NORMAL GENOMIC

• DeWard AD, Eisenmann KM, Matheson SF, Alberts AS
• The role of formins in human disease.
• Biochim Biophys Acta. 2010; 1803: 226-33
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• Formins are a conserved family of proteins that play key roles incytoskeletal remodeling. They nucleate and processively elongatenon-branched actin filaments and also modulate microtubule dynamics.Despite their significant contributions to cell biology and development,few studies have directly implicated formins in disease pathogenesis. Thisreview highlights the roles of formins in cell division, migration,immunity, and microvesicle formation in the context of human disease. Inaddition, we discuss the importance of controlling formin activity andprotein expression to maintain cell homeostasis.

• Sewell W, Williams T, Cooley J, Terry M, Ho R, Nagy L
• Evidence for a novel role for dachshund in patterning the proximalarthropod leg.
• Dev Genes Evol. 2008; 218: 293-305
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• The branchiopod crustacean Triops longicaudatus has paddlelike thoracicappendages with few joints and multiple marginal lobes. Here, we explorethe degree to which the Triops limb is patterned by the same network ofgenes known to pattern the uniramous, multi-jointed insect appendage.Insect leg patterning proceeds through a process of subdividing the leginto proximal, intermediate, and distal regions by the activity of thetranscription factors hth/exd, dac, and Dll. The immature Triops limb issubdivided into large, discrete regional domains (proximal and distal) asdefined by nuclear-EXD and DLL. We show that HTH expression in Triopsoverlaps cell-to-cell with n-EXD expression. In addition, dac is expressedin two domains: (1) adjacent to and partially overlapping the distal Dlldomain and (2) along the medial margin of the developing leg. The DACdomain adjacent to the distal Dll domain supports the early establishmentof the expected intermediate domain of DAC expression. The medialexpression domain resolves over time into a series of reiterated stripeslocated on the lower side of each medial lobe. Later, this expressionpattern correlates with the sclerotized regions associated with limbflexion. We propose that these stripes of DAC expression play a role informing reiterated medial lobes. Unlike Drosophila, where the proximaldistal patterning of the leg is coincident with patterning of reiteratedstructures (segments), we hypothesize that the patterning in Triops mayreflect an ancestral state where the patterning of reiterated medialstructures was not coincident with proximodistal limb patterning.

• Yoshizaki H et al.
• Activity of Rho-family GTPases during cell division as visualized withFRET-based probes.
• J Cell Biol. 2003; 162: 223-32
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• Rho-family GTPases regulate many cellular functions. To visualize theactivity of Rho-family GTPases in living cells, we developed fluorescenceresonance energy transfer (FRET)-based probes for Rac1 and Cdc42previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki,and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added twotypes of probes for RhoA. One is to monitor the activity balance betweenguanine nucleotide exchange factors and GTPase-activating proteins, andanother is to monitor the level of GTP-RhoA. Using these FRET probes, weimaged the activities of Rho-family GTPases during the cell division ofHeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at theplasma membrane in interphase, and decreased rapidly on entry into Mphase. From after anaphase, the RhoA activity increased at the plasmamembrane including cleavage furrow. Rac1 activity was suppressed at thespindle midzone and increased at the plasma membrane of polar sides aftertelophase. Cdc42 activity was suppressed at the plasma membrane and washigh at the intracellular membrane compartments during cytokinesis. Inconclusion, we could use the FRET-based probes to visualize the complexspatio-temporal regulation of Rho-family GTPases during cell division.

