Profilin I is essential for cell survival and cell division in early mouse development

Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1(ko)) allele in mice. Homozygous pfn1(ko/ko) mice are not viable. Pfn1(ko/ko) embryos died as early as the two-cell stage, and no pfn1(ko/ko) blastocysts were detectable. Adult pfn1(ko/wt) mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1(ko/wt) embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.     

The murine N-ras gene is not essential for growth and development.

The mammalian ras gene family encodes key cell-signaling, cell growth-related proteins that have been highly conserved in species from yeast to man. Specific point mutations in the ras genes are associated with various mammalian tumors. To understand the developmental role of the N-ras protooncogene in the mouse, we have disrupted its gene function by homologous recombination in embryonic stem cells. Mice derived from these cells that are homozygous for the N-ras mutation do not produce any detectable N-Ras protein and are morphologically and histologically indistinguishable from their heterozygous and wild-type siblings. Since N-ras is expressed at high levels in hematopoietic cells, we examined different populations of cells in peripheral blood and found no differences between mutant and normal animals. Our results show that N-ras gene function is dispensable for normal mouse development, growth, and fertility.     

Cdx2 is essential for axial elongation in mouse development

Inactivation of Cdx2 leads to preimplantation embryonic lethality. Rescue of the implantation defect by tetraploid fusion established that Cdx2 is necessary for trophoblastic development, vasculogenesis in the yolk sac mesoderm, allantoic growth, and chorioallantoic fusion. "Rescued" Cdx2 mutants die at late gastrulation stages because of failure of placental development. Cdx2 is also needed for the completion of the normal process of gastrulation and tail bud elongation. Presegmental paraxial mesoderm is severely restricted in amount and somites posterior to somite 5 are abnormal. The Cdx2 mutation, like mutations impairing Wnt and Fgf signaling, causes posterior truncations and disturbs axial patterning of the embryonic structures, indicated by changes in the Hox expression domains. The gene appears to be important in the integration of the pathways controlling embryonic axial elongation, and anterior-posterior patterning.     

SCL/Tal-1 is essential for hematopoietic commitment of the hemangioblast but not for its development

In this report, we have defined the stage at which Scl functions in the establishment of the hematopoietic system and provide evidence that its primary role is in the generation of the hematopoietic lineages from a progenitor called the blast colony-forming cell (BL-CFC), a cell considered to be the in vitro equivalent of the hemangioblast. Using an embryonic stem (ES) cell line in which lacZ cDNA has been targeted to the Scl locus, we show that most of the BL-CFCs are detected in the SCL/lacZ- population, indicating that this progenitor does not express Scl. In the blast colony assay, Scl-/- cells initiate colony growth but are unable to generate endothelial and hematopoietic progeny and thus form colonies consisting of vascular smooth muscle cells only. The capacity to give rise to blast colonies can be rescued by retroviral transduction of a wild-type Scl gene into Scl-/- FLK-1+ cells, suggesting that the BL-CFC is generated in this population. Finally, we show that Scl-/- endothelial cells display a growth deficiency in monolayer cultures that can be partially overcome by maintaining this population as 3-dimensional aggregates indicating that specific cellular interactions are required for maintenance of the Scl-/- endothelial lineage in vitro.     

Tropomodulin1 is required in the heart but not the yolk sac for mouse embryonic development

