Mouse Models of Congenital Kidney Anomalies.

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects, which cause the majority of chronic kidney diseases in children. CAKUT covers a wide range of malformations that derive from deficiencies in embryonic kidney and lower urinary tract development, including renal aplasia, hypodysplasia, hypoplasia, ectopia, and different forms of ureter abnormalities. The majority of the genetic causes of CAKUT remain unknown. Research on mutant mice has identified multiple genes that critically regulate renal differentiation. The data generated from this research have served as an excellent resource to identify the genetic bases of human kidney defects and have led to significantly improved diagnostics. Furthermore, genetic data from human CAKUT studies have also revealed novel genes regulating kidney differentiation.

A2B5+/GFAP+ Cells of Rat Spinal Cord Share a Similar Lipid Profile with Progenitor Cells: A Comparative Lipidomic Study.

The central nervous system (CNS) harbors multiple glial fibrillary acidic protein (GFAP) expressing cell types. In addition to the most abundant cell type of the CNS, the astrocytes, various stem cells and progenitor cells also contain GFAP+ populations. Here, in order to distinguish between two types of GFAP expressing cells with or without the expression of the A2B5 antigens, we performed lipidomic analyses on A2B5+/GFAP+ and A2B5-/GFAP+ cells from rat spinal cord. First, A2B5+/GFAP- progenitors were exposed to the leukemia inhibitory factor (LIF) or bone morphogenetic protein (BMP) to induce their differentiation to A2B5+/GFAP+ cells or A2B5-/GFAP+ astrocytes, respectively. The cells were then analyzed for changes in their phospholipid, sphingolipid or acyl chain profiles by mass spectrometry and gas chromatography. Compared to A2B5+/GFAP- progenitors, A2B5-/GFAP+ astrocytes contained higher amounts of ether phospholipids (especially the species containing arachidonic acid) and sphingomyelin, which may indicate characteristics of cellular differentiation and inability for multipotency. In comparison, principal component analyses revealed that the lipid composition of A2B5+/GFAP+ cells retained many of the characteristics of A2B5+/GFAP- progenitors, but their lipid profile was different from that of A2B5-/GFAP+ astrocytes. Thus, our study demonstrated that two GFAP+ cell populations have distinct lipid profiles with the A2B5+/GFAP+ cells sharing a phospholipid profile with progenitors rather than astrocytes. The progenitor cells may require regulated low levels of lipids known to mediate signaling functions in differentiated cells, and the precursor lipid profiles may serve as one measure of the differentiation capacity of a cell population.

In vivo activation of the PI3K-Akt pathway in mouse beta cells by the EGFR mutation L858R protects against diabetes.

AIMS/HYPOTHESIS: EGF receptor (EGFR) signalling is required for normal beta cell development and postnatal beta cell proliferation. We tested whether beta cell proliferation can be triggered by EGFR activation at any age and whether this can protect beta cells against apoptosis induced by diabetogenic insults in a mouse model. METHODS: We generated transgenic mice with doxycycline-inducible expression of constitutively active EGFR (L858R) (CA-EGFR) under the insulin promoter. Mice were given doxycycline at various ages for different time periods, and beta cell proliferation and mass were analysed. Mice were also challenged with streptozotocin and isolated islets exposed to cytokines. RESULTS: Expression of EGFR (L858R) led to increased phosphorylation of EGFR and Akt in pancreatic islets. CA-EGFR expression during pancreatic development (embryonic day [E]12.5 to postnatal day [P]1) increased beta cell proliferation and mass in newborn mice. However, CA-EGFR expression in adult mice did not affect beta cell mass. Expression of the transgene improved glycaemia and markedly inhibited beta cell apoptosis after a single high dose, as well as after multiple low doses of streptozotocin. In vitro mechanistic studies showed that CA-EGFR protected isolated islets from cytokine-mediated beta cell death, possibly by repressing the proapoptotic protein BCL2-like 11 (BIM). CONCLUSIONS/INTERPRETATION: Our findings show that the expression of CA-EGFR in the developing, but not in the adult pancreas stimulates beta cell replication and leads to increased beta cell mass. Importantly, CA-EGFR protects beta cells against streptozotocin- and cytokine-induced death.

Missing-in-metastasis MIM/MTSS1 promotes actin assembly at intercellular junctions and is required for integrity of kidney epithelia.

