dysfunction. Looper mice display an otosclerosis-like fusion of the stapes footplate to the cochlear oval window and blepharoconjunctivitis but not coloboma. Looper mice are hyperactive and have vestibular dysfunction but do not display motor impairment.

A new mouse model of Canavan leukodystrophy displays hearing impairment due to central nervous system dysmyelination.

Canavan disease is a leukodystrophy caused by mutations in the ASPA gene. This gene encodes the enzyme that converts N-acetylaspartate into acetate and aspartic acid. In Canavan disease, spongiform encephalopathy of the brain causes progressive mental retardation, motor deficit and death. We have isolated a mouse with a novel ethylnitrosourea-induced mutation in Aspa. This mutant, named deaf14, carries a c.516T>A mutation that is predicted to cause a p.Y172X protein truncation. No full-length ASPA protein is produced in deaf14 brain and there is extensive spongy degeneration. Interestingly, we found that deaf14 mice have an attenuated startle in response to loud noise. The first auditory brainstem response peak has normal latency and amplitude but peaks II, III, IV and V have increased latency and decreased amplitude in deaf14 mice. Our work reveals a hitherto unappreciated pathology in a mouse model of Canavan disease, implying that auditory brainstem response testing could be used in diagnosis and to monitor the progression of this disease.

Two ENU-induced alleles of Atp2b2 cause deafness in mice.

Over 120 loci are known to cause inherited hearing loss in humans. The deafness gene has been identified for only half of these loci. With the aim of identifying some of the remaining deafness genes, we performed an ethylnitrosourea mutagenesis screen for deaf mice. We isolated two mutants with semi-dominant hearing loss, Deaf11 and Deaf13. Both contained causative mutations in Atp2b2, which encodes the plasma membrane calcium ATPase 2. The Atp2b2 (Deaf11) mutation leads to a p. I1023S substitution in the tenth transmembrane domain. The Atp2b2 (Deaf13) mutation leads to a p. R561S substitution in the catalytic core. Mice homozygous for these mutations display profound hearing loss. Heterozygotes display mild to moderate, progressive hearing loss.

Genetic Modifier Screens in Mice.

ENU mutagenesis is a forward genetics strategy in which random mutagenesis and phenotypic screening is used to identify genes based on the phenotype induced when they are mutated. A modifier screen is a type of screen in which mice with a pre-existing phenotype are utilized to identify mutations that can enhance or suppress this phenotype. This approach has the potential to uncover missing pathway members, reveal novel genetic interactions, and pinpoint new drug targets. Considerations when planning a suppressor screen include current knowledge, genomic footprint, penetrance, variance, robustness, latency of the starting phenotype, viability, fertility, genetic background and ENU tolerance of starting strain, screening assay, mouse numbers required, and mutation identification strategy. Practical advice on each of these is provided in this review. Curr. Protoc. Mouse Biol. 2:75-87 © 2012 by John Wiley & Sons, Inc.

Vitamin D-deficient diet rescues hearing loss in Klotho mice.

Klotho-deficient mice exhibit a premature aging syndrome, a feature of which is mild hearing loss. In the present study, the hearing phenotype of Klotho mice was characterized to better determine how well this phenotype resembles presbycusis in humans. It was demonstrated that Klotho animals have auditory-evoked brainstem response (ABR) threshold shifts of 14-18 dB in response to pure tone stimuli of 4, 8, 16 and 32 kHz, and similarly, in response to clicks; however, cochlear histology and spiral ganglion neuron density appeared normal in these mice. It was further demonstrated that a vitamin D-deficient diet normalizes serum calcitriol (1,25(OH)(2)D(3)) levels and prevents hearing loss in Klotho mice. It is concluded that hearing loss in Klotho mice is caused by elevated renal 1α-hydroxylase expression and consequent excessive production of calcitriol. These findings implicate the vitamin D metabolic pathway in hearing loss and pose questions as to the mechanism by which elevated calcitriol levels mediate such hearing loss.

Regulation of hematopoietic stem cells by their mature progeny.

Thrombopoietin (TPO), acting through its receptor Mpl, has two major physiological roles: ensuring production of sufficient platelets via stimulation of megakaryocyte production and maintaining hematopoietic stem cell (HSC) quiescence. Mpl also controls circulating TPO concentration via receptor-mediated internalization and degradation. Here, we demonstrate that the megakaryocytosis and increased platelet mass in mice with mutations in the Myb or p300 genes causes reduced circulating TPO concentration and TPO starvation of the stem-cell compartment, which is exacerbated because these cells additionally exhibit impaired responsiveness to TPO. HSCs from Myb(Plt4/Plt4) mice show altered expression of TPO-responsive genes and, like HSCs from Tpo and Mpl mutant mice, exhibit increased cycling and a decline in the number of HSCs with age. These studies suggest that disorders of platelet number can have profound effects on the HSC compartment via effects on the feedback regulation of circulating TPO concentration.

Critical roles for c-Myb in lymphoid priming and early B-cell development.

c-Myb is a transcription factor with functions in many hematopoietic lineages. c-Myb-deficient mice display reduced numbers of B cells; however, it is unknown what role c-Myb plays in B lymphopoiesis because no critical target genes have been identified in the B-cell lineage. We demonstrate that conditional deletion of c-Myb in B-cell progenitors completely abolishes B-cell development. c-Myb is required for lymphoid progenitors to respond to the cytokines interleukin-7 and thymic stromal lymphopoietin; in the absence of sufficient c-Myb activity, mice display a B lymphopenia that closely resembles that observed in interleukin-7 receptor alpha-deficient animals. Analysis of the multipotent progenitor compartment indicates that c-Myb is also required for up-regulation of multiple lymphoid-associated genes, including Il7r, and for the subsequent development of the common lymphoid progenitor population. These data show that c-Myb plays a critical role in the regulatory pathways governing lymphoid specification and early B-cell differentiation.

