(Dres): a comparative and topographic

1998 Oxford University Press 1997–2006Human Molecular Genetics, 1998, Vol. 7, No. 13

ARTICLE

The embryonic expression pattern of 40 murinecDNAs homologous to Drosophila mutant genes(Dres): a comparative and topographic approachto predict gene functionAlessandro Bulfone , Claudio Gattuso , Anna Marchit iello , Celia Pardini 1, Edoardo Boncin elli 1, Giuseppe Borsani , Sandro Banfi and Andrea B allabio*

Telethon Institute of Genetics of Medicine (TIGEM), San Raffaele Biomedical Science Park, via Olgettina 58,Milan 20132, Italy and 1Dibit, San Raffaele Biomedical Science Park, Milan, Italy

Received July 24, 1998; Revised and Accepted September 29, 1998

Nature often utilizes the same metabolic ‘core groups’ of interacting genes or ‘pathways’ in completely differentorgans, tissues and cellular compartments. Deciphering the physiological role of a particular gene in a livingorganism is therefore critical to understanding not only how a gene/protein works, but also where (in whichtissue/organ) and when (at what developmental stage) it functions. We have performed systematic RNA in situhybridization on a subset of murine genes homologous to Drosophila mutant genes, called Drosophila -relatedexpressed sequences (Dres). This approach combines functional information derived from cross-speciessequence comparisons and biochemical, physiological and pathological studies performed in the fly withknowledge of the spatial and temporal distribution of gene expression. Forty murine Dres were tested by RNAin situ hybridization on sagittal, coronal and transverse sections at three developmental stages, E10.5, E12.5 andE17.5. For some of them, whole mount in situ hybridization was performed at earlier stages. These data arevaluable for establishing how the function of these genes and the genetic programs underlying the developmentof a particular tissue or organ have evolved during evolution. For example, six Dres genes showed restrictedexpression domains within the murine retina, suggesting a different role for each of these genes in eyedevelopment and functioning. Furthermore, the information derived from this combined approach will beinstrumental in predicting the phenotypic consequences of gene dysfunction in both mouse mutants and humangenetic diseases.

INTRODUCTION

We are rushing into an exciting era in which tens of thousands ofnovel human genes are being identified by large scale cDNA andgenomic sequencing projects. Over 1 000 000 human expressedsequence tags (ESTs) generated from a large variety of humantissue sources (1–7) and possibly corresponding to >40 000human genes are available in a public database (dbEST) (8). Thisdatabase is, in fact, the major source of novel genes withpotentially important biological functions. Nevertheless, a diffi-cult task arises from this abundance: how to systematicallyanalyze genes in order to begin unraveling their function. The gapbetween the amount of data generated by collective sequencingand mapping efforts and the capability of assigning a function toa novel sequence is widening, especially at the level of the whole

organism. For these reasons, a new and challenging period isapproaching (9,10).

Cross-species comparison represents an effective strategy toinvestigate the function of genes (11–15). For example, analysisof a number of genes conserved between yeast and man hasprovided striking insights into their function (12,16). Ourextensive knowledge of the function of Drosophila genes can alsofacilitate comprehension of the role of their mammalian homo-logs. Despite the remarkable evolutionary distance between manand Drosophila, a number of genes are highly conserved betweenhumans and the fruit fly and exert similar functions in the twospecies. The phenotypes caused by mutations in some of thesegenes can affect related systems and organs in both man and fly(17,18). For example, the human gene responsible for aniridia

*To whom correspondence should be addressed. Tel: +39 02 21560206; Fax: +39 02 21560220; Email: [email protected]

Human Molecular Genetics, 1998, Vol. 7, No. 131998

(PAX6) is homologous to the Drosophila eyeless gene (18).Recently, we performed a systematic identification of humancDNAs homologous to Drosophila mutant genes (19–21). Todate, we have identified >150 novel human genes which wenamed DRES (Drosophila-related expressed sequences). Thesignificant degree of conservation of these cDNAs throughoutevolution suggests that they encode molecules with key biologi-cal functions.

A detailed analysis of gene expression distribution is of crucialimportance in order to understand the physiological role of agiven gene/protein in a living organism. Most genes affectdifferent aspects of the cellular and tissue phenotype. Thispleiotropic effect originates when a protein or mRNA is necessaryat different sites and/or at different times. Consequently, we needa way to systematically investigate gene expression in developingorgans and tissues to document when and where genes arefunctioning. A detailed temporal and spatial investigation of geneexpression will also provide insight into gene product interactionsand perhaps indicate hierarchies in gene expression programs.Furthermore, a complete identification of expression domainscould be useful for the development of new therapeutic tools andto help estimate their potential side effects.