MIM/MTSS1 is a tissue-specific regulator of plasma membrane dynamics, whose altered expression levels have been linked to cancer metastasis. MIM deforms phosphoinositide-rich membranes through its I-BAR domain and interacts with actin monomers through its WH2 domain. Recent work proposed that MIM also potentiates Sonic hedgehog (Shh)-induced gene expression. Here, we generated MIM mutant mice and found that full-length MIM protein is dispensable for embryonic development. However, MIM-deficient mice displayed a severe urinary concentration defect caused by compromised integrity of kidney epithelia intercellular junctions, which led to bone abnormalities and end-stage renal failure. In cultured kidney epithelial (MDCK) cells, MIM displayed dynamic localization to adherens junctions, where it promoted Arp2/3-mediated actin filament assembly. This activity was dependent on the ability of MIM to interact with both membranes and actin monomers. Furthermore, results from the mouse model and cell culture experiments suggest that full-length MIM is not crucial for Shh signaling, at least during embryogenesis. Collectively, these data demonstrate that MIM modulates interplay between the actin cytoskeleton and plasma membrane to promote the maintenance of intercellular contacts in kidney epithelia.

Ultrahigh-risk group within the high-risk neuroblastoma category.

Children with high-risk neuroblastoma (NBL) constitute a heterogenous group, but little attention has been paid to further subdivision of the high-risk group. Although the current therapies including multiple high-dose consolidations have neared their efficacy and tolerability limits, alternative therapies are needed. We wanted to define an ultrahigh-risk group among high-risk NBL patients, to be potential candidates for novel therapies given up-front. Children with high-risk NBL (n=59) treated at a single institution during 1987 to 2010 were evaluated for upfront prognostic factors at diagnosis and response to induction therapy. The overall outcome was not different during 1987 to 1994 versus 1995 to 2010. Therapy consisted of induction chemotherapy, surgery, and high dose-consolidation (single, tandem, or triple) with autologous stem cell rescue, followed by local irradiation and cis-retinoic acid. MYCN amplification and bone metastases were powerful upfront prognostic factors, and a combination of these determined an ultrahigh-risk group with a 5-year event-free survival of 0.125±0.083. The combination of MYCN amplification and bone metastases overruled the intensity of the therapy given and remained the only significant predictor (P<0.019) in a multiple step-wise forward Cox regression analysis. We conclude that high-risk NBL patients can be categorized into prognostic subgroups based on MYCN status and bone metastases. MYCN amplification and bone metastases combined determined an ultrahigh-risk group of patients being suitable candidates for novel alternative therapies.

More than nervous: the emerging roles of plexins.

Plexins are the receptors for semaphorins, a large family of axon guidance cues. Accordingly, the role of plexins in the development of the nervous system was the first to be acknowledged. However, the expression of plexins is not restricted to neuronal cells, and recent research has been increasingly focused on the roles of plexin-semaphorin signalling outside of the nervous system. During embryogenesis, plexins regulate the development of many organs, including the cardiovascular system, skeleton and kidney. They have also been shown to be involved in immune system functions and tumour progression. Analyses of the plexin signalling in different tissues and cell types have provided new insight to the versatility of plexin interactions with semaphorins and other cell-surface receptors. In this review we try to summarise the current understanding of the roles of plexins in non-neural development and immunity.

Thiotepa and melphalan based single, tandem, and triple high dose therapy and autologous stem cell transplantation for high risk neuroblastoma.

BACKGROUND: Outcome of high risk neuroblastoma (NBL) remains unsatisfactory in spite of intensive treatment efforts. Consolidation with high-dose (HD) chemotherapy and autologous stem cell transplantation (ASCT) has been intensified with tandem and triple cycles with promising results. Our purpose was to improve the outcome with two or three HD-consolidations. METHODS: Thirty six children with high risk NBL, diagnosed 1995-2010, had intensive induction and surgery, and were stratified to single, tandem or triple HD-therapy and ASCT, followed by local irradiation and cis-retinoic acid. In inoperable patients surgery was facilitated by preoperative HD-melphalan. Long-term outcome of our old cohort from 1987-1994 was updated. RESULTS: Ten year event-free survival (EFS) from diagnosis was 0.44+/-0.10 of the old and 0.43+/-0.085 of the new cohort. EFS from the last ASCT was 0.53 +/-0.12 and 0.48+/-0.091, respectively. Preoperative HD-melphalan rendered 73% of bulky primaries operable in the new cohort. The 5-yr EFS from ASCT was 0.46+/-0.15 for single and 0.73+/-0.15 for tandem ASCT (P = 0.19). All triple ASCT patients, selected by poor/slow response, relapsed or died. CONCLUSIONS: Thiotepa- and melphalan based HD regimens, with or without total body irradiation (TBI), appeared to give an outcome comparable to major NBL study groups with acceptable toxicity. Tandem HD therapy gave a 5-year EFS of 73%, whereas a third HD consolidation did not offer any additional advantage for ultra high risk patients with slow response. Pediatr Blood Cancer 2012; 59: 1190-1197. © 2012 Wiley Periodicals, Inc.

