expression ofthenontransmembrane tyrosine phosphatase gene...

Vol. 4, 849-859, October 1993 Cell Growth & Differentiation 849

Expression of the Nontransmembrane TyrosinePhosphatase Gene erp during Mouse0 rganogenesis

Daniel Carrasco and Rodrigo Bravo1

Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical

Research Institute, Princeton, New Jersey 08543-4000

Abstrad

We have studied the expression of thenontransmembrane tyrosine phosphatase gene erpduring mouse development using in situ hybridizationanalysis. The results show that during the earlypostimplantational stages of development, erp expressionis observed only in maternally derived decidual cellssurrounding the developing embryo. At day 1 0.5, erp isweakly expressed in the embryo in the neural tube, hindgut, and other embryonic strudures. However, in1 2.5-day embryos, erp is present in most organs, withthe highest expression restrided to the developingneural system. During later development, at day 1 7.5,the levels of erp decline in some neural struduresbut remain high in others, like the dorsal root ganghia.High levels of erp expression are maintained in severalparts of adult brain, such as cortical layers, thalamus,hypothalamus, and hippocampus. High levels of erptranscripts are also observed in the cerebellar cortex, inthe Purkinje cell layer, and in the granular cell layer. Inall tissues analyzed, the expression of erp corresponds toregions undergoing terminal cell differentiation and/orregions where cell proliferation has declined.

Introdudion

Protein tyrosine phosphorylation is one ofthe major mecha-nisms of cellular signaling, and it is of vital importance foreukaryotic cell proliferation and differentiation (for a review,see Ref. 1). Cellular phosphotyrosine levels are regulated bythe activities of PTPs2 and PTKs. The PTPs comprise a familyof enzymes that can be categorized as transmembrane(CD45, LAR, PTP/3) and nontransmembrane (PTP 1 B, PTP i,TC-PTP), also referred to as receptor-like and nonreceptormolecules, respectively (2-6). Deregulated expression ofsome nontransmembrane tyrosine phosphatases has beenshown to affect cell growth and to increase the proportionof multinucleated cells, which, in part, may result from theirfailure to undergo cytokinesis (7, 8). Also, it has been shownthat Xenopus oocyte maturation induced by insulin is in-hibited by microinjection of purified PTP 1 B protein (9) and

Received 5/25/93; revised 7/29/93; accepted 8/3/93.

1 To whom requests for reprints should be addressed, at Department of Mo-

Iecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O.Box 4000, Princeton, NJ 08543-4000.2 The abbreviations used are: PTP, protein tyrosine phosphatase; PTK, proteintyrosine kinase; CNS, central nervous system; PNS, peripheral nervous sys-tem; SSC, standard saline citrate; DTT, dithiothreitol; PBS, phosphate bufferedsaline; cDNA, complementary DNA.

that the activity of a 37 kibodalton membrane associated PTPis enhanced in growth arrested fibroblasts (i 0), suggesting

that certain PTPs may function as negative regulators of cellproliferation. This is supported by the observations thatvanadate, an inhibitor of PTPs, can induce changes in cellmorphology similar to those detected in transformed cells(ii ), and by the fact that it can mimic the mitogenic effectsofseveral growth factors in intact cells (1 2, 1 3). Furthermore,it has been demonstrated that overexpression of a nontrans-membrane PTP can revert the transformed phenotype ofv-src and of human neu oncogene transformed cells (8). Onthe other hand, it has also been shown that overexpressionofa transmembrane PTP can have growth stimulatory effects(1 4); therefore, a fine balance between these activities must

exist in the cell to ensure proper growth control. The studies

of Howard et a/. (i 5) in Dictyoste/ium indicate that PTPs canalso play an important role during development.

The protein product of the mouse immediate early geneerp, also known as 3CHi34 (16, 17), has been shown toencode a nontransmembrane tyrosine phosphatase (i 7). Thederegulated expression of erp in NIH 3T3 cells reduces the

rate of cell proliferation, suggesting that it may play an im-portant role in the maintenance of the normal growth prop-erties of the cell (1 7). In order to better understand the physi-ological role of erp, we have studied its pattern of expressionby in situ hybridization during mouse development. The re-suIts show that erp is widely expressed throughout mousedevelopment, with the highest levels detected in maternaltissues in postimplantation embryos and in neural structuresduring late organogenesis. The expression of erp in the dif-ferent tissues analyzed corresponds to regions where termi-nal differentiation is ongoing or where cell proliferation hasstrongly declined.