Tropomodulin (Tmod)1 caps the pointed ends of actin filaments in sarcomeres of striated muscle myofibrils and in the erythrocyte membrane skeleton. Targeted deletion of mouse Tmod1 leads to defects in cardiac development, fragility of primitive erythroid cells, and an absence of yolk sac vasculogenesis, followed by embryonic lethality at embryonic day 9.5. The Tmod1-null embryonic hearts do not undergo looping morphogenesis and the cardiomyocytes fail to assemble striated myofibrils with regulated F-actin lengths. To test whether embryonic lethality of Tmod1 nulls results from defects in cardiac myofibrillogenesis and development or from erythroid cell fragility and subsequent defects in yolk sac vasculogenesis, we expressed Tmod1 specifically in the myocardium of the Tmod1-null mice under the control of the alpha-myosin heavy chain promoter Tg(alphaMHC-Tmod1). In contrast to Tmod1-null embryos, which fail to undergo cardiac looping and have defective yolk sac vasculogenesis, both cardiac and yolk sac morphology of Tmod1(-/-Tg(alphaMHC-Tmod1)) embryos are normal at embryonic day 9.5. Tmod1(-/-Tg(alphaMHC-Tmod1)) embryos develop into viable and fertile mice, indicating that expression of Tmod1 in the heart is sufficient to rescue the Tmod1-null embryonic defects. Thus, although loss of Tmod1 results in myriad defects and embryonic lethality, the Tmod1(-/-) primary defect is in the myocardium. Moreover, Tmod1 is not required in erythrocytes for viability, nor do the Tmod1(-/-) fragile primitive erythroid cells affect cardiac development, yolk sac vasculogenesis, or viability in the mouse.     

Alpha-fetoprotein,the major fetal serum protein,is not essential for embryonic development but is required for female fertility

The alpha-fetoprotein gene (Afp) is a member of a multigenic family that comprises the related genes encoding albumin, alpha-albumin, and vitamin D binding protein. The biological role of this major embryonic serum protein is unknown although numerous speculations have been made. We have used gene targeting to show that AFP is not required for embryonic development. AFP null embryos develop normally, and individually transplanted homozygous embryos can develop in an AFP-deficient microenvironment. Whereas mutant homozygous adult males are viable and fertile, AFP null females are infertile. Our analyses of these mice indicate that the defect is caused by a dysfunction of the hypothalamic/pituitary system, leading to anovulation.     

Insulin-like growth factor-I is essential for embryonic bone development

Although IGF-I has been identified as an important growth factor for the skeleton, the role of IGF-I on embryonic bone development remains unknown. Here we show that, in IGF-I-deficient (IGF-I(-/-)) mice, skeletal malformations, including short-limbed dwarfism, were evident at days post coitus (dpc) 14.5 to 18.5, accompanied by delays of mineralization in the spinal column, sternum, and fore paws. Reduced chondrocyte proliferation and increased chondrocyte apoptosis were identified in both the spinal ossification center and the growth plate of long bones. Abnormal chondrocyte differentiation and delayed initiation of mineralization was characterized by small size and fewer numbers of type X collagen expressing hypertrophic chondrocytes and lower osteocalcin expression. The Indian hedgehog-PTHrP feedback loop was altered; expression of Indian hedgehog was reduced in IGF-I(-/-) mice in long bones and in the spine, whereas expression of PTHrP was increased. Our results indicate that IGF-I plays an important role in skeletal development by promoting chondrocyte proliferation and maturation while inhibiting apoptosis to form bones of appropriate size and strength.     

Oxytocin is required for nursing but is not essential for parturition or reproductive behavior.

Oxytocin, a neurohypophyseal hormone, has been traditionally considered essential for mammalian reproduction. In addition to uterine contractions during labor and milk ejection during nursing, oxytocin has been implicated in anterior pituitary function, paracrine effects in the testis and ovary and the neural control of maternal and sexual behaviors. To determine the essential role(s) of oxytocin in mammalian reproductive function, mice deficient in oxytocin have been generated using embryonic stem cell technology. A deletion of exon 1 encoding the oxytocin peptide was generated in embryonic stem cells at a high frequency and was successfully transmitted in the germ line. Southern blot analysis of genomic DNA from homozygote offspring and in situ hybridization with an exonic probe 3' of the deletion failed to detect any oxytocin or neurophysin sequences, respectively, confirming that the mutation was a null mutation. Mice lacking oxytocin are both viable and fertile. Males do not have any reproductive behavioral or functional defects in the absence of oxytocin. Similarly, females lacking oxytocin have no obvious deficits in fertility or reproduction, including gestation and parturition. However, although oxytocin-deficient females demonstrate normal maternal behavior, all offspring die shortly after birth because of the dam's inability to nurse. Postpartum injections of oxytocin to the oxytocin deficient mothers restore milk ejection and rescue the offspring. Thus, despite the multiple reproductive activities that have been attributed to oxytocin, oxytocin plays an essential role only in milk ejection in the mouse.     

