Genetics of signal transduction: tales from the mouse

Genetics of signal transduction: Akira Imamoto, Fred Hutchinson

tales from the mouse

Philippe Soriano and Paul 1 Stein Cancer Research Center, Seattle, USA

Recent progress in understanding signal transduction owes much to new genetic approaches, first by unraveling the molecular basis of classic mutations, and then .by the use of gene targeting. Recent studies have examined mammalian signal transduction from cell surface to nucleus, especially ligand-receptor systems and cytosolic signal transducers. Current Opinion

in Genetics

1994,

4:40-46

receptors and ligands, while others encode intracellular signaling molecules and transcription factors.

Introduction In some respects, the study of signal transduction genetics in m ammals began 100 years ago with mouse fanciers who accumulated stocks of mice exhibiting unusual pigmentation patterns. Over time, more mutants were collected, that displayed other phenotypes such as dwarfism or curly fur. These collections have been proven to be a valuable repository. Phenotypic analysis of some of the loci has been extensive, and a number of loci have multiple alleles, exhibiting a range of phenotypes that give clues to the biochemical properties of the gene product. Recent advances in mapping of the mouse genome have facilitated identification of the molecular defects underlying certain mutations. Our understanding of the signal transduction network has been aided by the generation of mice that carry mutations in a cloned gene following homologous recombination in embryonic stem cells. This is a more directed approach than the analysis of pre-existing mutants, as the molecule of interest has often already been analyzed in vitro. A significant advantage of this approach is the identification of mutants that do not exhibit an overt phenotype, and which therefore would not have been identified by classical genetics. It is possible to design specific tests of gene function using biochemical data. In this review, we will focus primarily on mutations, both old and new, that affect intracellular signaling pathways.

Mutants

and Development

in search of a gene

By studying the inheritance of coat color, it has been possible to identify loci that affect melanocyte growth and function. Several of the genes at these loci encode

The mouse mutations at the White spotting ( w) or Steel
Abbreviations CNTF-ciliary neurotrophic factor; CSF-l-macrophage colony-stimulating factor; &-dwarf; EGF-epidermal growth factor; e-extension; g/-grey lerhal; CRF-growth hormone releasing factor; HCP-hematopoietic cell protein phosphatase; IGF-insulin-like growth factor; LIF-leukemia inhibitory factor; lit--little; me-motheaten; mi-microphthalmia; aMSHmelanocyte-stimulating hormone; NGF-nerve growth factor; oc-osteosclerotic; op-osteoperrosis; S/-&x$ TGFa-transforming growth factor-a; W-Whire spotring; wa-waved XLA-X-linked agammaglobulinemia.

40

8 Current

Biology

Ltd ISSN 0959-437X

Genetics

Table 1. Mutations

affecting

Classic mutations: Locus (abbrev) White

spotting

intracellular

signaling

Chromosome

Gene product

5

( wl

dwarf (dw) XLA xid motheaten (me)

10 8 6 16 X X 6

waved-l waved-2

6 11

(e)

(wa-1) (~a-21

Targeted mutations: Gene product

c-Kit

Description tyrosine

kinase.

Fyn

Src-family

tyrosine

kinase.

gpl30

tyrosine

kinase.

*notype

Src-family

CNTF

receptor

Ligand for c-Kit receptor. a-MSH receptor. Growth hormone releasing factor receptor. Pit-l transcription factor. Human Bruton tyrosine kinase. Mouse Bruton tyrosine kinase. PTP-1 C; hematopoietic cell protein tyrosine phosphatase. TGF-a. ECF receptor.

Src

Yes Lck Csk TGFa ICF-I, ICF-II, and IGF-IR TrkB LIF

Imamoto. Soriano and Stein

transduction

pathways.

sIeel(sfl extension Me (lit)

of simal

Src-family tyrosine kinase. Src-family tyrosine kinase. Regulator of Src-family kinases. Growth factor. Insulin-like growth factors and receptor. NGF receptor member. Growth factor. Ciliary neurotrophic factor. Subunit common to several receptor complexes.

The exfension (e) locus on chromosome 8 also affects pigmentation and controls the relative amount of eumelanin (black pigment) and phaeomelanin (yellow pigment) in mouse hair. The ratio of these pigments is controlled by tyrosinase: the higher the tyrosinase level, the more eumelanin is produced. The enzyme activity in melanocytes is regulated by a melanocytestimulating hormone CaMSH). Recently, a gene encoding a G protein coupled receptor for aMSH [7,81 was mapped to the e locus [PI. Point mutations within or around the second transmembrane domain were found in two dominant alleles characterized by black pigmentation, leading to a constitutive activation or hyper-responsiveness of the receptor, respectively. In contrast, the recessive yellow allele (e) carries a point mutation between the fourth and fifth transmembrane domain resulting in a frameshift and consequently non-functional aMSH receptor. Despite the absence of a functional receptor, why do homozygous recessive yellow (e/e mice still have black eyes? Apparently, retinal pigment cells are capable of synthesizing eumelanin in the absence of aMSH signal. The same may be true for dermal melanocytes, as eumelanin is found in the skin,