• Barrow JR et al.
• Ectodermal Wnt3/beta-catenin signaling is required for the establishmentand maintenance of the apical ectodermal ridge.
• Genes Dev. 2003; 17: 394-409
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• The formation of the apical ectodermal ridge (AER) is critical for thedistal outgrowth and patterning of the vertebrate limb. Recent work in thechick has demonstrated that interplay between the Wnt and Fgf signalingpathways is essential in the limb mesenchyme and ectoderm in theestablishment and perhaps the maintenance of the AER. In the mouse,whereas a role for Fgfs for AER establishment and function has beenclearly demonstrated, the role of Wnt/beta-catenin signaling, althoughknown to be important, is obscure. In this study, we demonstrate thatWnt3, which is expressed ubiquitously throughout the limb ectoderm, isessential for normal limb development and plays a critical role in theestablishment of the AER. We also show that the conditional removal ofbeta-catenin in the ventral ectodermal cells is sufficient to elicit themutant limb phenotype. In addition, removing beta-catenin after theinduction of the ridge results in the disappearance of the AER,demonstrating the requirement for continued beta-catenin signaling for themaintenance of this structure. Finally, we demonstrate thatWnt/beta-catenin signaling lies upstream of the Bmp signaling pathway inestablishment of the AER and regulation of the dorsoventral polarity ofthe limb.

• Han MJ, An JY, Kim WS
• Expression patterns of Fgf-8 during development and limb regeneration ofthe axolotl.
• Dev Dyn. 2001; 220: 40-8
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• Fgf-8 is one of the key signaling molecules implicated in the initiation,outgrowth, and patterning of vertebrate limbs. However, it is not clearwhether FGF-8 plays similar role in development and regeneration ofurodele limbs. We isolated a Fgf-8 cDNA from the Mexican axolotl(Ambystoma mexicanum) through the screening of an embryo cDNA library. Thecloned 1.26-kb cDNA contained an open reading frame encoding 212 aminoacid residues with 84%, 86%, and 80% amino acid identities to those ofXenopus, chick, and mouse, respectively. By using the above clone as aprobe, we examined the temporal and spatial expression patterns of Fgf-8in developing embryos and in regenerating larval limbs. In developingembryos, Fgf-8 was expressed in the neural fold, midbrain-hindbrainjunction, tail and limb buds, pharyngeal clefts, and primordia of maxillaand mandible. In the developing axolotl limb, Fgf-8 began to be expressedin the prospective forelimb region at pre-limb-bud and limb bud stages.Interestingly, strong expression was detected in the mesenchymal tissue ofthe limb bud before digit forming stages. In the regenerating limb, Fgf-8expression was noted in the basal layer of the apical epithelial cap (AEC)and the underlying thin layer of mesenchymal tissue during blastemaformation stages. These data suggest that Fgf-8 is involved in theorganogenesis of various craniofacial structures, the initiation andoutgrowth of limb development, and the blastema formation and outgrowth ofregenerating limbs. In the developing limb of axolotl, unlike in Xenopusor in amniotes such as chick and mouse, the Fgf-8 expression domain waslocalized mainly in the mesenchyme rather than epidermis. The uniqueexpression pattern of Fgf-8 in axolotl suggests that the regulatorymechanism of Fgf-8 expression is different between urodeles and otherhigher species. The expression of Fgf-8 in the deep layer of the AEC andthe thin layer of underlying mesenchymal tissue in the regenerating limbssupport the previous notion that the amphibian AEC is a functionalequivalent of the AER in amniotes.

• Butler H et al.
• Map position and expression of the genes in the 38 region of Drosophila.
• Genetics. 2001; 158: 1597-614
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• With the completion of the Drosophila genome sequence, an important nextstep is to extract its biological information by systematic functionalanalysis of genes. We have produced a high-resolution genetic map ofcytological region 38 of Drosophila using 41 deficiency stocks thatprovide a total of 54 breakpoints within the region. Of a total of 45independent P-element lines that mapped by in situ hybridization to theregion, 14 targeted 7 complementation groups within the 38 region.Additional EMS, X-ray, and spontaneous mutations define a total of 17complementation groups. Because these two pools partially overlap, thecompleted analysis revealed 21 distinct complementation groups defined bypoint mutations. Seven additional functions were defined bytrans-heterozygous combinations of deficiencies, resulting in a total of28 distinct functions. We further produced a developmental expressionprofile for the 760 kb from 38B to 38E. Of 135 transcription unitspredicted by GENSCAN, 22 have at least partial homology to mobile geneticelements such as transposons and retroviruses and 17 correspond topreviously characterized genes. We analyzed the developmental expressionpattern of the remaining genes using poly(A)(+) RNA from ovaries, earlyand late embryos, larvae, males, and females. We discuss the correlationbetween GENSCAN predictions and experimentally confirmed transcriptionunits, the high number of male-specific transcripts, and the alignment ofthe genetic and physical maps in cytological region 38.