Several systematic strategies have been developed to establishtime, location and level of gene expression. ‘Digital northerns’ or‘transcript profiles’ are either produced by sequencing thousandsof cDNA clones from different tissues and expression iscorrelated with variation in the relative frequency of sequencetags of the transcript of interest (4,22,23) or else they are basedon hybridization to arrayed oligonucleotides (24) or to cDNAlibraries (25–27). These ‘low resolution’ expression screensbased on EST programs lack the necessary resolution to definethe expression profile in the context of a developing or fullydifferentiated tissue, organ or organism. In fact, they provide acombined expression profile of all cells in the sample under study.These methods are then more appropriate to delineate expressionin cultured cell lines, for example in two cellular differentiationsystems inducible in vitro. If expression is to be viewed in originaltissues and in cell populations that are spatially restricted, or, onthe other hand, if simultaneous expression must be studied indistant regions or structures, then low resolution expressionscreens are not appropriate. A high resolution analysis of geneexpression can be performed by RNA in situ hybridization. Theadvantage of this technique is that it allows the localization ofgene expression at the cytological level, which is confined to afew cells or cell layers at a specific time and simultaneously in theentire developing organism. In other words, it allows a veryaccurate analysis of the spatial and temporal pattern of distribu-tion of gene transcripts (28).

To combine the value of comparative analysis with the contribu-tion of gene expression studies, we performed a detailed andsystematic high resolution expression study by RNA in situhybridization on a subset of murine homologs of human Dres genes.

RESULTS

General strategy

A searchable database termed the DRES search engine has beencreated and is accessible through the TIGEM DRES home page(http://www.tigem.it/dros.html ). The database, periodically up-

dated, contains a collection of TBlastN outputs and can beextensively searched in several ways: (i) with Drosophila proteinaccession numbers or IDs; (ii) with the progressive index number;or (iii) with a range of P values for the best matching ESTsequence in the output (19–21). Until now, >150 DRES geneshave been identified using the DRES search engine. In terms ofsequence conservation, all DRES show a high degree of similaritywith the corresponding Drosophila homologs, with P valuesranging from 1.4e–70 to 1.1e–6.

Murine homologs of DRES genes (Dres) were systematicallysearched by sequence database screening and by standardexperimental approaches. Bioinformatic searches were per-formed using the BlastN algorithm against dbEST, which nowcontains >300 000 mouse ESTs, mostly generated by theWashU-HHMI Mouse EST Project (6,7). Over 80% of murinecDNAs were identified by bioinformatic analysis, while theothers were isolated by screening of murine cDNA libraries andby PCR amplification of murine genomic DNA. Overall, we haveidentified >80 murine homologs of DRES genes. The level ofnucleotide homology between human and murine Dres rangedfrom 82 to 97% identity.

More than 30 murine Dres were also mapped and the positionof each was found to be syntenic to that of human homologs, thusconfirming that the murine genes were indeed the orthologs oftheir human counterparts. In Table 1, Dres genes analyzed in thisstudy are arbitrarily divided into five subgroups, based onphenotypic information of the corresponding Drosophila mu-tants: segmentation and development, eye-related, neural devel-opment, sex determination and reproduction and others. For eachDres, the name and the sequence accession number of thecorresponding full-length cDNA (if known) is reported, as wellas the sequence accession number of the probes used for the in situexperiments.

To systematically and comprehensively document expressionpatterns of Dres murine homologs, we collected a standard seriesof sections of mouse embryos at three developmental stages,E10.5, E12.5 and E17.5. These stages, which correspond to thelate embryonic and fetal human period, cover the time when mostmajor organs and body regions are organized and becomefunctional. Each slide series consists of sections of embryos cutin the sagittal, coronal and transverse planes.