Osteopetrosis due to defective osteoclast function. Hippocampal defects; T-cell receptor signaling alterations. No discernable phenotype. Early block in thymic development. Embryonic lethality between E9.5-10.5. Curly fur; allelic with wa-I. Growth retardation and additional complex phenotypes. Loss of selected neuronal populations Required to prepare uterine environment for implantation. Loss of selected motor Embryonic

neuronal lethality.

populations.

but not in the hair follicles, of e4e mice. Although several effects of aMSH besides that on melanocytes have been reported, de mice appear normal apart from their coat color. This suggests that other pathways, such as the one involving proopiomelanocortins and their receptor family, may transduce signals to produce eumelanin in cells other than hair follicle melanocytes, and that there may be a compensatory mechanism for the other actions of aMSH. A mouse mutation in another G protein coupled transmembrane receptor has been mapped to the little (lit) locus on chromosome 6 1101. The mutation is recessive, and homozygotes are characterized by anterior pituitary hypoplasia, in which there are few secretory granules in growth hormone-producing cells. As mutants are responsive to growth hormone but resistant to growth hormone releasing factor (GRF), it has been suggested that the mutation affects signaling of the GRF pathway. A point mutation was .found in the extracellular domain of the GRF receptor that could not transduce GRF signals. Interestingly, Snell dwu~f(dw) or Jackson dwurf(dud) mice carry a mutation on chromosome 16 in the gene that encodes the Pit-l transcrip

41

42

Oncoeenes

and cell Droliferation

tion factor, which normally controls expression of the GRF receptor 110-131. Thus, dwurfand-little mutations both affect the same pathway: however, dw/dw has a more severe phenotype, presumably because Pit-l also regulates other growth factor receptors 1101. Mutations in both humans and in mice have also shown the importance of intracellular signal modulators. Human X-linked agammaglobulinemia (XI& is caused by a mutation in a gene for cytoplasmic tyrosine kinase, which has a domain organization similar to, but distinct from, that of the Src family 114*,15*1. This mutation leads to an accumulation in the bone marrow of pre-B cells, which fail to expand and mature into functional B cells. Vetrie et al. 114.1 found two different point mutations within the kina.se domain in two XLA patients. Tsukada et al. 115.1 reported that B cell lines derived from other XLA patients had no detectable transcript for the kinase. Subsequently, it has been found that the mouse X-linked immunodeficiency (xia’) locus carries the mouse homolog of the human XLA gene, and the gene product has been termed Bruton’s tyrosine kinase (Btk) 116,171. A point mutation was found within the amino-terminal ‘pleckstrin homology’ (PH) 1181region of Btk, although the mutant Btk could autophosphorylate 116,171. The mice exhibit a milder phenotype than the human disease, perhaps because the kinase no longer interacts properly with the signaling network but is still catalytically active. Loss of tyrosine phosphatases also causes immunological problems. The recessive mutations motheaten (me> and viable motbeaten (me”), on chromosome 6 of mice, cause severe autoimmunity and immunodeficiency 111.Recently, it has been demonstrated that the motheaten mutations are within the hematopoietic cell protein phosphatase (HCP) gene (Hcpb in mice; P7PNG in humans) 119**,20’1. The gene product (HCP, PTPlC, SHPTP-1 or SH2 phosphatase; citations in 119.*1> contains two Src homology 2 domains (SH2). Point mutations in me or W cause a frameshift mutation leading to a truncated protein or a splicing variant retaining some residual catalytic activity, respectively 11~1. The severity of me phenotype correlates well with the molecular nature and expression of mutant protein. Biochemical analyses of normal HCP suggest that the protein may associate with the receptor-type tyrosine kinase c-Kit after stimulation with Steel factor, and that the association is mediated by its two SH2 domains [21l. Hematopoietic cells cultured from me mice are hypersensitive to growth factors. Macrophages can grow independently of macrophage colony-stimulating factor (CSF-11, and some erythroid precursor populations require no erythropoietin for proliferation (citations in 119”l). As c-Kit, CSF-1 receptor and erythropoietin receptor induce tyrosine phosphorylation, it is tempting to speculate that HCP dephosphorylates activated tyrosine kinases to turn them ‘off’. Because these cells can acquire growth factor independence in vitro, and me heterozygotes have an increased risk of lymphoid neoplasms, it is possible that the gene encoding HCP may act as a tumor suppressor [lyl.