Results

erp mRNA Is Present in the Developing Embryo andPlacenta

To determine whether erp is expressed during mouse em-

bryogenesis, total RNA from embryos of day 6.5 to day i 7.5of gestation and from placenta of 1 0.5 days were examined

by Northern blot analysis, using as a probe the full length erpcDNA. An erp mRNA of about 1 .8 kibobase pairs was de-tected in all of the tissues analyzed (Fig. 1). This mRNA wasobserved as early as in 6.5-day embryos and was expressedthroughout development until day 1 7.5. No significantchanges in expression levels were detected during this pe-nod. Furthermore, during the later stages of embryogenesis

(day 14.5 to 1 7.5), erp mRNA was detected at similar levelsin head, trunk, and limb sections. In placenta, erp mRNA isabundant at day 1 0.5; however, higher levels are seen at laterstages of development (data not shown).

Although Northern blot analysis indicates that the erpgene is expressed during embryonic development, it does

850 erp Expression Pattern during Organogenesis

�; is weakly expressed in the neural tube, hindgut, spinal cord,and other embryonic structures that later contribute to themature body.

erp Expression in the Developing Neural System

In situ hybridization with an erp probe performed againstsagittal, parasagittab, and transverse sections of whole em-bryos demonstrates that its transcripts are present in mostorgans. The areas of maximal erp expression correspond tothe developing neural system (Figs. 3 and 4). This is ilbus-trated by the pattern of erp hybridization in sagittal sectionsof 1 2.5- (Figs. 3, A and B) and i 4.5- (Fig. 3, D and F) dayembryos, where erp appears to be present in large parts ofthe developing CNS and PNS. In the CNS, the highest levelsof erp transcripts are detected along the spinal cord, teben-cephabon, mesencephabon, and myelencephabon (Fig. 3,A-F). In the PNS, high erp expression is detected in the dor-

sal root ganglia and the fifth cranial ganglion (Fig. 3, B andF). Additionally, erp hybridization is found within the form-ing neural retina (Fig. 3, A and B) and olfactory epithebium(Fig. 3, C and D), both components of the central nervoussystem. The transverse sections from a 1 3.5-day embryo,depicted in Fig. 4, highlight the presence of erp transcriptsin neural structures. In addition, Figs. 4, A and B, and SAshow that higher levels of erp transcripts are detected in theexternal layers of the developing telencephabon and mes-encephalon, but not in the most internal or the ventricularlayers of the still proliferating neuroepitheliab cells. The ep-endyma of the spinal cord does not display erp gene ex-pression (Figs. 4, C and D, and SB). During later develop-ment, the pattern of erp gene expression presents somedifferences from that already described. For example, in ai 7.5-day embryo, the bevels of erp transcripts seem to de-dine in some neural structures (Fig.3, G and H) but remainat high levels in others, like the dorsal root ganglia (Fig. 9,A and B). During the same time, other tissues in the embryo,such as the muscles ofthe neck and tongue, show high levelsof erp transcripts (Fig. 3, G and H).

erp Expression Is Maintained in the Adult Brain

The above observations prompted us to investigate whethererp expression is sustained in the adult brain. in situ hy-bridization experiments confirmed thatexpression ofthe erpgene is prolonged in the adult brain and that its mRNA isfound in various structures of the forebrain. A coronal sec-tion though the rostral region of the forebrain demonstratesthe presence of erp transcripts throughout the cortical layers,thabamus, hypothalamus, and hippocampus (Fig. 6, A andB). Regions of strongest hybridization include the cerebralcortex, thalamic nuclei, and the arcuate nucleus of the hy-pothalamus. A sagittal brain section shows that erp tran-scripts are also detected at very high levels in the cerebeb barcortex (Fig. 6, C and D). However, in contrast from its ap-parent generalized expression within the brain, erp tran-scripts are very low or undetectable in the corpus calbosumand in the dentate gyrus (Fig. 6, A-D), indicating cell typespecific expression of erp. This is underlined by the obser-vation that a sagittal serial section hybridized with c-jun an-tisense riboprobe shows strong hybridization in the dentategyrus (Fig. 6F), where erp expression is absent (Fig. 6D). Therestricted expression of erp in the adult brain is also evidentafter observation at higher magnification ofthe cerebral (Fig.7, A and B) and cerebellar cortex (Fig. 7, C and D). In the

