Development Of Mutant Mice Research Articles

Abstract P2-08-01: Loss of function of Brca1 or Gata3 induces basal-like breast cancer

Abstract BACKGROUND: Breast cancer is mainly divided into estrogen receptor (ER)-positive luminal and ER-negative basal-like tumors. Luminal-type tumors are associated with better survival and respond to hormone therapies whereas basal-like tumors are more aggressive and associated with a poor prognosis. Mammary epithelia are mainly composed of luminal and basal cells that are maintained by luminal and basal progenitors, respectively. The maintenance of luminal cell fate is orchestrated by networks of transcription factors, including BRCA1 and GATA3. Functional loss of BRCA1 by germline or somatic mutation or by promoter methylation is associated with more than one third of basal-like breast cancers. GATA3 expression is reduced in basal-like breast cancers and cancers that metastasize. Overexpression of GATA3 in cancer cells inhibits tumor formation. Deletion of Brca1 or Gata3 in mice results in early lethality or growth defects. How BRCA1 and GATA3 suppress breast cancer remains elusive. METHODS: We generated mice lacking Brca1 or Gata3 in mammary epithelia. Due to the proliferative defects and induction of p18Ink4c (p18), an inhibitor of CDK4/6, in mammary epithelial cells of these mice, we then generated mice lacking Brca1 or Gata3 in p18 deficient mammary epithelia. We determined spontaneous mammary tumor development in mutant mice and the mechanisms underlying the role of Brca1 and Gata3 in suppressing tumorigenesis and progression. RESULTS: Depletion of Brca1 or Gata3 led to growth defects of mammary epithelial cells, which was rescued by loss of p18. Depletion of Brca1 or Gata3 in a p18 null background induced heterogeneous mammary tumors with less luminal and more basal-like features and accelerated metastasis. Deletion of Brca1 eliminated Gata3 expression in human and mouse mammary tissues and cells. How Brca1 interacts with Gata3 to control mammary tumor development and progression is currently under investigation. CONCLUSION: Our results suggest that loss of function of either Brca1 or Gata3 induces basal-like mammary tumors in p18 deficient background. Citation Format: Pei X-H, Chan HL, Zhang L, Wang C, Robbins DJ, Capobianco T, Bai F. Loss of function of Brca1 or Gata3 induces basal-like breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-08-01.

Abstract 4802: Immune dysfunction and fungal infection contribute to esophageal carcinogenesis

Abstract Human esophageal cancer is the sixth leading cause of cancer death worldwide. More than 90% of esophageal cancer is esophageal squamous cell carcinoma (ESCC). While the etiological causes remain unclear, esophageal mucosal fungal infection is very common in esophageal cancer patients, including patients with autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) who have more than 20-fold risk of ESCC when compared to the healthy population. Here, we report that kinase-dead Ikka knock-in mice (hereafter referred to as mutant mice) develop APECED-like autoimmune disease due to developmental defects in thymus and lack of central tolerance. Similar to human APECED patients who develop esophageal mucosal fungal infection on average at the age of five, mutant mice develop fungal infection in oral cavity and esophagus as early as seven weeks old. About 20% of mutant mice develop ESCC during aging, which exhibits specific molecular signatures observed in human ESCC, such as p16 gene silencing, elevated EGFR phosphorylation and PD-L1 expression, etc. Autoreactive T cell depletion, or central tolerance reconstitution and/or normal T cell transfer prevents fungal infection and ESCC development in mutant mice. Importantly, antifungal drug treatment inhibits inflammation and ESCC development in mutant mice. However, oral inoculation of Clasdosporium, one of the major fungal species isolated from mutant mice, increases the tumor incidence from 20% to 63% in mutant mice. These results reveal that immune dysfunction and fungal infection is associated with ESCC development, which sheds new light on ESCC etiological factors, prevention and treatment. Citation Format: Feng Zhu, Jami Willette-Brown, Na-young Song, Peilin Zhang, Yinling Hu. Immune dysfunction and fungal infection contribute to esophageal carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4802. doi:10.1158/1538-7445.AM2017-4802