Genes in search of function The ability to disrupt any gene by homologous recombination has led to new approaches in the study of gene function in vivo, and in the testing of models of the physiological role of any given protein (see Table 1). In many cases the phenotypes have not been entirely predictable from the expression patterns. This suggests that several of the gene products serve unique roles in some cell types, but have overlapping functions in other tissues. One of the first genes to be studied using homologous recombination was the one that encoded the nonreceptor tyrosine kinase pp6oC-“rc. Despite widespread expression of the gene and the fact that Src has been implicated in many processes, including growth-stimulating signals from cell surface receptors to their substrates, no apparent defects were noted in Src-deficient mice, except in bone 1221. Mutant mice suffered from osteopetrosis, a bone modeling disorder due to a defect in osteoclast function. By a combination of cell culture techniques and fetal liver transplantation, the defect could be ascribed to an autonomous defect in osteoclast function, rather than the surrounding microenvironment 123’1. There are now six mutations that lead to osteopetrosis in mice, although the disease is rare in humans. Molecular characterization has indicated that several of them are components of signal transduction cascades. In osteopetrosis (op/opI mutants, a mis-sense mutation in the gene encoding CSF-1, the ligand for a receptor-type tyrosine kinase c-Fms, leads to a nonautonomous defect 124,251. CSF-1 appears to be necessary for normal proliferation and differentiation of osteoclast precursors 1261. However, CSF-1 may not be an absolute requirement for induction of osteoclasts [27], suggesting compensation by other signaling components. Three other mouse loci have been found to carry spontaneous mutations that cause osteopetrotic disorders: grey letbul
Genetics

dominantly found in hematopoietic cells. Lck expression is restricted primarily to thymocytes and T cells, and has been studied intensively by both transgenic and loss of function approaches. In the absence of Lck, thymocytes are blocked early in the developmental program and very few T cells emerge in the periphery [34]. This defect was mimicked by overexpression of a ‘dominant-negative’ form of the enzyme in transgenic mice. In the mutants, thymoblasts were arrested at a point where the T-cell receptor b-chain had undergone rearrangement but the a-chain remained in the germline configuration [351. In contrast, overexpression of active Lck resulted in thymocytes that lacked functional T-cell receptor because the &chain failed to rearrange. The developmental program could be restored by supplying a functionally rearranged @chain as a transgene 1361.These processes occur independently of CD4 and CD8, as overexpression of a mutant Lck that fails to interact with these surface molecules results in the same phenotype [37**1. The mechanism controlling T-cell receptor chain rearrangements is exquisitely sensitive to levels of active enzyme, and suggests that Lck is a component of the apparatus controlling allelic exclusion. Additional evidence that Lck can function through signaling molecules other than CD4 and CD8 comes from the study of yS T cells, a lineage not expressing these surface markers. In order to determine whether thymic development of these cells depended on Lck, mice expressing a transgene encoding a functional yreceptor were bred on a Lck- background. Most of the thymocytes expressed the transgene but never matured and entered the periphery [38], indicating that thymic-derived cells depend on Lck for maturation. Another study has focused on the activation, rather than loss, of Src family kinases, by creating a null mutation in the csk gene. Csk is a cytoplasmic protein-tyrosine kinase that is structurally similar to the Src family [3!91. In vitro data have demonstrated that Csk negatively regulates the activity of Src family kinases by phosphorylating the carboxy-terminal tyrosine (Tyr527 for Src) i39-411. Disruption of the es/z gene in mice leads to embryonic lethality between 7.5 and 10.5 days of gestation, with defects in the notochord, neural tube and allantois [42’,43’1. The kinase activity of the Src family members increased IO-fold in cells derived from these mutants [42*,43*1. Interestingly, peptide mapping of Src in the absence of Csk revealed residual phosphorylation at Tyr527, and increased phosphorylation at Tyr416, the site usually found phosphorylated in activated variants of Src [42*1. These findings suggest that Csk is the major regulator for the Src family kinases in vivo; nonetheless, autophosphorylation or other kinases also appear capable of phosphorylating the regulatory Tyr527. Crossing of Csk- mutants with mice deficient in Src, Fyn or Yes should reveal whether loss of a member of the Src family can suppress the Csk-deficient phenotype, and provide direct evidence that the phenotype is due to uncontrolled activity of the kinase family. Two research groups have created mice deficient in transforming growth factor-a (TGFa), one of the lig-