cerebral cortex, erp transcripts are distributed in a gradient

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Fig. 1. Northern blot analysis of erp expression in placenta and duringmouse development. Approximately 20 pg of total RNA of the embryonic

stages indicated were hybridized with the complete nick translated erpcDNA.F, embryo; H, head; L, limb and tails; P, placenta; T, trunk. Glyceraldehyde

3-phosphate dehydrogenase (GAPDH( was used as a control.

not give any insight into the tissue specificity. Therefore, wehave further investigated erp expression by in situ hybrid-ization.

erp Gene Expression during Early EmbryonicDevelopment

Serial cryostat sections prepared at different stages of em-bryogenesis were hybridized with erp sense or antisenseriboprobes. Only antisense erp riboprobes could be de-tected after 3 to 4 weeks exposure to photographic emulsion

as specific labeling above background in all tissues exam-med. During early postimplantation stages of development,

expression of erp is observed only in maternally derived de-cidual cells surrounding the developing embryo. This can beseen in Fig. 2, A and B, where a section of a 6.5-day embryoembedded in the decidua and hybridized with erp antisenseriboprobe is shown. The maternal decidua that immediatelysurrounds the embryo and which is believed to be under-going differentiation (18), shows the highest levels of erpexpression, whereas the more peripheral, and still mitotic,decidual cells exhibit bower levels of erp mRNA. No erptranscripts are detected in the embryo itself nor in embryonicderived tissues, bike the ectoplacentab cone or the extraem-bryonic ectoderm (Fig. 2, A to C, and data not shown). Theerp mRNA detected in the 6.5-day embryo by Northern blotanalysis most probably corresponds to contaminating de-ciduab cells carried during the process of embryonic dissec-tion. By 1 0.5 days of gestation, the ectoplacental cone andchorionic ectoderm have fused with the abbantois to generatethe chorioablantoic placenta. As shown in Fig. 2, D and F,erp transcripts are still most abundant in maternally derivedtissues. erp expression is strong in mesenchymab cells form-ing the vascular zone near the central artery of the maternalpart of the placenta. Examination at higher magnification ofthis region revealed that the strongest signal originated fromendothelial cells (data not shown). In our studies, erp geneexpression could first be detected in a 1 0.5-day embryo sec-tion. At this stage, erp transcripts are present in elements ofthe embryonic placenta and in the embryo itself (Fig. 2, Dand F). In the embryonic placenta, erp transcripts are con-fined to some cells of the mural trophoblast giant cell layerand to the labyrinthine trophobbast. Other embryonic de-rived tissues such as the yolk sac and the amnion are devoidof erp gene expression. In the embryo itself, at day 1 0.5, erp

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Fig. 2. Upperpanel, 6.5-day embryos; lowerpanel, 10.5-day embryos. Embryos were sectioned within their decidua and show the distribution of erp transcripts

detected by in situ hybridization. Sections were hybridized with antisense (B and E) or sense (C and F) erp riboprobes and photographed under dark-fieldillumination. A and 0, the tissue sections of B and E, respectively, after hematoxylin staining and photographed under bright-field illumination. am, amnion; e,

embryo; epc, ectoplacental cone; hg, hindgut; Ia, labyrinthine trophoblast; mca, maternal central artery; md, maternal decidua; nt, neural tube; sc, spinal cord;

tgc, trophoblastic giant cell; ys, yolk sac. Microphotographs were taken at 20X (A-C) and 12X (0-F) magnification.

Cell Growth & Differentiation 851

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with the highest bevels in the occipital region. In the cer-ebebbar cortex, high transcriptional levels are observed in thePurkinje and in the granular cell layers, whereas hybridiza-tion to other cerebellar neurons is not detectable. The dra-matic difference in erp expression between layer VI of thecerebral cortex and the corpus calbosum is illustrated inFig. 8A.