Recombinant interferon-alpha,but not interferon-gamma is effective therapie for essential thrombocythemia

Summary Recombinant interferon-gamma with a starting dose of 0.5 mg 3×/week subcutaneously, was administered to 6 patients with essential thrombocythemia (median platelet count 1172×10⁹/1, range 602–1564). Four of the patients had received alkylating agents previously. Hematological remission, defined as a decrease in platelet counts to 350×10⁹/l, was observed in none of these patients. Subsequently 4 of these 6 patients, supplemented by 2 others were treated with interferonalpha_2c at a dose of 5×10⁶ U daily subcutaneously. Five patients showed hematological remission. In case of hematological remission the interferonalpha dosis was reduced to 5× an thereafter to 3× weekly 5×10⁶ U. During an observation period ranging from 12–41 weeks platelet counts remained normal in all patients. Side-effects were mild and consisted of fever, myalgias, malaise and itching occurring mainly during the first month of treatment. No dose adaptation was required. The patients treated previously with interferon-gamma experienced the side effects from this drug less tolerably than those from the alpha-compound. These observations suggest that recombinant interferonalpha may be an effective drug in treating essential thrombocythemia resulting in a sustained response.     

Fyn is not essential for Bcr-Abl-induced leukemogenesis in mouse bone marrow transplantation models

The Bcr-Abl oncogene causes human Philadelphia chromosome-positive (Ph(+)) leukemias, including B-cell acute lymphoblastic leukemia (B-ALL) and chronic myeloid leukemia (CML) with chronic phase (CML-CP) to blast crisis (CML-BC). Previous studies have demonstrated that Src family kinases are required for the induction of B-ALL, but not for CML, which is induced by Bcr-Abl in mice. In contrast, it has been reported that Fyn is up-regulated in human CML-BC compared with CML-CP, implicating Fyn in the blast crisis transition. Here, we aimed to delineate the exact role of Fyn in the induction/progression of Ph(+) leukemias. We found that Fyn is expressed in mouse hematopoietic cells at varying stages of development, including c-kit(+)Sca-1(+)Lin(-) cells. Notably, Fyn is highly expressed in some of human lymphomas, but not in human Ph(+) leukemias including CML-BC. In mouse bone marrow transplantation models, mice transplanted with wild-type or Fyn-deficient bone marrow cells transduced with Bcr-Abl showed no differences in the development of B-ALL or CML-like diseases. Similarly, Fyn deficiency failed to impact the development of myeloid CML-BC induced by Bcr-Abl and Hes1. Elevated expression of Fyn was not found in mouse samples of Bcr-Abl-mediated CML- and CML-BC-like diseases. Thus, Fyn is not required for the pathogenesis of Bcr-Abl-mediated leukemias.     

Ras membrane targeting is essential for glucose signaling but not for viability in yeast.

Ras proteins are small GTP binding proteins that serve as critical relays in a variety of signal transduction pathways in eukaryotic cells. Like most metazoan Ras proteins, yeast Ras is post-translationally modified by addition of a farnesyl and a palmitoyl moiety, and these modifications are required for targeting the protein to the cytoplasmic face of the plasma membrane and for biological activity of the protein. We have constructed mutants of the yeast (Saccharomyces cerevisiae) Ras that are farnesylated in vivo but are not palmitoylated. These mutant proteins are not localized to the plasma membrane but function in the cell as well as the wild-type protein. Such mutants are viable but fail to induce a transient increase in intracellular cAMP concentration in response to glucose addition, although this deficiency does not yield a marked growth phenotype. These results are consistent with the hypothesis that the essential role of the farnesyl moiety on yeast Ras is to enhance productive interaction between Ras and its essential downstream target, adenylyl cyclase, rather than to localize Ras to the plasma membrane.     