of sianal transduction

Imamoto, Soriano and Stein

ands for the epidermal growth factor (EGF) receptor [44’,45’1. Homozygotes did not show any remarkable disorder except wavy hair and whiskers. This finding was surprising, because TGFa has potent growth-stimulating effects on a wide variety of cell types and is expressed in several tissues as well as in embryos. The mild phenotype may be due to a functional compensation within the EGF family. The TGFa locus was found to be allelic with a recessive mutation on chromosome 6, waved-l (wa-11, which displays the same curly hair phenotype [44.,45’1. As another phenotypically similar mutation, waved-2 (wa-2J, maps close to the EGF receptor on chromosome 11, and is cell autonomous (within the hair follicle), it has been suggested that wa-2 and the EGF receptor are allelic 145.1. In fact, in wa-2 mice, a point mutation was found within the kinase domain of the EGF receptor which signif: icantly reduced its kinase activity (AR Dunn, personal communication; DC Lee, personal communication). Another ligand-receptor signaling system has been studied in depth using a series of mouse mutants. The system for insulin-like growth factors (IGFs) involves a receptor with intrinsic tyrosine kinase activity and is believed to promote cell proliferation. Mice lacking IGF-I, IGF-II or the IGF-I receptor (IGFlR) exhibit a complex phenotype including dwarfism and lethality 146,471. Mutants for either ligand are about 40% smaller than normal, whereas double mutants are 70% smaller. In contrast, animals failing to express the IGFlR are half the size of wild-type litter mates. Analysis of double mutants between the IGFlR and either of the two ligands suggested that IGFlR may be the sole receptor for IGF-I, but another receptor which is capable of binding IGF-II may also exist to transduce growth-promoting signal. The Trk family of receptor-type tyrosine kinases are high affinity receptors for the nerve growth factor (NGF) family of neutrophins. Three loci (trk, t&B and t&C) encode this family of kinases, and one of these loci, t&B, is known to generate multiple transcripts. The full-length transcript, which encodes gp145ti? transduces a signal upon binding of its ligands, such as brain-derived neurotrophic factor and neurotrophin-4. The analysis of mice deficient for gp145’M [48*1 shows a substantial loss of neurons due to cell death in the trigeminal ganglia, the facial nucleus, the dorsal root ganglia and the spinal cord. The mice die shortly after birth because of a feeding problem, most probably due to poor innervation of the motor neuron system, especially in the facial nucleus. Some questions remain to be answered. As trkB is expressed throughout the central nervous system, why are motor neurons affected more than anything else? Why do some neurons still survive in the affected region? What is the role of gp75 tnfLs! Interestingly, different defects in the peripheral sensory nervous system have been reported for mice lacking the low affinity NGF receptor, p75NGFH, which can bind four members of the NGF family (NGF, brain-derived neurotrophic factor, neurotrophin-3 and neutrophin-4) 1471. Intercrossing of gp145lrM and p75NGflt mutants, and the derivation

43

44

Oncogenes

and cell proliferation

of a mutation disrupting both gp9SM and gpl45m, might provide valuable information i48.1. In addition, the analysis of mice deficient for other t&genes might support the idea of functional compensation in this gene family. Like Steel factor, leukemia inhibitory factor (LIF) is a cytokine. that is &volved in many cell lineages. One of the fascinating effects of the factor is its ability to maintain stem cells in a pluripotent and undifferentiated state. The ptiysiological importance of the factor has been confirmed by the analysis of LIF mutant mice. Stewart ef al. 1501 have demonstrated that maternal expression of LIF is essential for implantation of embryos. Escary et al. [511 have shown that whereas stem cell populations in spleen and bone marrow have some dependency on LIF, the stem cells in the LIF- mice appear to remain pluripotent. These observations suggest that LIF deficiency may be partially compensated for by another member of the LIF family or by a parallel signaling pathway. The LIF-receptor consists of two transmembrane & components, gp130 and LIF receptor-& Although neither of the f&components has intrinsic kinase activity, a group of tyrosine kinases (including JAK kinases), upon binding of the ligand, can associate to form a functional receptor complex [52,531. The glycoprotein gp130 can either heterodimerize with LIF receptor-p or homodimerize 1531. The gp130 homodimer can associate with the IL-6a receptor on the cell surface to transduce the IL-~ signal [53,541. The heterodimer (gpl3GLIF receptor-b) can transduce a signal for LIF or oncostatin M. In addition, it can also associate with the a-component of ciliary neurotropic factor (CNTF) receptor [531. Therefore, oncostatin M, CNTF and IL6 may be able to induce signals qualitatively similar to that of LIE In culture, CNTF and LIF can induce nearly identical effects on sympathetic neurons [55]. Nonetheless, CNTF deficiency leads to a defect in only a subset of neural tissue, motor neurons in adult mutants [551, perhaps because of its restricted expression pattern. The loss of gp130, a key component of several receptor complexes, results in embryonic lethality (T Kishimoto, personal communication). It will be of interest to examine stem cells in the mutant embryos. As in the absence of gp130, none of the signals mentioned above should be transduced to maintain the stem-cell population.

Conclusions The rapid expansion in the analysis of both pre-existing and targeted mutants should help unravel signaling pathways, both by identifying genetic interactions and by generating a whole allelic series in specific genes. As exemplified by naturally occurring mutants at Wand Sl, we can anticipate a host of information regarding the subtleties of signal transduction. In addition, the creation of tissue-specific or temporal-specific muta-

tions using site-specific recombinases 156,571 might be especially useful in the study of genes that produce a lethal phenotype or pleiotropic effects. In the same way that one unties complex knots from both ends, these approaches may help untangle otherwise complicated biological phenomena.

Acknowledgements We wish to thank Ashley Dunn. Riidiger Klein, David Lee and Tadamitsu Kiihimoto for sharing valuable information prior to publication. We are grateful to Zhi Chen and Glenn Friedrich for critical reading of the manuscript. We would also like to apologize to colleagues whose work was not cited because of space restrdints.

References

and recommended

reading

Papers review,

of particular interest, published have been highlighted as: of special interestof outstanding interest

1.

GREEN MC: Catalog of Mutant Genes and Polymorphic In Genetic Vutianrs and Stnrlns of the Laboramy edn 2. Edited by Lyon MF, Searle AG. Oxford: Oxford versity Press; 1989112403.

. ..

within

the annual

period

of

Loci.

Mouse. Uni-

2.

FI.~%CHMAN 1~: From of the Kit Receptor Gener 1993, 9:28%290.