Programmed anuclear cell death delimits platelet life span.

Platelets are anuclear cytoplasmic fragments essential for blood clotting and wound healing. Despite much speculation, the factors determining their life span in the circulation are unknown. We show here that an intrinsic program for apoptosis controls platelet survival and dictates their life span. Pro-survival Bcl-x(L) constrains the pro-apoptotic activity of Bak to maintain platelet survival, but as Bcl-x(L) degrades, aged platelets are primed for cell death. Genetic ablation or pharmacological inactivation of Bcl-x(L) reduces platelet half-life and causes thrombocytopenia in a dose-dependent manner. Deletion of Bak corrects these defects, and platelets from Bak-deficient mice live longer than normal. Thus, platelets are, by default, genetically programmed to die by apoptosis. The antagonistic balance between Bcl-x(L) and Bak constitutes a molecular clock that determines platelet life span: this represents an important paradigm for cellular homeostasis, and has profound implications for the diagnosis and treatment of disorders that affect platelet number and function.

Thrombocytopenia and kidney disease in mice with a mutation in the C1galt1 gene.

An N-ethyl-N-nitrosourea mutagenesis screen in mice was performed to isolate regulators of circulating platelet number. We report here recessive thrombocytopenia and kidney disease in plt1 mice, which is the result of a severe but partial loss-of-function mutation in the gene encoding glycoprotein-N-acetylgalactosamine-3-beta-galactosyltransferase (C1GalT1), an enzyme essential for the synthesis of extended mucin-type O-glycans. Platelet half-life and basic hemostatic parameters were unaffected in plt1/plt1 mice, and the thrombocytopenia and kidney disease were not attenuated on a lymphocyte-deficient rag1-null background. gpIbalpha and podocalyxin were found to be major underglycosylated proteins in plt1/plt1 platelets and the kidney, respectively, implying that these are key targets for C1GalT1, appropriate glycosylation of which is essential for platelet production and kidney function. Compromised C1GalT1 activity has been associated with immune-mediated diseases in humans, most notably Tn syndrome and IgA nephropathy. The disease in plt1/plt1 mice suggests that, in addition to immune-mediated effects, intrinsic C1Gal-T1 deficiency in megakaryocytes and the kidney may contribute to pathology.

A mutation in the translation initiation codon of Gata-1 disrupts megakaryocyte maturation and causes thrombocytopenia.

We have generated mice from a N-ethyl-N-nitrosourea mutagenesis screen that carry a mutation in the translation initiation codon of Gata-1, termed Plt13, which is equivalent to mutations found in patients with acute megakaryoblastic leukemia and Down syndrome. The Gata-1 locus is present on the X chromosome in humans and in mice. Male mice hemizygous for the mutation (Gata-1Plt13/Y) failed to produce red blood cells and died during embryogenesis at a similar stage to Gata-1-null animals. Female mice that carry the Plt13 mutation are mosaic because of random inactivation of the X chromosome. Adult Gata-1Plt13/+ females were not anemic, but they were thrombocytopenic and accumulated abnormal megakaryocytes without a concomitant increase in megakaryocyte progenitor cells. Gata-1Plt13/+ mice contained large numbers of blast-like colony-forming cells, particularly in the fetal liver, but also in adult spleen and bone marrow, from which continuous mast cells lines were readily derived. Although the equivalent mutation to Gata-1Plt13 in humans results in production of GATA-1s, a short protein isoform initiated from a start codon downstream of the mutated initiation codon, Gata-1s was not detected in Gata-1Plt13/+ mice.

Anomalous megakaryocytopoiesis in mice with mutations in the c-Myb gene.

Mpl(-/-) mice bearing the Plt3 or Plt4 mutations in the c-Myb gene exhibit thrombopoietin (TPO)-independent supraphysiological platelet production accompanied by excessive megakaryocytopoiesis and defective erythroid and lymphoid cell production. To better define the cellular basis for the thrombocytosis in these mice, we analyzed the production and characteristics of megakaryocytes and their progenitors. Consistent with thrombocytosis arising from hyperactive production, the high platelet counts in mice carrying the c-Myb(Plt4) allele were not accompanied by any significant alteration in platelet half-life. Megakaryocytes in c-Myb mutant mice displayed reduced modal DNA ploidy and, among the excessive numbers of megakaryocyte progenitor cells, more mature precursors were particularly evident. Megakaryocyte progenitor cells carrying the Plt3 or Plt4 c-Myb mutations, but not granulocyte-macrophage progenitors, exhibited 200-fold enhanced responsiveness to granulocyte-macrophage colony-stimulating factor (GM-CSF), suggesting that altered responses to cytokines may contribute to expanded megakaryocytopoiesis. Mutant preprogenitor (blast colony-forming) cells appeared to have little capacity to form megakaryocyte progenitor cells. In contrast, the spleens of irradiated mice 12 days after transplantation with mutant bone marrow contained abundant megakaryocyte progenitor cells, suggesting that altered c-Myb activity skews differentiation commitment in spleen colony-forming units (CFU-S) in favor of excess megakaryocytopoiesis.