Sema4C-Plexin B2 signalling modulates ureteric branching in developing kidney.

Semaphorins, originally identified as axon guidance molecules, have also been implicated in angiogenesis, function of the immune system and cancerous growth. Here we show that deletion of Plexin B2 (Plxnb2), a semaphorin receptor that is expressed both in the pretubular aggregates and the ureteric epithelium in the developing kidney, results in renal hypoplasia and occasional double ureters. The rate of cell proliferation in the ureteric epithelium and consequently the number of ureteric tips are reduced in the kidneys lacking Plexin B2 (Plxnb2-/-). Semaphorin 4C, a ligand for Plexin B2, stimulates branching of the ureteric epithelium in wild type and Plxnb2+/- kidney explants, but not in Plxnb2-/- explants. As shown by co-immunoprecipitation Plexin B2 interacts with the Ret receptor tyrosine kinase, the receptor of Glial-cell-line-derived neurotrophic factor (Gdnf), in embryonic kidneys. Isolated Plxnb2-/- ureteric buds fail to respond to Gdnf by branching, but this response is rescued by Fibroblast growth factor 7 and Follistatin as well as by the metanephric mesenchyme. The differentiation of the nephrogenic mesenchyme, its morphology and the rate of apoptosis in the Plxnb2-/- kidneys are normal. Plexin B2 is co-expressed with Plexin B1 (Plxnb1) in the kidney. The double homozygous Plxnb1-Plxnb2-deficient mice show high embryonic lethality prior to onset of nephrogenesis. The only double homozygous embryo surviving to E12 showed hypoplastic kidneys with ureteric branches and differentiating mesenchyme. Taken together, our results show that Sema4C-Plexin B2 signalling regulates ureteric branching, possibly through modulation of Gdnf signalling by interaction with Ret, and suggest non-redundant roles for Plexin B1 and Plexin B2 in kidney development.

The GDNF target Vsnl1 marks the ureteric tip.

Glial cell line-derived neurotrophic factor (GDNF) is indispensable for ureteric budding and branching. If applied exogenously, GDNF promotes ectopic ureteric buds from the Wolffian duct. Although several downstream effectors of GDNF are known, the identification of early response genes is incomplete. Here, microarray screening detected several GDNF-regulated genes in the Wolffian duct, including Visinin like 1 (Vsnl1), which encodes a neuronal calcium-sensor protein. We observed renal Vsnl1 expression exclusively in the ureteric epithelium, but not in Gdnf-null kidneys. In the tissue culture of Gdnf-deficient kidney primordium, exogenous GDNF and alternative bud inducers (FGF7 and follistatin) restored Vsnl1 expression. Hence, Vsnl1 characterizes the tip of the ureteric bud epithelium regardless of the inducer. In the tips, Vsnl1 showed a mosaic expression pattern that was mutually exclusive with ß-catenin transcriptional activation. Vsnl1 was downregulated in both ß-catenin-stabilized and ß-catenin-deficient kidneys. Moreover, in a mouse collecting duct cell line, Vsnl1 compromised ß-catenin stability, suggesting a counteracting relationship between Vsnl1 and ß-catenin. In summary, Vsnl1 marks ureteric bud tips in embryonic kidneys, and its mosaic pattern demonstrates a heterogeneity of cell types that may be critical for normal ureteric branching.

CIP2A increases self-renewal and is linked to Myc in neural progenitor cells.