White Spots in Mammalian

3.

WILLIAMS SD: The

P. NAMEN AE, WII)MER MB, Lkv Eiol 1992, 151:361%376.

4.

MORRISON-GRAHAM K, TAKAHASHI Y: Steel Factor and c-Kit Receptor: from Mutants to a Growth Factor System. Eioessays 1993. 1577433.

5.

MARZIALI G, LAZARO D, SOKHEM’INO V: Binding of Germ Cells to Mutant SF Scrtoli Cells Is Dcfcctivc and Is Rcscued by Expression of the Transmcmbranc Form of the c-Kit L&and. &u Blof 1993, 157:182-190.

6.

BRANNAN CI, I~ED~I.L MA, KF~NICK JL, EPPIC MA, WILLIAMS DE, LYMAN SD, DONOVAN PJ, COPIXAND NG: Dcvclopmcntal Abnormalities Mice Result from a Splicing Dcfcct in the Cytoplasmic Tail. Genes &CJ 1992, 6:1832-1842.

7.

M~UNTJOY KG, ROI~BINS LS, MORIXUD MT, CONE RD: The Cloning of a Family of Gcncs that Encode the Mclanocortin Rcccptors. Scfence 1992, 257:124%1251.

8.

CHHAJLANI V, WIKREHC JES: Molecular Cloning pression of the Human Mclanocytc Stimulating Receptor. FFBS Letl 1992. 309:417-420.

DE, Steel

DEVRI~ Factor.

to Stem Cells: Dcvclopmcnt.

the

Role rrenl LYMAN

JJ, HANDEL JENKINS NA. in Sleell’fl Steel Factor

and ExHormone

9. ..

R~UI~INS LS, NADEAU JH, JOHNSON KR, KELLY MA, ROSELLIREH~USS L, DAACK E, MOUNTJOY KG, CONE RD: Pigmcntation Phcnotypcs of Variant ertenslon Locus Alleles RcsuIt from Point Mutations that AItcr MSH Rcccptor Function. Cell 1993, 728274334. Biological phenomena caused by r*len\‘ion alleles are well explained by the molecular nature of the mutations. The authors connect biochemistry of each aMSH receptor mutation to biological observations. Furthermore, the paper raises important questions concerning current knowledge In biochemistry. 10.

LIN S-C, LIN CR, GUKOVSKY I, LUSIS AJ, SAWCHENKO PE. ROSENPEI.D MG: Molecular Basis of the IIftle Mouse Phcne

Genetics type and implications 1993, 364208-213. 11.

for Cell Type-Specific

Growth.

Nurzrre

U S, CRENSHAW EB 111, RAWSON EJ, SIMMONS DM, SWANSON LW, ROS~NF~LD MC: dwutj locus Mutants Lacking Three Pituitary Cell Types Result from Mutations in the POU-Domain

Gene pitl.

Nutrrre 1990, 347:528-532.

12.

PF;~FFLE RW, DIMA-~~A GE, PARKS JS, BROWN MR, Wrr JM, JANSEN M, VAN DER NAT H, VAN DEN Brwul)e JL, ROS~NFELD MG, INGRAHAM HA: Mutation of the POU-Spccilic Domain of Pit-l and Hypopituitarism Without Pituitary Hypoplasia. Sc&nce 1992, 257:111l%l121.

13.

LIN C. LIN S-C, CHANC C-P, ROSENFELD MC: Pit-l-Dependent tiprcssion of the Receptor for Growth Hormone R&as ing Factor Mediates Pituitary Cell Growth. Nature 1992, 360:76%768.

14. .

VF~RIE D, VORECHOVSKY 1, SIIJERAS P, HOLLAND J. DAVIM A, FWM‘ER F, HAMMAKSI~M L, KINNON C. LEVINSKY II, BOUROW M. E-/’ AL.: The Gene Involved in X-Linked Agammaglobulinacmia Is a Member of the src Family of Protein-Tyrosinc Kinascs. Nutwe 1993. 361:226-233. This is one of two papers documenting the first example of a human genetic diseax (XLA) that is caused by a defect in a cytoplasmic tyrosine kinasc. This paper describes two patients who have point mutations within the kinase domain of the protein. Also see 115.1. TSUKADA S, SAFFRAN DC, RAWLINGS DJ, PAROLINI 0. ALLEN RC, KI.ISAK I. SPARK~S RS, KUI~AGAWA H, MOHANDAS T, QUAN S, 1:7‘ AL.: DcIicicnt Expression of a B Cell Cytoplasmic Tyrosine Kinasc in Human X-Linked Agammaglobulincmia. Cell 1993, 72~279-290. This is the second pdpcr describing the molecular nature of the XLA deficiency in man. In a search for novel B cell spccilic tyrosine kinases, thesr authors describe a new cytopllsmic kinasc that maps 10 the XLA locus. B cell lines from a number of XLA patients lack kinase-specific RNA or produce inactive kinzse. Also see 114.1.

15. .

16.

THOMAS JD. SIDERAS P, SMI’IH CIE, VORECHOVSKY I, CHAPMAN V, PAUL WE Colocalization of X-Linked Agammaglobulincmia and X-Linked lmmunodchcicncy Genes. Science 1993, 261:35%358.