Abstract 5511: Lineage-specific and shared cytokine-sensing genes respond distinctly to the master regulator STAT5

Abstract JAK/STAT signaling pathways are critical for cell response to cytokines across multiple tissues, genetic variation in this pathway could associate with neoplastic diseases. STAT5, the most common used STAT family member, differently activates two distinct classes of genes. It activates cell-specific genes up to two orders of magnitude higher than shared genes expressed across different cell types. Socs2 (Suppressors of cytokine signaling 2), a widely expressed STAT5 target gene and a negative regulator of cytokine feedback loops, contains STAT5 response element within its promoter region. In our study, we demonstrated that STAT5 preferentially binds to promoter sequence of shared STAT5 targeted genes and to distal putative enhancers of lineage-specific genes. DNase-seq demonstrated that chromatin accessibility of STAT5-based enhancers was greatly dependent on cytokine exposure, while common STAT5 promoter targets were constitutively accessible. CRIPSR /Cas9 gene editing was used to delete the generic STAT5 binding sites in Socs2 locus in mice. Prolactin induction of Socs2 was abrogated both in liver and mammary gland, and resulted in elevated STAT5 activation and precocious mammary development in mutant mice. Taken together, we demonstrated that promoter-bound STAT5 modulates cytokine responses of universal STAT5 target genes, while enhancer-bound STAT5 is necessary for linage-specific target gene activation. This study provides new insight into the mechanism of used by a generic cytokine response element controlling lineage-specific transcription. Citation Format: xianke zeng, Michaela Willi, Ha Youn Shin, Chaochen Wang, Lothar Hennighausen. Lineage-specific and shared cytokine-sensing genes respond distinctly to the master regulator STAT5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5511. doi:10.1158/1538-7445.AM2017-5511

Impaired development of hippocampal mossy fibre synapses in mouse mutants for the presynaptic scaffold protein Bassoon

Bassoon, a protein highly concentrated at the synaptic active zone, is thought to participate in the organization of the cytomatrix at the site of neurotransmitter release. Bassoon is amongst the first proteins to accumulate at newly formed synaptic junctions, raising the question of the functional role of this protein in the early stages of synaptic development. Here we show that the course of synaptic maturation of hippocampal mossy fibre (MF) synapses (glutamatergic synapses with multiple release sites) is markedly altered during the first 2 weeks of postnatal development in mutant mice lacking the central region of Bassoon (Bsn(-/-) mice). At postnatal day 7 (P7), Bsn(-/-) mice display large amplitude MF-EPSCs with decreased paired pulse ratios, an abnormality which may be linked to deficits in the organization of the presynaptic active zone. Surprisingly, 1 week later, decreased MF-EPSCs amplitude is observed in Bsn(-/-) mice, consistent with the inactivation of a subset of synaptic release sites. Finally, at more mature states a decreased posttetanic potentiation is observed at MF-synapses. These results support the notion that Bassoon is important for organizing the presynaptic active zone during the postnatal maturation of glutamatergic synapses.

Development of Vestibular Organ and Cochlea

Inner ear is an important organ that is responsible for balance and hearing. It is composed of two complicated major subunits, vestibular organ and cochlea. During development, both functional units require complex genetic interactions to achieve proper patterning and morphology as its purpose. This review comprises three parts. In the first part, general information about technical tools in studying mouse ear development is introduced. In the second part, it is shown how semicircular canals are formed and what genetic interaction is involved in canal formation. In the last part, genetic interactions required for proper development of cochlea are elaborated, focusing on the length of cochlea and morphology of organ of Corti. The study about development of mutant mice provides us useful information about genetic interaction involved in the formation of structures for hearing and balance. The data acquired in the development field could provide a breakthrough to hair cell regeneration and stem cell therapy fields in stuck, which are directly applicable to human. Korean J Otorhinolaryngol-Head Neck Surg 2010;53:265-74 Key WordsZZDevelopment·Mouse·Semicircular canal·Cochlea·Organ of Corti·Hair cell.