Antisense and sense radioactive riboprobes, mostly originatingfrom the 3′-untranslated region (the least conserved among genefamilies) of murine EST clones, were used for hybridization.Whole mount in situ hybridization was also systematicallyperformed at earlier stages (E8.5, E9.5 and E10.5) whentranscripts were detected at E10.5 by radioactive in situ hybrid-ization on sections. In a few cases, tissues that have a prolongedperiod of development, such as the brain and eye, were alsoanalyzed postnatally. The slides and stained embryos were thensubjected to a detailed analysis. Panels of images for any relevantin situ hybridization experiment were generated for each Dresgene and stored in a local database.

Dres were selected on the basis of an intriguing phenotype ofthe corresponding Drosophila mutants, an attractive human ormurine mapping assignment and an interesting type of encodedprotein. Particular importance was given to Dres homologs ofDrosophila mutant genes with an eye-related or developmentalphenotype (Table 1).

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Table 1. Forty murine Dres selected for the expression analysis

Drosophila MouseDres Gene Type of protein Gene Accession no. In situ probe

Developmental

115 BarH1 (mouse) homeodomain containing protein – – W36375

46 Bicaudal C putative RNA binding domain – – AA028383

45 Bithorax complex adenosylhomocysteinase – – AA103383

44 bithoraxoid – – – AA403393

129 caupolican homeodomain containing protein iroquois homeobox protein 3 Y15001 W41873

93 empty spiracles homeodomain containing protein – – Y18247

14 fat facets ubiquitin C-terminal hydrolase fat facets homolog (Fam) U67874 AA260311

48 fringe cell-signalling molecule radical fringe AF015770 W83195

42 frizzled transmembrane domain frezzled U68058 AA278065

124 muscleblind ring-finger protein – – AA250561

52 pumilio – – – AA144200

56 rotund GTPase activating protein – – AA124923

30 Serrate Notch ligand jagged2 Y14495 Y14331

118 sister of odd and bowel C2H2 zinc finger – – AA003288

55 Suppressor of hairless J Kappa recombination signal binding protein – – Y18248

144 trithorax protein ASH2 – – – AA089069

Eye Related

91 CDS=photoreceptor-specific CDP-diacylglycerol synthase – – AA170489

92 CDS=photoreceptor-specific CDP-diacylglycerol synthase – – W30593

155 eyes absent – Eya1 Y10263 W34432

67 eyes absent – Eya3 U61112 AA049689

119 eyes absent – – – Y17115

16 nemo serine/treonine kinase nemo-like kinase (Nlk) AF036332 AA764140

156 norpA phospholipase C – – W50364

17 prune GTPase activating protein – – AA547423

8 retinal degeneration A diacylglycerolkinase – – AA472764

9 retinal degeneration B phosphatidylinositol transfer protein Dres9 Y08922 X98655

10 retinal degeneration C phosphoprotein Ppef – Y08234

Neural development

32 bendless ubiquitin-conjugating enzyme – – AA982879

27 minibrain serine/threonine kinase – – AA589241

65 Ras opposite vesicular trafficking protein unc-18 homologue D45903 AA049343

Sex determination and reproduction

23 male lethal 3 – – AA575543

147 Rga transcriptional regulator – – W36804

140 angel – – – AA050900

131 Des-1 transmembrane protein Mdes transmembrane protein Y08460 AA035819

25 diaphanous FH1-FH2 domains – – AA920816

86 diaphanous FH1-FH2 domains p140mDia U96963 W67065

Others

61 Eag-like K[+] channel putative potassium channel – – W51365

139 kekkon-2 (mouse) immunoglobulin superfamily – – AA153559

134 Past1 ATP/GTP binding site – – AA051670

132 TBP-associated factor 150kD TATA-binding protein associated factor – – AA028778

For each Dres the name and the sequence accession number of the corresponding full-length cDNA, if known, and the sequence accession number of the probesused for the experiments are reported.


Figure 1. Examples of Dres expression in multiple organs. (A) Complex Dres118 expression in the craniofacial mesenchyme and digestive tract in an E12.5 embryo,sagittal section. (B) Transverse hand plate section of E12.5 embryo showing the expression of Dres118 in the mesenchyme surrounding the condensating cartilage.(C–G) Dres139 expression. (C) Sagittal section of the head of an E17.5 embryo. (D–G) High magnification views in panels showing strong expression in the vibrissae(D), in the tooth buds (E), in the tip of the developing digits (F) and in the sinus hair follicles (G). CC, condensating cartilage; T, telencephalon; E, eya; TB, tooth bud;VF, vibrissae follicles; SHF, sinus hair follicle; BB, basisphenoid bone.