17.

KAWI.INGS DJ, SAFFRAN DC, TSUKAIIA S, LARCA~PAI)A DA, GRIMALI)I JC, COHEN L, MOHR RN. BA~AN JF, HOWARD M, COP~~NI, NG, /:r AI..: Mutation of Unique Region of Bruton’s Tyrosinc Kinasc in Immunodcficicnt XlD Mice. Sctence 1993. 261:358-361.

18.

MUSA~CHIO A, GII~~ON T, ltic~ M: The PH Domain: a Common Patchwork of Signaling Proteins. 18:343348.

P, THOMPSON J, SAI~ASI% Piece in the Structural Trenrls BIocbern Scl 1993,

SHUL’I’Z LD, SCHWEI’I’ZER PA, RAJAN TV, YI T. IHLE JN, MA’I-IHEWS RJ, THOMA.S ML, BEIER DR: IMutations af the Murinc motbeufen Locus Arc within the Hcmatopoictic Cell Protein-Tyrosinc Phosphatasc (Hcpb) Gene. Cell 1993, 73:1445-1454. This paper studies the molecular nature of mutliions at the melocus. Lack of the functional phoSphJtJsc leads 10 aberrant proliferation of hematopoietic cells. The potential role of the phosphatase in signal transduction in hematopoietic cells is discussed in detail. The scenario described here could be one of the model systems for control of intracellular signaling. Also see I20.1.

19. ..

TSUI HW, SIMINOVITCH ICA, Ix DOLJ~A L, TSUI FWL: mot&eaten and viable motbeulen &Mice Have Mutations in the Hacmatopoictic Cell Phosphatasc Gene. Nufure Gener 1993, 4:124-129. This paper describes the molecular characterization of the me mutations, and draws similar, if not identical, conclusions 10 [19”1. 20. .

22.

YI T, 1HI.E JN: Association of Hcmatopoictic Cell Phosphatasc with c-Kit tier Stimulation with c-Kit Ligand. Mel Cell Blol 1993. 13:3350-33513.

Imamoto,

Soriano

and Stein

SORIANO P, MONI’GOMERY C, GE~KE R, BRADLEY A: Targeted Disruption of the c-src ProtoOncogcnc Leads to Ostcopctrosis in ~Micc. CeU 1991, 64693702.

LOWE C, YONEDA T, BOYCE BF, CHEN H, MUNDY GR. SORIANO P: Osteopctrosis in Src-Dcficicnt Mice 1s Due to an Autonomous Dcfcct of Ostcoclasts. froc Nut1 Acud Scl USA 1993, 90:448%489. These mix-and-match and fetal liver tnnspluntotion experiments identified a cell-autonomous defect in osteoclast function. The authors also discuss why other Src-related kinascs cannot substitute for the absence of Src in osteoclasts.

23. .

24.

YOSHIDA H, HAYASHI S-I, KUNISADA T, OCAWA M, NISHIKAWA S, OKLIMURA H, SUDO T, SH~JLI~ LD, NISHIKAWA S-I: The Murinc IMutation Ostcopctrosis is in the Coding Region of the IMacrophagc Colony Stimulating Factor Gene. Nature 1990, 345442-444.

25.

TAKAHASHI N. UDAGAWA N, AKA’ISU T, I.~~GAI Y, SUDA T: Dcficicncy of Ostcoclasts in Ostcopctrotic Mice Is Due to a Defect in the Local Microcnvironmcnt Provided by Ostcoblastic Cells. Endocrinolvgy 1991, 128~1792-1796.

26.

TANAKA S. TAKAHASHI N, UDAGAWA N, TAMURA T, Ax~?su T, S~‘ANLEY ER, K~ROKAWA T, SUDA T: Macrophagc ColonyStimulating Factor is lndispcnsiblc for both Proliferation and Diicrcntiation of Ostcoflast Progenitors. / C&z Intkzr 1993, 91:257-263.

27.

BEGG SK. RADLEY JM, POLLARD JW, CHISHOLM OT, STANLEY EH, BER’I’ONCELLO 1: Dclaycd Hcmatopoictic Dcvclopmcnt in Cktcopotrotic (op/op) Mice. J lhp Med 1993, 177:237-242.

28. ..

HODGKINSON CA, MOORE KJ, NAKAYAMA A, STRINGRiMSSON E, COPEWND NG, JENKINS NA, ARNHE~IFR H: Mutations at the Mouse mlcropbtbalmiu Locus Arc Associated with Dcfccts in a Gene Encoding a Novel Basic-Helix-Loop-Hclppcr Protein. Cell 1993, 74:395-404. The mllocus has been identified as important for the development of osteoclasts. This paper describes the cloning of this locus and shows thar the gene product is a broadly expressed trunscription factor. The pattern of expression fits well with the multiple phenotypes observed in mice carrying different alleles. Molecular analysis of fwo ml alleles showed that one is due to a deletion in the region encoding the amino port of the protein. The other allele was the result of a single codon deletion that removed an arginine residue thought to be involved in DNA contact. a 29.