Comparison of Ethanol Preference and Neurochemical Measures of Mesolimbic Dopamine and Adenosine Systems Across Different Strains of Mice

To extend the known phenotype of strains commonly used in the development of mutant mice, ethanol, saccharin, and caffeine preferences were examined in C57Bl/6J, CD-1, and hybrid C57Bl/6J x CD-1 mice. As dopaminergic mechanisms are inherently involved in the neuronal processing of many drugs of abuse (including ethanol), and an important role for adenosine-dopamine interactions has also been reported, the dopaminergic and purinergic neurochemical profiles of mice were compared against the consummatory phenotype observed. Ethanol (5% v/v), saccharin (0.1% w/v), and caffeine (0.1% w/v) consumption and preference were examined using a 2-bottle free-choice paradigm. Dopamine and adenosine receptor and transporter mRNA and protein density were quantified using in situ hybridization histochemistry and in vitro autoradiography, respectively. C57Bl/6J and hybrid C57Bl/6J x CD-1 mice demonstrated a clear ethanol preference, voluntarily consuming large quantities of ethanol when given the choice between drinking vessels containing either ethanol or water. Conversely, CD-1 mice were characterized as ethanol-avoiding under the present paradigm. Differences in D(1) receptor mRNA between the strains were consistent with the observed behavioral differences in ethanol preference. The high ethanol-preferring phenotype of C57Bl/6J mice could not be directly linked to alterations in dopamine transporter neurochemistry and/or enkephalin levels as proposed by earlier researchers. Ethanol-seeking behavior appeared to correlate with D2 receptor expression, however, with evidence that ethanol-preferring mice also exhibit an increased density of D2 receptors within limbic dopaminergic projection nuclei. Interestingly, strain differences in the expression of the ethanol-sensitive nucleoside transporter paralleled differences in ethanol consumption, a novel finding consonant with purinergic involvement in dopamine-related behaviors. This study has highlighted the relevance of alterations in dopamine receptor expression and purinergic modulation within the mesolimbic pathway and predisposition toward the development of ethanol-seeking behavior.

Essential role of Shp2-binding sites on FRS2α for corticogenesis and for FGF2-dependent proliferation of neural progenitor cells

Mammalian corticogenesis occurs through a complex process that includes neurogenesis, in which neural progenitor cells proliferate, differentiate, and migrate. It has been reported recently that neurogenesis occurs in the subventricular zone (SVZ), a region previously thought to be the primary site of gliogenesis. It has been recognized that in the SVZ, intermediate progenitor cells, derived from radial glial cells that are multipotent neural stem cells, produce only neurons. However, the molecular mechanisms underlying the regulation of neural stem cells and intermediate progenitor cells as well as their contribution to overall corticogenesis remain unknown. The docking protein FRS2alpha is a major mediator of signaling by means of FGFs and neurotrophins. FRS2alpha mediates many of its pleiotropic cellular responses by recruiting the adaptor protein Grb2 and the protein tyrosine phosphatase Shp2 upon ligand stimulation. Here, we report that targeted disruption of Shp2-binding sites in FRS2alpha leads to severe impairment in cerebral cortex development in mutant mice. The defect in corticogenesis appears to be due at least in part to abnormalities in intermediate progenitor cells. Genetic evidence is provided that FRS2alpha plays critical roles in the maintenance of intermediate progenitor cells and in neurogenesis in the cerebral cortex. Moreover, FGF2-responsive neurospheres, which are cell aggregates derived from neural stem/progenitor cells (NSPCs), from FRS2alpha mutant mice were smaller than those of WT mice. However, mutant NSPCs were able to self-renew, demonstrating that Shp2-binding sites on FRS2alpha play an important role in NSPC proliferation but are dispensable for NSPC self-renewing capacity after FGF2 stimulation.