The oncogenic transcription factor Myc has an established role in the regulation of stem cell self-renewal and differentiation. However, the regulation of Myc activity or expression in stem and progenitor cells is not thoroughly understood. We studied the expression and function of the Myc stabilizing protein and a newly found oncogene, cancerous inhibitor of protein phosphatase 2A (CIP2A) in mouse neural progenitor cells (NPCs). We found intensive CIP2A expression in the neurogenic areas of the developing E13 as well as of the adult mouse brain. Here we also show that retroviral overexpression of CIP2A increases and siRNA silencing of CIP2A decreases NPC self-renewal and proliferation. Differentiation of the NPCs correlates with diminished CIP2A expression although overexpression of CIP2A does not prevent differentiation of neurons and astrocytes. Lastly, we demonstrate that both Myc and CIP2A enhance each other's expression and siRNA against CIP2A in Myc-overexpressing NPCs significantly reduces the ability of Myc to increase self-renewal and proliferation thus indicating a functional connection between CIP2A and Myc in NPCs.

Conservation, expression, and knockdown of zebrafish plxnb2a and plxnb2b.

In mice lacking Plexin B2, a receptor of the axon guidance molecules Semaphorin 4C and Semaphorin 4D, the closure of the neural tube and structural organization of the cerebellum are severely impaired. We cloned two Plexin B2 orthologs, plxnb2a and plxnb2b, in zebrafish, which is a widely used model for the development of the vertebrate central nervous system (CNS). The predicted proteins, Plexin B2a and Plexin B2b, contain all the conserved and functional domains of the plexin B-subfamily. During embryonic development, plxnb2a is expressed, e.g., in pharyngeal arches while plxnb2b expression is more confined to neuronal structures like the cerebellum. However, both plxnb2a and plxnb2b are expressed at the midbrain-hindbrain boundary, in the otic vesicles, facial ganglia, and pectoral fins. Knockdown of both plxnb2a and plxnb2b simultaneously (>95% and 45%, respectively) resulted in normal CNS structure, axon guidance and swimming performance of the morphants.

HLA-G expression correlates with histological grade but not with prognosis in colorectal carcinoma.

Trophoblast-specific expression of HLA-G induces immune tolerance for the developing fetus. Pathological HLA-G expression later in life might contribute to immune escape of various cancers. We studied the still controversial role of HLA-G in colorectal carcinoma (CRC) using the MEM-G/1 antibody and a tissue microarray series of CRC tumors (n = 317). HLA-G expression appeared in 20% of the tumors and showed high intratumoral heterogeneity. HLA-G positivity was associated with better differentiation (p = 0.002) and non-mucinous histology (p = 0.008). However, HLA-G expression alone showed no prognostic value: 5-years disease-specific survival among patients with HLA-G expression was 68.9% (95% CI: 62.7%-75.0%) compared to 74.8% (95% CI: 63.2%-86.3%) among those without expression. These results support a modulatory role of HLA-G in CRC.

STRN-ALK rearranged pediatric malignant peritoneal mesothelioma - Functional testing of 527 cancer drugs in patient-derived cancer cells.

Genetic rearrangements involving the anaplastic lymphoma kinase (ALK) gene create oncogenic drivers for several cancers, including malignant peritoneal mesothelioma (MPeM). Here, we report genomic and functional precision oncology profiling on a rare case of a 5-year old patient diagnosed with wide-spread and aggressive MPeM, driven by STRN-ALK rearrangement. We established genomically representative patient-derived cancer cells (PDCs) from the tumor sample and performed high-throughput drug sensitivity testing with 527 oncology compounds to identify potent inhibitors. As expected, the PDCs were overall sensitive to the ALK inhibitors, although the eight different inhibitors tested had variable efficacy. We also discovered other effective inhibitors, such as MEK/ERK inhibitors and those targeting pathways downstream of ALK as well as Bcl-xl inhibitors. In contrast, most cytotoxic drugs were not very effective. ALK inhibitors synergized with MEK and PI3K/mTOR inhibitors, highlighting potential combinatorial strategies to enhance drug efficacy and tackle drug resistance. Based on genomic data and associated functional validation, the patient was treated with the ALK inhibitor crizotinib in combination with conventional chemotherapy (cisplatin and gemcitabine). A complete disease remission was reached, lasting now for over 3 years. Our results illustrate a rare pediatric cancer case, and highlight the potential of functional precision oncology to discover pathogenetic drivers, validate dependency on driver signals, compare different inhibitors against each other and potentially enhance targeted treatments by drug combinations. Such real-time implementation of functional precision oncology could pave the way towards safer and more effective personalized cancer therapies for individual pediatric cancer patients with rare tumors.