To further confirm the expression oferp in the Purkinje celllayer in the cerebellum, immunohistochemicab staining wasperformed using rabbit polycbonal antibodies raised againsta peptide containing amino acids 219 to 230 of the ERPprotein.As shown in Fig.8, B and C, ERP staining isdetectedin both the Purkinje cells and the granular cell layer, thesame places where erp transcripts are found. Interestingly,

the staining is homogeneously distributed throughout thecell body, and no association with the cell membrane orother particulate fractions is observed. The activity of theanti-ERP antibody is blocked bythe immunizing ERP peptide(data not shown).

erp Expression in the Developing Axial andLimb Skeleton

A population of cells derived from the somatic mesoderm,the sclerotomes, differentiates into mesenchymab cell typesthat eventually form the vertebral column and ribs. Duringadvanced organogenesis, the scberotomes condense intoprevertebrae, and chondrogenesis of the vertebral body de-

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Fig. 3. In situ localization of erp

transcripts in 12.5- (A-C), 14.5-(0-F), and 17.5- (C-I) day em-

bryos. Cryosections were hybrid-ized with antisense (B, E, and H) orsense (C, F, and I) riboprobes. Ana-

tomical sites are indicated as a ref-

erence: cI, clavicule; h, heart; id,intervertebral disc; Ii, liver; lu,lung; m, myotome; me, mesen-

cephalon; mu, muscle; my, myel-encephalon; nc, nasal chamber; r,retina; s, sclerotome; sc, spinal

cord; sg, spinal ganglia; te, telen-

cephalon; tg, trigeminal ganglia;ye, vertebra. Microphotographs

were taken at 8X (A-F), and 4X

(C-I) magnification.

velops in an anteroposterior order. In the lumbar vertebralcolumn of a 1 2.5-day embryo, the sclerotomes still appear

as homogeneous blocks, and, as shown in Fig. 3, A and B,they display low levels of erp transcripts during this time. Atanterior positions, where axial development is more ad-

. vanced, vertebral and intervertebral discs form at this stage.In contrast to the thoracic intervertebral discs, which expresshigh levels of erp transcripts, the forming vertebral bodyshows no erpexpression (Fig. 3, A and B). By day 14.5, when

chondrogenesis is well under way along the vertebral cob-umn, it is evident that erp transcripts are expressed at sig-nificant levels in the densely packed cells of the interverte-bral tissues (Figs. 3, D and F, and SC). As these cells alternatewith the more loosely packed cells which later give rise tothe vertebrae, the developing backbone shows a periodic orstriped pattern of expression of erp throughout its length,extending from the thoracic to the tail vertebrae. In otherchondrification centers such as the sternum, the nasal cham-

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Fig. 4. In Situ expression analysis of erp at day 1 3.5 ofgestation. A, C, and F, bright-field images ofthe sections; B, 0, and F, the corresponding dark-field images.

G, schematic representation ofa 1 3.5-day embryo. Lines a, b, and c, the levels ofthe coronal sections shown in A, C, and F, respectively. aq, aqueduct; el, ependymallayer; h, heart; Ii, liver; Iv, lateral ventricle; me, mesencephalon; r, radius; s, sclerotome; Sc, spinal cord; sg, spinal ganglia; te, telencephalon. Microphotographs

were taken at lOx (A and B), 12x (Cand D(, and 14x (Eand F) magnification.

bers, and the growth plate of long bones, erp expression wasnot detected (Figs. 3, D-H; 4, Cand D; and data not shown).The above observations suggest that erpexpression is linkedto the development of structures which connect and sur-round prospective bones, bike the joints. In support of thisinterpretation, we detected erp mRNA in other structures, inaddition to intervertebral tissues, in which this process isoccurring. For example, erp transcripts were seen in the con-necting tissue ofbong bones such as the radius in the forelimb(Figs. 4, C and D; SD; and data not shown).