• Pizette S, Abate-Shen C, Niswander L
• BMP controls proximodistal outgrowth, via induction of the apicalectodermal ridge, and dorsoventral patterning in the vertebrate limb.
• Development. 2001; 128: 4463-74
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• Dorsoventral (DV) patterning of the vertebrate limb requires the functionof the transcription factor Engrailed 1 (EN1) in the ventral ectoderm. EN1restricts, to the dorsal half of the limb, the expression of the two genesknown to specify dorsal pattern. Limb growth along the proximodistal (PD)axis is controlled by the apical ectodermal ridge (AER), a specializedepithelium that forms at the distal junction between dorsal and ventralectoderm. Using retroviral-mediated misexpression of the bonemorphogenetic protein (BMP) antagonist Noggin or an activated form of theBMP receptor in the chick limb, we demonstrate that BMP plays a key rolein both DV patterning and AER induction. Thus, the DV and PD axes arelinked by a common signal. Loss and gain of BMP function experiments showthat BMP signaling is both necessary and sufficient to regulate EN1expression, and consequently DV patterning. Our results also indicate thatBMPs are required during induction of the AER. Manipulation of BMPsignaling results in either disruptions in the endogenous AER, leading toabsent or severely truncated limbs or the formation of ectopic AERs thatcan direct outgrowth. Moreover, BMP controls the expression of the MSXtranscription factors, and our results suggest that MSX acts downstream ofBMP in AER induction. We propose that the BMP signal bifurcates at thelevel of EN1 and MSX to mediate differentially DV patterning and AERinduction, respectively.

• Filippov V, Filippova M, Sehnal F, Gill SS
• Temporal and spatial expression of the cell-cycle regulator cul-1 inDrosophila and its stimulation by radiation-induced apoptosis.
• J Exp Biol. 2000; 203: 2747-56
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• Cul-1 protein is part of the ubiquitin ligase complex that is conservedfrom yeast to humans. This complex specifically marks cell-cycleregulators for their subsequent destruction. Two null mutations of thecul-1 gene are known, in budding yeast and in nematodes. Although in boththese organisms the cul-1 gene executes essentially the same function, themanifestation of its lack-of-function mutations differs considerably. Inyeast the mutation causes arrest at the G(1)/S-phase transition, whereasin nematodes excessive cell divisions occur because mutant cells areunable to exit the mitotic cycle. We isolated cul-1 orthologues from twomodel organisms, Drosophila melanogaster and mouse. We show that theDrosophila full-length cul-1 gene restores the yeast mutant's inability topass through the G(1)/S-phase transition. We also characterize expressionof this gene at the transcript and protein levels during Drosophiladevelopment and show that cul-1 gene is maternally supplied as a protein,but not as an RNA transcript. Zygotic transcription of the gene, however,resumes at early stages of embryogenesis. We also found an increase incul-1 transcription in cultured cells treated with a lethal dose ofgamma-irradiation.