Developmental changes in integrated ultrasound backscatter from embryonic blood in vivo in mice at high US frequency

Mouse blood imaged using high-frequency ultrasound (US) is more echogenic in embryos than in adults. Studying changes in blood echogenicity in embryos may be of fundamental interest in studies on the genetic regulation of normal and abnormal blood development in mutant mice. Embryonic red blood cells (RBCs) are large and nucleated in midgestation but decrease in size and become enucleated as they mature. We therefore hypothesised that these structural alterations are responsible for variations in echogenicity of embryonic blood with gestational age and development. The objective of the current study was to quantify these structural changes in echogenicity (echo brightness) and apparent integrated backscatter (AIB) from embryonic blood at high US frequencies in vivo in mice. Results from anaesthetised pregnant mice studied using transcutaneous US showed that echogenicity of embryonic blood in the heart, aorta and umbilical cord and AIB within the heart chambers peaked at embryonic day (ED) 13.5 and then decreased progressively toward term. Between EDs 13.5 and 17.5 (near term), RBC mean cell volume decreased from 133 to 109 fL, haematocrit increased from 12 to 34%, and the percentage of nucleated RBCs decreased from 59 to 2%. Relative to younger ages, RBC nuclei at ED 13.5 were small and dense (pyknotic) which may have contributed to the peak in echogenicity and AIB at this age. To calculate the AIB, radiofrequency (RF) signals with centre frequencies of 28 MHz and 35 MHz were integrated over the 16- to 35-MHz and 21- to 42-MHz frequency range, respectively. At 28 MHz, mean apparent integrated backscatter of blood in the embryonic heart increased significantly from 0.0023 ± 0.0004 Sr·cm −1 (mean ± SEM) at ED 12.5 to peak at 0.0037 ± 0.0005 Sr·cm −1 at ED 13.5. The mean AIB then decreased progressively with advancing gestation to 0.0002 ± 0.0001 Sr·cm −1 at ED 17.5. At 35 MHz, the mean AIB changed similarly with gestational age, except that values were lower than at 28 MHz at all ages. Higher attenuation of US at 35 MHz than at 28 MHz in tissue likely accounted for the lower AIB of blood insonified at 35 MHz. We speculate that developmental changes in red cell morphology are responsible for the observed changes in echogenicity and AIB of embryonic blood with gestational age in mice. (E-mail: lefloch@uniroma3.it)

Connective tissue growth factor expression and Smad signaling during mouse heart development and myocardial infarction.

Connective tissue growth factor (CTGF) is reported to be a target gene of transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) in vitro. Its physiological role in angiogenesis and skeletogenesis during mouse development has been described recently. Here, we have mapped expression of CTGF mRNA during mouse heart development, postnatal adult life, and after experimental myocardial infarction. Furthermore, we investigated the relationship between CTGF and the BMP/TGFbeta signaling pathway in particular during heart development in mutant mice. Postnatally, CTGF expression in the heart became restricted to the atrium. Strikingly, 1 week after myocardial infarction, when myocytes have disappeared from the infarct zone, CTGF and TGFbeta expression as well as activated forms of TGFbeta but not BMP, Smad effector proteins are colocalized exclusively in the fibroblasts of the scar tissue, suggesting possible cooperation between CTGF and TGFbeta during the pathological fibrotic response.

Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression.

Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.

Mutant mouse models of insulin-like growth factor actions in the central nervous system

Insulin-like growth factor-I (IGF-I) and its cognate receptor, the type 1 IGF receptor (IGF1R), as well as high-affinity IGF binding proteins (IGFBP) that modulate IGF-I actions, are expressed throughout the course of brain development. These observations, taken together with studies in cultured neural cells demonstrating a variety of IGF-I growth-promoting activities, provide a strong argument for IGF-I having a central role in the growth and development of the CNS. This report reviews studies of brain development in mutant mice with alterations of IGF-I expression or action. Transgenic (Tg) mice overexpressing IGF-I postnatally exhibit brain overgrowth characterized by increased neuron and oligodendrocyte number, as well as marked increases in myelination. Mutant mice with ablated IGF-I and IGF1R expression, as well as those with overexpression of IGFBPs capable of inhibiting IGF actions, exhibit brain growth retardation with a variety of growth deficits. These studies confirm a role for IGF-I in neural development, and indicate that IGF-I stimulates neurogenesis and synaptogenesis, facilitates oligodendrocyte development, promotes neuron and oligodendrocyte survival, and stimulates myelination. Evidence from experiments in these mouse models also indicates that IGF-I has a role in recovery from neural injury.