JOHNSON RS, SPIEGELMAN Effects of a Null Mutation 1992, 71:577-s.

30.

WANG Z-Q, Own C, GRIGORIADIS AE, MOHL&S’IFINI.EIN U, ROI’HER U, WAGNER EF: Bone and Hacmatopoictic Dcfccts in Mice Lacking c-jbs. Nutwe 1992, 360741-745.

31.

~PLEI~Y MW, GROIN JA, PERLMLII-IF.R RM: Defective Mice Lacking the Thymic 70:751-763.

32.

S~FIN PL, LEE H-M, RICH S, SORIANO P: pp5Wm Mutant Mice Display Dicrcntial Signaling in Thymoqtcs and Pcriphcnl T Cells. Cell 1992, 70:741-750.

BM, PAPAIOANNOU V: Plciotropic in the C-/OS Proto-Oncogcnc. Cell

CWKE MP, LEVIN SD, QIAN X, T Cell Receptor Signaling in Isoform of p59@. Cell 1992,

GHANI’ SGN, O’DEU TJ, KARL KA. S’I’EIN PL, SORIANO P, KANDEL ER: Impaired Long-Term Potcntiation, Spatial Learning, and Hippocampal Dcvclopmcnt in Fyn Mutant Mice. Scfence 1992. 258:19031910. The hippocdmpus WJS isolated from different mutant mice, and examined for effects on long-term potentiation. Only mice lacking Fyn exhibited an attenuated long-term potent&ion response. This correlated with evidence of impaired memory functions. 33.

.

34. 21.

of signal transduction

MOI.INA TJ, KISHIHARA K. SII)EROVSKI DP, VAN EWIJK W, NARENDHAN A. TIMMS E, WAKEHAM A. PAIGE CJ, HARTMANN KU, VEILI.EI~~ A, I:~‘AI..: Profound Block in Thymocytc Dcvclopmcnt in IMice Lacking p5tiCk. Nurwe 1992, 357:161-164.

45

46

Oncogenes 35.

.

LEVIN

and cell proliferation SD,

ANDER%N

SJ, FOHBUSH

.KA,

P~~ALMUITEH

RM:

A

Dominant-Negative Transgene DeIines a Role for p56rur in Thymopoiesis. EMBO / 1993, 12:1671-1680. 36.

ANDERSON PERLMIJIIFR

SJ,

ABRAHAM

KM,

NAKAYAMA

T,

SINGEH

A.

RM: Inhibition of TCcll Receptor whain Gene Rearrangement by Overexpression of the Non-Receptor Pro tcin Tyrosinc Kinase p5*. FM80 / 1992, 11:4877-@86.

LEVIN SD, ABRAHAM KM, ANDER.SON SJ, FOHBUSH KA, PERLMU~~EHKM: The Protein ‘Qrosinc p5@ Regulates Thymocytc Development Independently of Its Interaction with CD4 and CDS,Coreccptors. J Erp Med 1993, 178:245-255. This paper shows how transgenic mice can be used to study signaling. Using a transgene encoding a mutant form of Lck, the authors demonstmted that thymocyte matumtion depends on Lck but does not require binding to CD4 or CD& This provides evidence that Lck promotes signaling by interacting with other molecules. 37. ..

38.

39.

40.

41.

PENNINGEHJ, K~SHIH+~A K, MOLINA T, WALLACE VA, TIMMS E, HEDRICKSM, MAK TW: Requirement for Tyrosinc Kinase p56r* for Thymic Development of Transgenic y8 T Cells. Science 1993, 2603358-361. NADA S, OKADA M, MAC&II.EY A, Coorktt JA, NA~A~AWA H: Cloning of a Complementary DNA for a Protein-Tyrosinc Kinase That Specifically Phosphorylates a Negative Regulatory Site of p6OcF Nature 1991, 351:69-72. OKAIIA M, NADA S, YAMANMH~ Y. YAMAMOIU T, NA~A~AWA H: CSK: a Protein-Tyrosine Kinase Involved in Regulation of src Family Kinases. J Biol Cbem 1991. 266:24249-24252. MA~AUIXY A, O~AI,A M, NAIIA S. NAKAGAWA H, COO~EHJA: Phosphorylation of Src Mutants at Tyr-527 in Fibroblasts Does Not Correlate with in Vftm Phosphorylation by CSIC. Oncogmr

1993,

8~117-124.

42. .

IMAMOIU A, S~RIANO P: Disruption of the crk Gene, Encoding a Negative Regulator of Src Family Tyrosine Kinascs, Leads to Neural Tube Defects and Embryonic Lethality in Mice. Cdl 1993, 73:1117-1124. The authors constructed mice with activated Src family kinases by mutating the negative regulator Csk. Consequently, mutants died In rctrro with complex defects. One of the significant contributions of this ~x~per to the field is the finding that Csk is the major regulatory kinase In r&o, yet other kinases could also be responsible for the regulatory phosphoryhtion on &Oar. NAI~A S. YAGI T, TAKEDA H, T~KUNAGA T. NAKAGAWA H. IKAWA Y, OKADA M. AIZAWA S: Constitutive Activation of Src Family Kinases in Mouse Embryos That lack Csk. Cell 1993. 73:1125-l 135. In this work, another disruption of the cs& gene is described. The mutation resulted ln the same phenotype as described above. It w&s suggested that embryonic expression of Csk appears to precede that of Src during mid-gestation, presumably hecause Src kinase activity needs to be regulated by Csk.