• Scaal M et al.
• SF/HGF is a mediator between limb patterning and muscle development.
• Development. 1999; 126: 4885-93
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• Scatter factor/hepatocyte growth factor (SF/HGF) is known to be involvedin the detachment of myogenic precursor cells from the lateraldermomyotomes and their subsequent migration into the newly formed limbbuds. As yet, however, nothing has been known about the role of thepersistent expression of SF/HGF in the limb bud mesenchyme during laterstages of limb bud development. To test for a potential role of SF/HGF inearly limb muscle patterning, we examined the regulation of SF/HGFexpression in the limb bud as well as the influence of SF/HGF on directioncontrol of myogenic precursor cells in limb bud mesenchyme. We demonstratethat SF/HGF expression is controlled by signals involved in limb budpatterning. In the absence of an apical ectodermal ridge (AER), noexpression of SF/HGF in the limb bud is observed. However, FGF-2application can rescue SF/HGF expression. Excision of the zone ofpolarizing activity (ZPA) results in ectopic and enhanced SF/HGFexpression in the posterior limb bud mesenchyme. We could identify BMP-2as a potential inhibitor of SF/HGF expression in the posterior limb budmesenchyme. We further demonstrate that ZPA excision results in a shift ofPax-3-positive cells towards the posterior limb bud mesenchyme, indicatinga role of the ZPA in positioning of the premuscle masses. Moreover, wepresent evidence that, in the limb bud mesenchyme, SF/HGF increases themotility of myogenic precursor cells and has a role in maintaining theirundifferentiated state during migration. We present a model for a crucialrole of SF/HGF during migration and early patterning of muscle precursorcells in the vertebrate limb.

• Long AR, Yang M, Kaiser K, Shepherd D
• Isolation and characterisation of smallminded, a Drosophila gene encodinga new member of the Cdc48p/VCP subfamily of AAA proteins.
• Gene. 1998; 208: 191-9
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• Smallminded (smid) encodes a new member of the cdc48p/VCP subfamily of AAAproteins in Drosophila. The gene was isolated by plasmid rescue from aGAL4 enhancer trap line which shows reporter gene expression inneuroblasts, imaginal disks and a subset of sensory neurons. Larvaehomozygous for the insert arrest development as second instar larvae anddie without pupating. The most obvious defect in these larvae is asignificantly reduced CNS, hence the naming of the gene as smallminded.The deduced amino acid sequence of smid contains a tandem duplication ofthe AAA nucleotide binding domain characteristic of the cdc48p/VCPsubfamily. Overall, smid shares 33% identical residues with its closestrelative, yeast L0919-chrXII and 26-29% with other members of thecdc48p/VCP subfamily. The most highly conserved regions of the predictedprotein structure are found in and around the nucleotide binding domains.The gene is expressed at all developmental stages.

• Giansanti MG et al.
• Cooperative interactions between the central spindle and the contractilering during Drosophila cytokinesis.
• Genes Dev. 1998; 12: 396-410
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• We analyzed male meiosis in mutants of the chickadee (chic) locus, aDrosophila melanogaster gene that encodes profilin, a low molecular weightactin-binding protein that modulates F-actin polymerization. These mutantsare severely defective in meiotic cytokinesis. During ana-telophase ofboth meiotic divisions, they exhibit a central spindle less dense thanwild type; certain chic allelic combinations cause almost completedisappearance of the central spindle. Moreover, chic mutant spermatocytesfail to form an actomyosin contractile ring. To further investigate therelationships between the central spindle and the contractile ring, weexamined meiosis in the cytokinesis-defective mutants KLP3A and diaphanousand in testes treated with cytochalasin B. In all cases, we found that thecentral spindle and the contractile ring in meiotic ana-telophases weresimultaneously absent. Together, these results suggest a cooperativeinteraction between elements of the actin-based contractile ring and thecentral spindle microtubules: When one of these structures is disrupted,the proper assembly of the other is also affected. In addition to effectson the central spindle and the cytokinetic apparatus, we observed anotherconsequence of chic mutations: A large fraction of chic spermatocytesexhibit abnormal positioning and delayed migration of asters to the cellpoles. A similar phenotype was seen in testes treated with cytochalasin Band has been noted previously in mutants at the twinstar locus, a genethat encodes a Drosophila member of the cofilin/ADF family ofactin-severing proteins. These observations all indicate that proper actinassembly is necessary for centrosome separation and migration.