The in situ hybridization results are summarized in Table 2. Theexpression profile is schematically illustrated in the last column,where the different systems and parts of the mouse embryo arelisted. The distribution of expression data is represented by organsystems and not by embryonic regions or structures, as most of thesteps of morphogenesis and organogenesis have already occurredin the stages that were analyzed (E10.5–E17.5). Separatecolumns highlighting expression in developing limbs and headhave been added, mainly because these structures have complexdevelopmental processes involving large networks of interactinggene products. Two arbitrary levels of gene expression (low andhigh) are reported. It is also emphasized when regional domainsof expression are detected in particular organs, as in the case ofDres93, for which expression is found in defined areas of thedeveloping brain and retina.

Patterns of expression observed

The expression distribution of Dres genes reported and summar-ized in Table 2 can be compiled in several categories: undetect-able, ubiquitous, organ-specific and domain- or region-specific.Examples of the data described below are shown in Figures 1–4.The complete set of expression data will be deposited as videoimages in the DRES database (http://www.tigem.it/dros.html ).

Undetectable transcripts. The transcripts of ∼20% of the Dresstudied could not be detected by RNA in situ hybridization at threedifferent stages. To assess whether the genes were not expressed atthese stages or whether the results were due to technical limitations,hybridization experiments were repeated at least three times,utilizing different fragments of cDNAs as templates for generating

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Table 2. Schematic illustration of the expression profiles

Two arbitrary levels of gene expression are reported: low (gray box) and high(black box). White boxes indicate undetectable expression. Regional do-mains of expression in particular organs are represented by hatched boxes.

riboprobes. Among this group are the homologs of Bicaudal C(Dres46), frizzled (Dres42) (29), diaphanous (Dres25 andDres86), Suppressor of hairless (Dres55), eyes absent (Dres67)(30) and TBP-associated factor (Dres132).

Ubiquitous expression. Expression of 15% of the genes wasubiquitously distributed. In particular, Dres32, the homolog of theDrosophila bendless gene, is expressed at high levels in allstructures of the embryo. Conversely, transcripts relative to thehomologs of rotund (Dres56), fat facets (Dres14), Des-1(Dres131), minibrain (Dres27) and angel (Dres140) are ubiqui-tously present at lower levels.

Expression in multiple organs. Many Dres are expressed in a fewdistinct structures or tissues. Among those expressed simulta-neously in different organs are the homologs of Serrate (Dres30)and eyes absent (Dres155, Dres67 and Dres119). The Dres30transcript is detected at high levels in the limb buds, first branchialarch, central and peripheral nervous system, retina, tooth buds,thymus, submandibular glands and stomach (31). Dres155(eya-1) is instead expressed at high levels in the central andperipheral nervous system, craniofacial mesenchyme, precarti-lage tissue, olfactory epithelium, vibrissae follicles, prevertebrae,gut, kidney, eye and heart. Another homolog of eyes absent, eya-3(Dres67), is undetectable.

More restricted expression patterns were observed for: prune/Dres17 (central and peripheral nervous system, retina and limbbuds), Bithorax complex/Dres45 (central and peripheral nervoussystem and maxillary mesenchyme), sister of odd/Dres118(branchial arches, limbs, midgut, stomach, omentum and cranialmesenchyme; Fig. 1A and B), caupolican/Dres129 (central andperipheral nervous system, retina, inner ear, craniofacial mesen-chyme, ectoderm and lung), Past 1/Dres134 (central andperipheral nervous system, retina and gut) and kekkon-2/Dres139.In particular, Dres139 expression is first detected at E17.5 in toothbuds, vibrissae follicles, cranial and thoracic bones and the tip ofdeveloping digits (Fig. 1C–G).

Organ-specific and discrete domains of expression. Intriguingly,some Drosophila homologs are expressed exclusively in specificorgans. For example, Elk/Dres61 in the developing thyroid (Fig.2A), CDS/Dres92 in the thymic anlage (Fig. 2B) and Rasopposite/Dres65 in the entire neural tube (Fig. 2C–E).

Few Dres show discrete domains of expression in particularorgans. Dres115, the homolog of BarH1, and Dres93, the homologof empty spiracles, are expressed in highly defined regions of thecentral nervous system and neural retina, respectively.