Other Sites of erp Expression

TheLiver. Between days 10.5 and 14.5 ofembryonic life,erp expression can be detected at significant bevels in thefetal liver (Figs. 3, D and F; 4, Cand D; and data not shown).Most cells ofthe developing liver were labeled, although theintensity of the hybridization signal was heterogeneouslydistributed. The endodermal prehepatocytes were appar-ently more strongly labeled than the mesodermab hemato-poietic precursor cells within the blood forming islands ofthe embryonic liver. A rapid decline of erp expression oc-curred after day 1 4.5, and almost no erp mRNA was detectedby day 1 7.5 (Fig. 3, G and H). This decrease in erp gene

Fig. 5. erp expression in neural and bone structures. A and B, higher mag-nification of the telencephalon and spinal cord shown in Fig. 4, B and F,respectively. C and 0, higher magnification of the backbone and forelimbshown in Figs. 3Eand 4D, respectively. ct, connecting tissue; el, ependymal

layer; id, intervertebral disc; h, humerus; lv, lateral ventricle; r, radius; sc,

spinal cord; te, telencephalon; ye, vertebrae. Microphotographs were takenat 25X (A, B, and 0), and 50x (C) magnification.


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Fig. 6. erp expression in the adult brain. Coronal (A and B) and sagittal (C and 0) adult brain sections were hybridized with antisense erp riboprobes and

photographed under bright- (A and C) and dark-field illumination (B and 0). A serial sagittal section was hybridized with a c-jun antisense riboprobe andphotographed under bright-field (El and dark-field (F) illumination for comparison. arc, arcuate hypothalamic nucleus; cb, cerebellum; cx, cortex; dg, dentategyrus; h, hippocampus; pir, piriform cortex; t, thalamus. Microphotographs were taken at 7x magnification.

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expression coincides with the further differentiation of pre-hepatocytes, which is characterized by morphologicalchanges of the cells from steblate to polygonal, and with aconcomitant expansion of the endoplasmic reticulum andincreased glycogen storage (i 9).

The Intestine. By day 1 7.5 of mouse development, thetypical structures of the intestine, the finger-bike intestinalvilli, and thetubular invaginations called crypts have alreadyformed. At this stage of development, erp transcripts are de-tected in the differentiated cells covering the villi, whereasthe proliferating epitheliab cells within the intestinal cryptsdo not express this transcript (Fig. 9, C and D).

The Thymus. The embryonic thymus contains a hetero-geneousby distributed population of stromal cells and 1-cellprecursors at various stages of maturation. By day i 5 of fetallife, the thymus begins to show corticomedullary differen-tiation, and after day i 7.5, the cortex, the largest region ofthe thymus, can be identified as a dark staining region tightlypacked with small thymocytes, whereas the medulla can beidentified as a paler staining central region more looselypacked with slightly larger thymocytes. Hybridization of ai 7.5-day embryo with an erp probe clearly demonstratesthat the expression of erp is confined to the thymic cortex(Fig. 9, Eand F).

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Fig. 7. erp expression in brain cortex. Pairs A, B and C, 0 correspond to enlargements of A, B and C, 0 in Fig. 5, showing erp expression in the cerebral andcerebellar cortex, respectively. I, II, Ill, IV, V, and VI, the different neuronal layers in the cerebral cortex. CA 1, CA2, and CA3, the different fields of Ammon’shorn in the hippocampus. cc, corpus callosum; dg, dentate gyrus; gI, granular cell layer; ml, molecular cell layer; pur, Purkinje cell layer; wm, white matter.

Microphotographs were taken at 1 8x magnification.


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The Nasal Chamber. By day 1 4.5, erp expression wasclearly detectable in the olfactory pits (Fig. 3, D and F). Atday 1 7.5 (Fig. 3, G and H) and in newborn animals (Fig. 9,C and H), the nasal gland was strongly labeled, and mod-erate levels of erp mRNA were detected in the olfactory epi-thelia. The nasal cartilage was devoid of erp expression.

The Spleen. The spleen displays a complex pattern of he-matopoietic activity in which myebopoiesis and erythropoi-

esis occur during late embryogenesis, whereas lymphopoi-esis takes place after birth upon arrival of the bymphoidprecursors (20). No erp mRNA was detected in the embry-onic spleen (data not shown). However, hybridization of aspleen section prepared from a 5-week adult mouse revealedmoderate levels of erp transcripts in the white pulp, the sitewhere lymphoid cells reside in this organ (Fig. 9, I and j).

erp transcripts were not detected in the red pulp or in themarginal zone.