• Wang CC, Chan DC, Leder P
• The mouse formin (Fmn) gene: genomic structure, novel exons, and geneticmapping.
• Genomics. 1997; 39: 303-11
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• Mutations in the mouse formin (Fmn) gene, formerly known as the limbdeformity (ld) gene, give rise to recessively inherited limb deformitiesand renal malformations or aplasia. The Fmn gene encodes manydifferentially processed transcripts that are expressed in both adult andembryonic tissues. To study the genomic organization of the Fmn locus, wehave used Fmn probes to isolate and characterize genomic clones spanning500 kb. Our analysis of these clones shows that the Fmn gene is composedof at least 24 exons and spans 400 kb. We have identified two novel exonsthat are expressed in the developing embryonic limb bud as well as adulttissues such as brain and kidney. We have also used a microsatellitepolymorphism from within the Fmn gene to map it genetically to a 2.2-cMinterval between D2Mit58 and D2Mit103.

• Miklos GL, Yamamoto M, Burns RG, Maleszka R
• An essential cell division gene of Drosophila, absent from Saccharomyces,encodes an unusual protein with tubulin-like and myosin-like peptidemotifs.
• Proc Natl Acad Sci U S A. 1997; 94: 5189-94
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• Null mutations at the misato locus of Drosophila melanogaster areassociated with irregular chromosomal segregation at cell division. Theconsequences for morphogenesis are that mutant larvae are almost devoid ofimaginal disk tissue, have a reduction in brain size, and die before thelate third-instar larval stage. To analyze these findings, we isolatedcDNAs in and around the misato locus, mapped the breakpoints ofchromosomal deficiencies, determined which transcript corresponded to themisato gene, rescued the cell division defects in transgenic organisms,and sequenced the genomic DNA. Database searches revealed that misatocodes for a novel protein, the N-terminal half of which contains a mixtureof peptide motifs found in alpha-, beta-, and gamma-tubulins, as well as amotif related to part of the myosin heavy chain proteins. The sequencecharacteristics of misato indicate either that it arose from an ancestraltubulin-like gene, different parts of which underwent convergent evolutionto resemble motifs in the conventional tubulins, or that it arose by thecapture of motifs from different tubulin genes. The Saccharomycescerevisiae genome lacks a true homolog of the misato gene, and thisfinding highlights the emerging problem of assigning functional attributesto orphan genes that occur only in some evolutionary lineages.

• Maeda T, Watanabe Y, Kunitomo H, Yamamoto M
• Cloning of the pka1 gene encoding the catalytic subunit of thecAMP-dependent protein kinase in Schizosaccharomyces pombe.
• J Biol Chem. 1994; 269: 9632-7
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• We have isolated Schizosaccharomyces pombe genes that confer sterility tothe fission yeast cell when expressed from a multicopy plasmid. One ofthese genes strongly hybridized to a probe carrying the open reading frameof Saccharomyces cerevisiae TPK1, which encodes a catalytic subunit of thecAMP-dependent protein kinase (protein kinase A). This S. pombe gene,named pka1, has a coding potential of 512 amino acids, and the deducedgene product is 60% identical with the S. cerevisiae Tpk1 protein in theC-terminal 320 amino acids. Disruption of pka1 slows cell growth but isnot lethal. The resultant cells, however, are highly derepressed forsexual development, readily undergoing conjugation and sporulation in theabsence of nitrogen starvation. They are, thus, phenotypicallyindistinguishable from the adenylyl cyclase-defective (cyr1-) cellspreviously characterized, except that the pka1- spores are retarded ingermination, whereas the cyr1- spores are not. Disruption of pka1 isepistatic to a defect in cgs1, which encodes the regulatory subunit ofprotein kinase A. These results strongly suggest that the product of pka1is a catalytic subunit of protein kinase A and, furthermore, that S. pombehas only one gene encoding it. This situation contrasts with the case ofS. cerevisiae, in which three genes encode the catalytic subunits.

• Kobozieff N, Lucotte G, Gemahling E
• [Systemic competition in the case of hemimelia in mice].
• C R Seances Soc Biol Fil. 1976; 170: 940-2
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• In the mice hemimely, data suggest some systemic competitive phenomena,described in terms of surplus "digit material" with some contraints on itsdistribution between limbs.