BAFFling No More

A number of tumor necrosis factor (TNF)-related ligands and receptors have recently been identified that regulate B cell development and function. One of these is the B cell-activating factor, BAFF, which interacts with two receptors [transmembrane activator and calcium-modulating cyclophilin ligand interacting protein (TACI) and B cell maturation antigen (BCMA)] to promote survival and proliferation of splenic B cells (see the Perspective by Waldschmidt and Noelle ). Scheimann et al. observed a profound defect in B cell development in mutant mice that lack expression of BAFF that was only partially reiterated in TACI- and BCMA-deficient animals. These results implicate the existence of another receptor for BAFF. Thompson et al. describe a candidate receptor that binds BAFF but not any other of the identified TNF-like ligands. The A/WySnJ strain of mice harbors a truncation in the BAFF-R gene that leads to a B cell deficit similar to that observed in BAFF-deficient mice. Together, these studies suggest that the interaction of BAFF with BAFF-R mediates major effects on B cell development. T. J. Waldschmidt, R. J. Noelle, Long live the mature B cell--a BAFFling mystery resolved. Science 293 , 2012-2013 (2001). [Full Text] B. Schiemann, J. L. Gommerman, K. Vora, T. G. Cachero, S. Shulga-Morskaya, M. Dobles, E. Frew, M. L. Scott, An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science 293 , 2111-2114 (2001). [Abstract] [Full Text] J. S. Thompson, S. A. Bixler, F. Qian, K. Vora, M. L. Scott, T. G. Cachero, C. Hession, P. Schneider, I. D. Sizing, C. Mullen, K. Strauch, M. Zafari, C. D. Benjamin, J. Tschopp, J. L. Browning, C. Ambrose, BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science 293 , 2108-2111 (2001). [Abstract] [Full Text]

Development of the neuromuscular junction: Genetic analysis in mice

Formation of the skeletal neuromuscular junction is a multi-step process that requires communication between the nerve and muscle. Studies in many laboratories have led to identification of factors that seem likely to mediate these interactions. ‘Knock-out’ mice have now been generated with mutations in several genes that encode candidate transsynaptic messengers and components of their effector mechanisms. Using these mice, it is possible to test hypotheses about the control of synaptogenesis. Here, we review our studies on neuromuscular development in mutant mice lacking agrin αCGRP, rapsyn, MuSK, dystrophin, dystrobrevin, utrophin, laminin α5, laminin β2, collagen α3(IV), the acetylcholine receptor ε subunit, the collagenous tail of acetycholinesterase, fibroblast growth factor-5, the neural cell adhesion molecule, and tenascin-C.

The MAP Kinase Pathway Controls Differentiation from Double-Negative to Double-Positive Thymocyte

T cell development is regulated at two major control points where maturation, proliferation, and antigen receptor gene rearrangement are coordinated. Progression through these developmental control points is dependent upon the expression of different forms of the T cell receptor. Here we show that the MAP kinase cascade is a regulator of the differentiation of immature thymocytes from double-negative to double-positive cell, most probably acting as a transducer of pre-T cell receptor signaling. Furthermore, this study demonstrates the use of retrovirus-mediated gene transfer in fetal thymic organ culture in the analysis of thymic development in mutant mice, an alternative to transgenesis by oocyte injection.

Lymphocyte development and function in T-cell receptor and RAG-1 mutant mice.

We have used gene targeting in embryonic stem cells to generate mice with mutations in T-cell receptor (TCR) gene-alpha, TCR-beta, TCR-delta or the Recombination Activating Gene-1 (RAG-1). TCR-alpha or TCR-beta mutant mice are deficient in alpha beta T cells, but still contain gamma delta T cells and B cells. TCR-delta mutant mice are deficient in gamma delta T cells, but still contain alpha beta T cells and B cells. Mice doubly mutant for TCR-beta and TCR-delta do not have any mature T cells, but still have B cells. RAG-1 mutant mice are totally deficient in both mature T cells and B cells. Here, I describe recent studies of thymocyte development in the mutant mice, and I review experiments addressing the function of the immune system of the mutant mice.