Discussion

erp is an immediate early gene which encodes a nontrans-membrane member of the phosphotyrosine phosphatasegene family. Protein tyrosine phosphorybation/dephosphor-ylation has been widely accepted as a major mechanism ofcellular signaling (1-6); however, the physiological role, aswell as the regulation and target specificity of PTPs, is stillpoorly understood. To gain insight into the potential bio-logical robe of ERP, we have examined the expression patternof the erp gene during mouse development.

erp Expression during Embryonic Neural Development.The expression pattern of PIP family members varies widelyin different adult tissues that have been examined by North-em blot analysis. For example, two transmembrane PTPshave been found ubiquitously in all tissues and cells tested(21 , 22). Other transmembrane PTPs are expressed only incells of hematopoietic lineage (23) or predominantly in thebrain (24), suggesting a more cell type specific function.

Some nontransmembrane PTPs have also been identified ina limited number of tissues and cell lines (25, 26).

With the exception of Syp, which encodes a nontrans-membrane SH2 containing phosphotyrosine phosphatase

(27), little is known about the expression pattern of otherphosphotyrosine phosphatase family members during mam-mabian embryonic development. Syp mRNA was found to bebroadly distributed in both embryonic and extraembryonictissues and in maternal tissues. Expression of Syp transcriptsis widespread in all of the embryonic stages examined

(between 7.5 and 15.5 days), with small variations in theamount of expression among different tissues.

In this study, we show that the expression ofthe erp gene,which encodes a nontransmembrane PTP, is devebopmen-tally regulated and predominantly distributed in tissues ofneuroectodermab origin. The highest levels of erp transcriptsare detected in neural structures during mouse embryogen-esis, and this expression is sustained in the adult brain. Earlyafter implantation, erp is expressed in the maternal placentabut not in tissues of embryonic origin. Low bevels of erp

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Fig. 8. A, in siti.i hybridization localization of erp mRNA in the cerebral

cortex. Enlargement of the interline between the sixth cortical layer and thecorpus callosum. B and C, immunohistochemical localization of ERP protein

in the cerebellar cortex. Different magnifications ofthe cerebellar region froman adult brain section stained with anti-ERP antibody are shown. cc, corpuscallosum; gc, granular cell layer; pur, Purkinje cell layer; VI, sixth cerebrallayer. Microphotographs were taken at lox (A), lOOX (B), and 250x (C)magnification.

transcripts first appear in the embryo at 10.5 days and al-ready at multiple sites, with the highest levels detected in thedeveloping neural system. In this study, we have observedsignificant erp expression in cells of the CNS (brain and spi-nab cord) and PNS, including cranial and spinal ganglia atthe later stages here investigated (1 2.5-, 14.5-, and 1 7.5-dayembryos).

A large part ofthe mammalian central and peripheral ner-vous system development does not start until midgestation,and some processes of neural development, such as my-elination and dendritic cell elaboration, occur largely duringthe prenatal and early postnatal period (28, 29). Our findingsindicate that erp is present at the inception ofthis final periodof neural system maturation. Moreover, erp transcripts aredetected in the adult brain, in postmitotic, terminally dif-ferentiated cells, such as the pyramidal cell layer in the hip-

pocampus, cerebral cortex, and Purkinje and granular celllayers in the cerebellum, suggesting that the ERP protein

does not function primarily in a mitogenic pathway.The level ofcellular phosphotyrosine is determined by the

relative activities of protein tyrosine kinases and protein ty-rosine phosphatases. The physiological substrates for most

of the tyrosine phosphatases, including ERP, are still un-

known. We can speculate that the coexpression of erp with

members of the tyrosine kinase gene family in a particulartissue may indicate that both enzymes share common sub-

strates. Interestingly, the expression of several genes encod-ing phosphotyrosine kinases overlaps in some degree with

that observed for erp. Among the receptor tyrosine kinases,the products ofthe trkfamily ofgenes display a very specific

pattern ofexpression in the developing and adult neural sys-

tem (30-32). c-kitand c-fms are both expressed in maternalplacenta; however, transcripts of c-fms tend to surround the

embryo, and in contrast to c-kit, they are not expressed in theembryo proper even at later stages of development (33, 34).