Fetal HLA-G mediated immune tolerance and interferon response in preeclampsia.

BACKGROUND: Fetal immune tolerance is crucial for pregnancy success. We studied the link between preeclampsia, a severe pregnancy disorder with uncertain pathogenesis, and fetal human leukocyte antigen G (HLA-G) and other genes regulating maternal immune responses. METHODS: We assessed sex ratios and regulatory HLA-G haplotypes in population cohorts and series of preeclampsia and stillbirth. We studied placental mRNA expression of 136 genes by sequencing and HLA-G and interferon alpha (IFNα) protein expression by immunohistochemistry. FINDINGS: We found underrepresentation of males in preeclamptic births, especially those delivered preterm or small for gestational age. Balancing selection at HLA-G associated with the sex ratio, stillbirth, and preeclampsia. We observed downregulation of HLA-G, its receptors, and many other tolerogenic genes, and marked upregulation of IFNA1 in preeclamptic placentas. INTERPRETATION: These findings indicate that an evolutionary trade-off between immune tolerance and protection against infections at the maternal-fetal interface promotes genetic diversity in fetal HLA-G, thereby affecting survival, preeclampsia, and sex ratio. We highlight IFNA1 as a potential mediator of preeclampsia and a target for therapeutic trials. FUNDING: Finnish Medical Foundation, Päivikki and Sakari Sohlberg Foundation, Karolinska Institutet Research Foundation, Scandinavia-Japan Sasakawa Foundation, Japan Eye Bank Association, Astellas Foundation for Research on Metabolic Disorders, Japan Society for the Promotion of Science, Knut and Alice Wallenberg Foundation, Swedish Research Council, Medical Society Liv och Hälsa, Sigrid Jusélius Foundation, Helsinki University Hospital and University of Helsinki, Jane and Aatos Erkko Foundation, Academy of Finland, Finska Läkaresällskapet, Novo Nordisk Foundation, Finnish Foundation for Pediatric Research, and Emil Aaltonen Foundation.

GDNF promotes tubulogenesis of GFRalpha1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase.

Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor alpha1 (GFRalpha1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRalpha1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRalpha1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRalpha1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRalpha1-positive and GFRalpha1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.

Simple 3D culture of dissociated kidney mesenchyme mimics nephron progenitor niche and facilitates nephrogenesis Wnt-independently.

Kidney mesenchyme (KM) and nephron progenitors (NPs) depend on WNT activity, and their culture in vitro requires extensive repertoire of recombinant proteins and chemicals. Here we established a robust, simple culture of mouse KM using a combination of 3D Matrigel and growth media supplemented with Fibroblast Growth Factor 2 (FGF2) and Src inhibitor PP2. This allows dissociated KM to spontaneously self-organize into spheres. To reassess the requirement of WNT activity in KM self-organization and NPs maintenance, cells were cultured with short pulse of high-dose GSK3ß inhibitor BIO, on a constant low-dose or without BIO. Robust proliferation at 48 hours and differentiation at 1 week were observed in cultures with high BIO pulse. Importantly, dissociated KM cultured without BIO, similarly to that exposed to constant low dose of BIO, maintained NPs up to one week and spontaneously differentiated into nephron tubules at 3 weeks of culture. Our results show that KM is maintained and induced to differentiate in a simple culture system. They also imply that GSK3ß/WNT-independent pathways contribute to the maintenance and induction of mouse KM. The robust and easy 3D culture enables further characterization of NPs, and may facilitate disease modeling when applied to human cells.

Development of the urogenital system is regulated via the 3'UTR of GDNF.

Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial cell-line derived neurotrophic factor (GDNF) induced RET signaling is critical for ureteric bud outgrowth, but the function of endogenous GDNF in further renal differentiation and urogenital system development remains discursive. Here we analyzed mice where 3' untranslated region (UTR) of GDNF is replaced with sequence less responsive to microRNA-mediated regulation, leading to increased GDNF expression specifically in cells naturally transcribing Gdnf. We demonstrate that increased Gdnf leads to short ureters in kidneys located in an abnormally caudal position thus resembling human pelvic kidneys. High GDNF levels expand collecting ductal progenitors at the expense of ureteric trunk elongation and result in expanded tip and short trunk phenotype due to changes in cell cycle length and progenitor motility. MEK-inhibition rescues these defects suggesting that MAPK-activity mediates GDNF's effects on progenitors. Moreover, Gdnf   hyper mice are infertile likely due to effects of excess GDNF on distal ureter remodeling. Our findings suggest that dysregulation of GDNF levels, for example via alterations in 3'UTR, may account for a subset of congenital anomalies of the kidney and urinary tract (CAKUT) and/or congenital infertility cases in humans and pave way to future studies.

GDNF maintains mouse spermatogonial stem cells in vivo and in vitro.

Spermatogonial stem cells (SSCs) produce sperm throughout the post-pubertal life of a male. Transgenic loss- and gain-of-function mouse models have shown that their self-renewal and differentiation are controlled in vivo by glial-cell-line-derived neurotrophic factor (GDNF) in a dose-dependent manner. After this in vivo observation, the culture conditions for mouse SSCs were rapidly developed. GDNF together with other growth factors, hormones, and vitamins maintain proliferation and self-renewal of SSCs for years in vitro. Both serum-supplemented and serum-free culture methods have been described. The cells are cultivated either on feeder layer or laminin-coated dishes. First reports from random and targeted mutagenesis of SSCs have been published. Some cells in the spermatogonial stem cell culture transform to embryonic stem cell-like cells and form teratomas in nude mice. In general, the spermatogonial stem cells maintain their germline identity in long-term culture. The mechanism for transformation to embryonic stem cell-like cells is not known, but the data suggest that germline and embryonic stem cells are closely related. We describe in detail the culture system of SSCs developed by Dr. Takashi Shinohara in 2003.

An adenosine triphosphatase of the sucrose nonfermenting 2 family, androgen receptor-interacting protein 4, is essential for mouse embryonic development and cell proliferation.

An adenosine triphosphatase of the sucrose nonfermenting 2 protein family, androgen receptor-interacting protein 4 (ARIP4), modulates androgen receptor activity. To elucidate receptor-dependent and -independent functions of ARIP4, we have analyzed Arip4 gene-targeted mice. Heterozygous Arip4 mutants were normal. Arip4 is expressed mainly in the neural tube and limb buds during early embryonic development. Arip4-/- embryos were abnormal already at embryonic d 9.5 (E9.5) and died by E11.5. At E9.5 and E10.5, almost all major tissues of Arip4-null embryos were proportionally smaller than those of wild-type embryos, and the neural tube was shrunk in some Arip4-/- embryos. Dramatically reduced cell proliferation and increased apoptosis were observed in E9.5 and E10.5 Arip4-null embryos. Mouse embryonic fibroblasts (MEFs) isolated from Arip4-/- embryos ceased to grow after two to three passages and exhibited increased apoptosis and decreased DNA synthesis compared with wild-type MEFs. Comparison of gene expression profiles of Arip4-/- and wild-type MEFs at E9.5 revealed that putative ARIP4 target genes are involved in cell growth and proliferation, apoptosis, cell death, DNA replication and repair, and development. Collectively, ARIP4 plays an essential role in mouse embryonic development and cell proliferation, and it appears to coordinate multiple essential biological processes, possibly through a complex chromatin remodeling system.

RET tyrosine kinase signaling in development and cancer.

The variety of diseases caused by mutations in RET receptor tyrosine kinase provides a classic example of phenotypic heterogeneity. Gain-of-function mutations of RET are associated with human cancer. Gene rearrangements juxtaposing the tyrosine kinase domain to heterologous gene partners have been found in sporadic papillary carcinomas of the thyroid (PTC). These rearrangements generate chimeric RET/PTC oncogenes. In the germline, point mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Both MEN 2 mutations and PTC gene rearrangements potentiate the intrinsic tyrosine kinase activity of RET and, ultimately, activate the RET downstream targets. Loss-of-function mutations of RET cause Hirschsprung's disease (HSCR) or colonic aganglionosis. A deeper understanding of the molecular signaling of normal versus abnormal RET activity in cancer will enable the development of potential new treatments for patients with sporadic and inherited thyroid cancer or MEN 2 syndrome. We now review the role and mechanisms of RET signaling in development and carcinogenesis.