As shown in Figure 3, we found an intriguing and specificdistribution of Dres transcripts in neural retina at E12.5: Dres16and Dres9 are expressed in the entire neural retina; Dres65,Dres45 and Dres129 are expressed in the inner layer ofdifferentiating neuroblasts; Dres93 expression is confined to theinferior portion.

Early expression. The analysis was also extended to early stagesof development for Dres genes expressed at E10.5. These wholemount in situ hybridization experiments turned out to be veryinformative for Dres129, Dres115 and Dres93. Dres129 tran-scripts are detected at E8.5 in the cephalic neural folds andunderlying mesenchyme (Fig. 4A). The anlage of the diencepha-lon and the most dorsal part of the hindbrain and spinal cord arethe only regions where Dres115 is expressed at E9.0 (Fig. 4C). Notranscript was detected for Dres93 before E9.5, the stage in whichexpression is limited to the optic cup (Fig. 4B).

DISCUSSION

Expression analysis of Dres genes

The sequence of a significant number of human genes can be foundin publicly available EST databases. One of the major challenges ofbiomedical research for the next decade will be to identifysystematic approaches to help understand their function (10). Anobvious and productive shortcut to this goal is to start characterizinghuman genes sharing sequence similarities with those for which a


Figure 2. (A and B) Dres expression in specific organs. (A) Dres61 transcriptis specifically detected in the developing thyroid, as shown on this E10.5coronal section. (B) Sagittal section at E12.5 showing Dres92 expression in thethymic anlage. (C–F) Examples of expression conservation betweenDrosophila and mouse: Dres65 and Dres91. (C) Sagittal section of an E12.5embryo showing Dres65 expression in the entire central nervous system. Highmagnification showing expression in the post-mitotic neurons of thetelencephalic mantle (D), nasal epithelium and vomero-nasal organ (E). (F andG) Sagittal section of an E16.5 embryo showing the expression of Dres91 in thecentral nervous system and specifically in the inner neuroblast layer of theneural retina. TR, thyroid primordium; TM, thymic primordium; T,telencephalon; D, diencephalon; M, mesencephalon; Rh, rhombencephalon;CC, cerebral cortex; TV, telencephalic vesicle; OP, olfactory epithelium; VO,vomero-nasal organ; ONL, outer neuroblastic layer; INL, inner neuroblasticlayer.

functional characterization has already been performed in otherspecies. This was the idea behind the DRES project: to study humangenes showing significant sequence identity to Drosophila mutantgenes (19–21). We wanted to take advantage of the functionalstudies (e.g. biochemical analyses and characterization of mutantphenotypes) previously performed in another organism to study thefunction of an interesting subset of human genes.

Sequence similarity often corresponds to the structural rela-tionship of the predicted protein products, suggesting that thebiochemical functions performed are also similar. However, dueto the major changes that have occurred during evolution, leadingto striking anatomical and physiological differences between manand fly, the physiological roles of related genes/proteins can becompletely different in the two species, even if their biochemicalfunction has been conserved. Nature often uses the same ‘coregroups’ of interacting genes or ‘pathways’ in completely differentorgans, tissues or cellular compartments. It is, therefore, criticalto understand not only how a gene/protein works but also where(i.e. in which tissue/organ) and when (i.e. at what developmentalstage) it exerts its function. One way to achieve this is to analyzegene expression patterns at different stages during development.This study demonstrates both the feasibility and value of usingRNA in situ hybridization for the systematic analysis of geneexpression. Obviously, optimization of the speed of each stepinvolved in the process and the use of automation wouldsignificantly increase the number of genes tested. Exploring thecomplexity of the spatial and temporal distribution of expressionof Dres genes has provided us with very useful information onseveral aspects of gene function, which are discussed below.