45. .

LU~KE NC, QIU TH, PEIFFEHRL, OLIVER P, SMITHIES0. LEE DC: TGFa Deficiency Results in Hair Follicle and Eye Abnormalities in Targeted and tuawd-1 Mice. cell 1993, 73:263-278. This paper also describes a null mutation at the TGFa locus. As w&l mapped close to this locus, the authors crossed both mutants and found that uw-1 is allelic to the TGFa null mutation. Also see 144’1. 46. LIU JP, BAKER J, PERKINS AS, ROBEH~~~N EJ, EFSTRATIADIS A: Mice Carrying Null Mutations of the Genes Encoding, Insulin-Like Growth Factor I (Igf-1) and Type 1 IGF Receptor (IgfIr). Cell 1993, 75:59-72. 47. BAKER J, LIU JP, ROBERTSONEJ, E~~~~RAI~ADISA: Role of Insulin-Like Growth Factors in Embryonic and Postnatal Growth. Cell 1993, 75:73-82. 48. KLEIN R, SMEYNE RJ, WUH?;I’ W, LONG LK, AUERBUCHDA, . JOYNER AL, BARI~ACI~ M: Targeted Disruption of the tnhE Ncurotrophin Receptor Gene Results in Nervous System Lesion and Neonatal Death. Cefl 1993. 75:113-122. In this work, the authors focused on gpl45’% The paper also discusses the complexity of signaling by the NGF family of ligands, as well as the high affinity Trk family receptors and the low affinity receptor gp75Norg. 49. LEE K-F, LI E, HUIIER J, L+NDIS SC. SHARPEAH. CHAO MV. JAENISCHR: Targeted Mutation of the Gene Encoding the Low Afllnity NGF Receptor p75 Leads to Deficits in the Peripheral Sensory Nervous System. Cell 1992, 69:737-749. 50. S~FWARI’ CL, KASPAH P, BHUN~YI’LJ, BHAIY H, GA~I I, K~)NXXN F, ABBONDANZO SJ: Blastocyst Implantation Depends on Maternal Expression of Leukemia Inhibitory Factor. Nurtrre 1992. 359~7679. 51. E.XARY J-L, PERH~AU J, DUMI?NII. D, EZINE S, BR~~LFI‘ P: Leukemia Inhibitory Factor Is Necessary for Maintenance of Haematopoietic Stem Cells and Thymocytc Stimulation. Nature

52. 53.

54.

43. .

44. .

MANN GB, FOWLEHKJ. GAI~HIEI.A, NICE EC, WILLIAMS RL, DUNN AR: Mice with a Null Mutation of the TGFa Gene Have Abnormal Skin Architecture, Wavy Hair, and Curly Whiskers and Often Develop Comeal Inflammation. Cell 1993. 73:24%261. The results of this paper were unexpected. The mild phenotype called the authors attention to existing mouse mutations with a similar phenotype. Thus, t&u-I is found to be allelic to the TGFa mutation. The physiological role of TGFa is discussed. Also see [45*1.

1993.

363:361-364.

N, YANCOPOIJLOS GD: The Alphas, Betas, and Kinases of Cytokine Receptor Complexes. Cell 1993. 74587-590. DAVIS S. AI.I)KICH TH, S’I’AHI. N, PAN L, TAGA T, KISHIM~I~ T, IP NY, YANCOPOUI.OS GD: LIFg and gp130 as Hetcrodimcrizing Signal Transducers of the Tripartite CNTF Receptor. Scfence 1993, 260:1&%-180%

STAHL

MUHAKAMI M. Hlni M, NAKA~AWA N, NAKAGAWA T, YASUMASA K. YAMANISHI K. TAGA T, KISHIMOI’~ T: IL&Induced Homo

dimerization of gp130 and Associated Activation of a Tyro sine Kinase. Science 1993, 260:1&W-1810. 55. 56.

MAS~J Y, WOLF E, HOI.TMANN THOENEN H: Disruption of the

Cells.

57.

B,

SENIXNEH

M,

BREht

G,

CNTF Gene Results in IMotor Neuron Degeneration. Naflrrr 1993, 365:27-32. O’GORMAN S. FOX DT, WAHI. GM: Rccombinase-Mediated Gene Activation and Site-Specitic Integration in Mammalian ORI~AN

Science

1991,

MARTH JD: Tissue- and Site-SpeciIic DNA in Transgenic Mice. f%c Nat1 Acad Sci USA

PC, CH~JI 0.

Recombination 1992,

251:1351-1355.

89f1861-6865.

A Imamoto, P Soriano and PL Stein, Fred Hutchinson Cancer Research Center, Progmm in Molecular Medicine, Seattle, Washington 98104, USA.