Vitronectin is not essential for normal mammalian development and fertility.

Vitronectin (VN) is an abundant glycoprotein present in plasma and the extracellular matrix of most tissues. Though the precise function of VN in vivo is unknown, it has been implicated as a participant in diverse biological processes, including cell attachment and spreading, complement activation, and regulation of hemostasis. The major site of synthesis appears to be the liver, though VN is also found in the brain at an early stage of mouse organogenesis, suggesting that it may play an important role in mouse development. Genetic deficiency of VN has not been reported in humans or in other higher organisms. To examine the biologic function of VN within the context of the intact animal, we have established a murine model for VN deficiency through targeted disruption of the murine VN gene. Southern blot analysis of DNA obtained from homozygous null mice demonstrates deletion of all VN coding sequences, and immunological analysis confirms the complete absence of VN protein expression in plasma. However, heterozygous mice carrying one normal and one null VN allele and homozygous null mice completely deficient in VN demonstrate normal development, fertility, and survival. Sera obtained from VN-deficient mice are completely deficient in "serum spreading factor" and plasminogen activator inhibitor 1 binding activities. These observations demonstrate that VN is not essential for cell adhesion and migration during normal mouse development and suggest that its role in these processes may partially overlap with other adhesive matrix components.     

Signaling but not trafficking function of HIV-1 protein Nef is essential for Nef-induced defects in human intrathymic T-cell development

The HIV-1 gene nef is important for progression toward AIDS and cellular depletion of the infected thymus. Expression of the Nef protein alone impairs human thymopoiesis. Here, we performed a structure-function analysis of the Nef protein by comparing the effect on T-cell development of different nef alleles, either wild type or defective for selected functions, expressed by human thymocytes. We show that Nef-mediated impaired thymopoiesis is not due to altered surface marker trafficking, nor dependent on oligomerization of Nef. By contrast, mutations in the myristoylation site and in signaling sites of Nef, ie, sites important for interaction with phosphofurin acidic cluster sorting protein-1 (PACS-1), Src homology domain 3 (SH3) domains, and p21-activated kinase 2 (PAK2), were found to be critical for its effect on T-cell development. These results point to sites in Nef to target therapeutically for restoration of thymopoiesis in HIV infection.     

Thyrotropin is not a growth factor for human thyroid cells in culture

Thyroid cells, obtained from both normal human tissue and benign nodular goiter, were cultured and maintained in vitro in 4-18 passages. Cultures with confluent cells accumulated cyclic AMP (10-150 times the basal amount) upon addition of bovine thyrotropin (100 milliunits/ml), indicating that the cells in culture maintained a thyrotropin-sensitive adenylate cyclase system. Addition of high doses of thyrotropin also induced a characteristic and reversible change in the morphology of the cells.The effect of thyrotropin on cell growth was studied in short- and long-term experiments. Thyrotropin reduced [(3)H]thymidine incorporation in a dose-dependent fashion in all cultures of thyroid cells. The maximal inhibition over a 24-hr period was about 50%. The thyroid cells were notably sensitive, and the half-maximal effect occurred at about 100 milliunits of thyrotropin per ml. In contrast, the hormone had no effect on [(3)H]-thymidine incorporation into human glial cells. Low doses of thyrotropin also had no effect on human fibroblasts and, at high doses, a stimulation of [(3)H]thymidine incorporation was seen. Thyroid cell cultures grown in the presence of 10 milliunits of thyrotropin per ml for 7-14 days had a slower growth rate and 24-36% lower cell numbers at saturation density than control dishes, indicating that the hormone also had a long-term effect on cell proliferation. The data agree with in vitro studies by others of the effects of corticotropin and lutropin on target cells and suggest that in vivo the primary action of pituitary trophic hormones on endocrine tissues is not stimulation of growth.