Expression pattern conservation

For some Dres genes, we found a clear conservation of expressionpattern in related structures between mouse and Drosophila. Forexample, the Drosophila gene Ras opposite (32,33) is highlyexpressed in the nervous system, where its product acts as amodulator of neurotransmitter release. Similarly, Dres65, highlyhomologous to Ras opposite, is exclusively expressed in neuraltissue in the mouse (Fig. 2C–E). Along these lines, Dres91 andDres92 are significantly homologous to the Drosophila CDP-diacylglycerol synthase (CDS) gene, involved in light-inducedphotoreceptor degeneration (34). The Dres92 transcript has beenfound in the anlage of thymus at E12.5 (Fig. 2B) and in adultretina, while Dres91 is also expressed in the developing neuralretina (Fig. 2F and G), indicating that these murine genes mightbe the real functional CDS homologs and may represent candidategenes for human retinopathies (35). Another striking example offunctional conservation found was Dres30, a murine homolog ofthe Drosophila Serrate gene that is involved in the dorso-ventralpatterning of the wing (36–38). Dres30, subsequently renamedJagged2, is expressed from E9.0 in the surface ectoderm ofbranchial arches and in the apical ectodermal ridge of limb buds,confirming that similar molecular mechanisms are involved inlimb development in insects and vertebrates (31). Noticeably,mutations in Jagged2 were recently found to be responsible forabnormal digit formation in the mouse mutant syndactylism (sm),displaying hyperplasia of the apical ectodermal ridge (39).

It is indeed remarkable that some Dres genes, which are definedby a highly significant sequence homology with Drosophila,often have a similar pattern of expression and exert relatedbiological functions in the development of the metazoan body

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Figure 3. Specific distribution of Dres transcripts in the developing neural retina. Sagittal sections of E12.5 embryos. Dres16 (A) and Dres9 (B) are expressed in theentire neural retina; but while Dres16 transcript distribution is uniform, Dres9 shows prevalent expression in the outer proliferative neuroblast layer. Dres93 (C)expression is peculiarly confined to the whole inferior portion of the neural retina. Dres65 (D), Dres45 (E) and Dres129 (F) are exclusively expressed in the inner layerof differentiating neuroblasts along the gradients of differentiation, from the inner to the outer layers and from the center to the periphery. Dres45 (E) is also expressedalong radial columns of cells in the outer, proliferative neuroblast layer. OpS, optic stalk; ONL, outer neuroblastic layer; INL, inner neuroblastic layer; L, lens.

Figure 4. Early expression assessed by whole mount in situ hybridization. (A) Dorsal view of Dres129 expression at the neural plate stage (E8.0): specific signal isdetected in the neural fold apposition, in the lower cervical region; in a restricted domain of the neuroepithelium in the prospective hindbrain region and in the underlyingcephalic mesenchyme. (B) Dres93 expression is confined at E9.0 to the inferior part of the optic cup. (C) Dres115 is specifically expressed at E9.0 in a regioncorresponding to the anlage of the diencephalon and in the most dorsal part of the alar plate along the hindbrain and the spinal cord. CM, cephalic mesenchyme; Hb,hindbrain; NFA, neural fold apposition; OC, optic cup; PV, prosencephalic vesicle; D, dincephalon; M, mesencephalon; Rh, rhombencephalon; SP, spinal cord.

plan. On the other hand, we found several examples of Dres geneswith expression patterns that are strikingly different betweenmouse and Drosophila. For example, the Drosophila retinaldegeneration (rdg)A, rdgB and rdgC genes are strongly expressedin the retina and are involved in the degeneration of photorecep-tors (40–42). Expression patterns observed for their respectivemurine homologs, Dres8, Dres9 and Dres10, suggest differentroles for these genes in mammalian development. Dres9, beingpredominantly expressed in the neural retina, has an expression

pattern remarkably similar to rdgB (43). Instead, Dres8 andDres10 have patterns of expression unequivocally different fromtheir relatives rdgA and rdgC. The Dres8 transcript is detected ina variety of tissues, including the nervous system, gut andvibrissae follicles and Dres10 is restrictively expressed in theperipheral nervous system, within sensory neurons of neural crestorigin (44). The fact that the expression patterns of Dres8 andDres10 are not conserved suggests that their functions have beenrecruited in other tissues for a novel specialized use.


Expression analysis and genetic programs

A systematic, high resolution expression analysis of develop-mentally regulated genes can also contribute to the definition ofcell types and stages and to the definition of the genetic programunderlying the development of complex structures, such as theeye. Among the collection of Dres genes analyzed in this study,several were found to be expressed in the neural retina. However,the spatial distribution of gene expression observed was diverse,in some cases showing layer-specific expression patterns and thepresence of boundaries of gene expression. Figure 3 shows thedifferent spatial distribution of six Dres cDNAs in the neuralretina at E12.5. Dres45, Dres65 and Dres129 are expressed alongthe major gradients of differentiation that proceed from the innerto the outer layers and from the center toward the periphery of theretina. In particular, while Dres65 and Dres129 transcripts areexclusively detected in the differentiated ganglionic cell layer,Dres45 is also expressed in some cells of the outer proliferativeneuroblast layer. These cells expressing Dres45 seem to bealigned in radial columns, in accordance with the radial pattern ofcellular differentiation of the retina. In contrast, Dres16 isuniformly expressed in the entire neural retina and Dres9 ismainly expressed in the outer proliferative neuroblast layer. Themost striking pattern of expression was observed for Dres93,which shows a domain of expression spatially restricted to theinfero-nasal portion of the retina. These few examples ofdifferential spatial distribution of gene expression in the neuralretina demonstrate how broad is the spectrum of genes involvedin the development of a single structure and how fine is the tuningof gene expression that needs to be unraveled.

Expression analysis and disease gene identification

Several genes responsible for human disease have already beenidentified through the screening of EST databases (11,45,46). Inthese cases, disease gene identification is usually based on theso-called positional candidate gene approach, which relies on theassociation of positional and functional data (47), i.e. theco-localization of a disease locus and a gene with a suggestivepathogenetic role in the disease phenotype. On the same basis,DRES can be considered potential candidate genes for humandiseases. Some DRES genes have already been found to beinvolved in human disorders, such as the RIEG gene (DRES112),a novel homeobox gene similar to the Drosophila orthodenticleand aristaless genes, found to be mutated in Rieger syndrome(48).

Even if there are examples in which the disease phenotypereflects only a subset of the tissues which express the gene, suchas the CBP gene mutated in Rubinstein–Taybi syndrome (49), theknowledge of gene expression patterns contributes significantlyto the positional candidate approach by pointing to a dysfunctionof a particular organ or tissue. For example, a promising‘positional candidate’ disease gene identified in this study isDres119, a fourth human homolog of the Drosophila eyes absentgene. In the mouse we found Dres119 to be expressed incraniofacial mesenchyme, external auditory meatus, dermomyo-tome, kidney and limbs. Its human counterpart, DRES119, mapsto chromosome 6q23, making it a suitable candidate foroculodentodigital syndrome (ODD), an autosomal dominantdisorder previously mapped to the same region and affectingdevelopment of the face, eyes, limbs and dentition (50). Another

candidate disease gene is Dres139, the homolog of the Drosophilakekkon gene, which may be involved in Cornelia de Langesyndrome, characterized by distinctive facial features such asmicrognathia, hirsutism, synophrys and bushy eyebrows, lowanterior hairline and small teeth, in association with growth andmental retardation, upper limb and digital defects (i.e. shorttapering fingers) and delayed skeletal maturation. The transcriptdistribution for Dres139 is highly restricted to developing cranialand thoracic bones, vibrissae and sinus hair follicles, tooth budsand in the tip of the digits (Fig. 1C–G). In addition, Dres139 mapsto mouse chromosome 3, within the human syntenic region for3q26–qter, where the locus for de Lange syndrome is located(MIM 122470).

A possible involvement of certain DRES genes in particulardisorders can also be hypothesized solely on the basis of theirexpression, especially if expressed in specific organs, such asDres61, Dres92 and Dres93. The Dres61 transcript, an Eag-likepotassium channel, could be detected exclusively in the primor-dium of the thyroid (Fig. 2A), suggesting an important role in thedevelopment of this organ and possibly in the pathogenesis ofthyroid dysgenesis. Dres92, homologous to the Drosophila geneCDS, is selectively expressed in the thymic rudiment at E12.5(Fig. 2B), while a novel homolog of empty spiracles, Dres93, hasa restricted domain of expression in the developing neural retina(Fig. 3C).

Expression analysis and mouse models

The data obtained from systematic high resolution expressionstudies will certainly add important biological information to thecreation and use of the preferred model for mammalian develop-ment, the laboratory mouse. Recently, there has been dramaticprogress in the efficiency of methods addressing gene function inmouse. Positional cloning of mouse mutant genes, induction ofnew mutants by chemical mutagenesis, targeted mutation byhomologous recombination, analysis of polygenic traits andcomparative gene mapping are some of the strategies being usedto study the phenotypic effects of mutation of specific murinegenes (51). A study of the spatio-temporal transcript distributionis the first requirement in order to fully comprehend which celltypes, developmental stages and cellular processes can be alteredby the mutation, as well as the compensatory effects that may bedue to expression of related genes.

Furthermore, systematic RNA in situ hybridization mayprovide very useful information in order to choose which mousegenes would be more interesting targets for gene inactivationstrategies. Recently, systematic targeted mutagenesis approacheshave made possible the creation and storage of several thousandES cell lines each carrying a knock-out of a different gene (52).Many of these genes are unknown. Therefore, knowing theexpression patterns of these genes would be of great value beforedeciding on the generation of null mutant mice.

Expression database

To store, group and search the data obtained in this initialexpression study and especially in light of the next phase of largescale data collection, we will deposit the complete expressionprofiles as edited panels of captured in situ images in the existingDRES database (20), accessible through a World Wide Webinterface (http://www.tigem.it/dros.html ). To effectively inte-grate the high resolution expression data with the extensive

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structural documentation on genes available in public reposi-tories, we are also planning to submit the expression profiles tothe comprehensive Mouse Gene Expression Information Re-source (MGEIR) through the mouse GXD Gene ExpressionDatabase, both of which are currently under development(53–55).

The comparative approach initiated with the DRES genes, instructural (sequence homology) and functional (expressionanalysis) terms, represents an effective strategy to obtain usefulinformation on gene function. Moreover, the results haveimportant implications regarding both mouse developmentalbiology and the evolutionary conservation of genes that orches-trate the development of the metazoan body plan. It is conceivablethat all metazoans have a set of core biochemical processes andthat duplicated mammalian pathways have adapted to specializedbiological functions. If high resolution expression studies areapplied on a large scale to ESTs and the information iscomprehensively collected in databases, it will then be possibleto scrutinize their sequence and/or expression patterns forparticular motifs. These data will provide an essential directoryto compare sequence similarities with temporal and spatialpatterns of gene expression. Ultimately, this information may beof considerable help in predicting clinical consequences of genedysfunction.

MATERIALS AND METHODS

Bioinformatics

EST entries, including homology data, were retrieved from dbEST(8). Computational analysis of EST sequences was performedusing the Blast basic local alignment search tool (56), either atTIGEMNet (http://www.tigem.it ) or at the National Center forBiotechnology Information (NCBI), National Library of Medicine(http://www.ncbi.nlm.nih.gov/Recipon/bs_seq.html ).

Murine cDNA clone retrieval and characterization

EST cDNA clones were obtained from IMAGE Consor-tium Clone Distributors (http://www-bio.llnl.gov/bbrp/image/idist_add.html/ ). Automated fluorescent DNA sequencing wasperformed using Perkin Elmer 377 Prism machines with both thedye terminator and dye primer cycle sequencing chemistries ondouble strand plasmid templates.

In situ hybridization

Expression was detected in tissue sections of E10.5, E12.5 andE17.5 mouse embryos using radioactive in situ hybridizationaccording to a published protocol (57). Embryos, embedded inparaffin, were fixed in 4% paraformaldehyde in phosphate-buffered saline. Sets of serial sections cut in three different planes(sagittal, coronal and transverse) covering the entire embryoswere hybridized with [35S]UTP-labeled antisense or senseriboprobes. The probes were transcribed from the 3′-untranslatedregion of the murine ESTs. Sense probes did not show anyspecific hybridization. Slides were counterstained in Hoechst33258 dye to stain the cell nuclei. The resulting red colorrepresented the hybridization signal.

The whole mount in situ hybridization experiments on E8.0,E9.5 and E10.5 mouse embryos were performed as previously

described using digoxygenin-labeled RNA antisense and senseprobes (58). Panels for Figures 1–4 were obtained by capturingand processing images using Nikon SMZ-U dissecting and ZiessAxiophot microscopes and Adobe Photoshop software.

ACKNOWLEDGEMENTS

We wish to thank Gyorgy Simon and Alessandro Guffanti forbioinformatic support, Massimo Zollo and the TIGEM Sequen-cing Core and Melissa Smith for preparation of this manuscript.We thank Dado Boncinelli, Anna Stornaiuolo, Giovanni Lavorg-na, Branca Dabovic, Massimo Zollo, Loris Bernard, GiorgioCasari, Annibale Puca and Cinzia Sala for support and sharingunpublished data. The financial support of the Italian TelethonFoundation (grant no. B.37), the Merck Genome ResearchInstitute (grant no. 37 to A.B.) and the EC (grant no.BMH4-CT97-2341 to A.B.) are gratefully acknowledged.

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