At least three nonreceptor PTK genes, c-src, fyn, and c-yes,

are expressed preferentially, although not exclusively, in thenervous system (35-38). Moreover, c-yes, bike erp, is ex-

pressed in cerebellar Purkinje cells (38).

Cellular Differentiation and erp Expression. A number of

oncogenes encode PTKs and transform by virtue of elevatingtyrosine phosphorylation. Because PTPs catalyze the reversereaction, it is postulated that these molecules are also in-volved in the control ofcelb proliferation. Evidence indicates

that PTPs can have a negative or a positive effect in cellproliferation (2-4). In particular, we have previously shown

that the deregulated expression of ERP in NIH 313 cells hasa negativeeffecton cell proliferation (1 7). Thework reported

here allows this notion to be examined within the context of

the developing embryo and placental precursors. Our results

indicate that high expression of erp transcripts in the embryocorrelates well with areas undergoing terminal cell differ-

entiation and/or regions where cell proliferation has sign ifi-cantly decreased. erp is expressed at very low or undetect-

able levels in known sitesofcelb proliferation, indicating thatthe erp product does not function primarily in a mitogenic

pathway. In postimplantation embryos, a differential expres-sion of erp in the maternal decidua was observed. The layerof maternal decidua that immediately surrounds the con-

ceptus, which is believed to be undergoing differentiation(1 8), shows the highest levels of erp expression, whereas the

more peripheral region, containing actively dividing de-

cidual cells, exhibits little erpexpression. At 6.5 days of ges-tation, erp mRNA is absent throughout the conceptus in-

cluding the most highly proliferative tissue of the embryo,

the primitive ectoderm (39). erp is expressed abundantly in

primary giant cells, which are invasive but are not prolifer-ating. Primary giant cells derive from the trophectoderm andmay duplicate their genomes by up to 500-fold without un-

dergoing cellular division (40). These data are particularlyinteresting in lightoftheobservation thaterptransfected NIH

313 cells are significantly larger than the parental cell line

and in general are mubtinucleated (1 7).erp expression was also undetectable in other sites of cel-

lular proliferation bike the ventricular zone of the telen-

cephabon, the basal layer of the skin, and crypts in the in-testine. The strong correlation of erp expression inpostmitotic cells suggests a role for this gene in cellular dif-ferentiation and/or as a negative effector of cell growth.

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k

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Fig. 9. erpexpression in different

mouse tissues. The bright-field (A,C, F, C, and I) and the correspond-

ing dark-field photomicrographs(B, 0, F, H, and I) are shown. A to

Fcorrespond to sections from a 1 7.

5-day embryo hybridized with an

erp antisense riboprobe and over-exposed in order to see low ormoderate levels of erp expression.

The three selected tissues are: dor-sal root ganglia (A and B); intestine

(Cand 0); and thymus (Eand F).C and H, sagittal section of thehead of a newborn animal hybrid-

ized with erp antisense riboprobe.I and I, sagittal spleen section pre-pared from a 5-week adult mouse

and hybridized with erp antisense

riboprobe. c, cartilage; co, cortex;

me, medulla; ng, nasal gland; oe,

olfactory epithelium; rp, red pulp;

Sg, spinal ganglia; ye, vertebra; vi,

villi; wp, white pulp. Microphoto-

graphs were taken at 18x (A andB), sox (Cand D(, 25x (Eand F),14x (C and H), and 7x (I and))magnification.

A,-.�‘

ftflIL�.1d� I

� ‘

� .

\� .&�.


Materials and Methods

RNA Extradion and Northern Blot Analysis

Embryos were obtained from natural matings of B6C3F1mice. The time of pregnancy was established by the pres-ence of a vaginal plug and was regarded as day 0.5 ofgestation.

Mouse embryos were dissected under the microscope,frozen in liquid nitrogen, and stored at -70#{176}Cuntil used.Total RNA was extracted using RNAzol (Cinna/Biotecx) fob-lowing the manufacturer’s recommendations. For Northernbbotanalysis, 20 pgoftotal RNAwere run in 1% agarose gelscontaining 6% formaldehyde (41 ) and blotted onto a Gene-Screen Plus membrane (NEN-DuPont). The purified insert ofthe erp clone was labeled by nick translation (42) to a spe-cific activity of 1 to 5 x i 08 cpm/pg. The hybridization wascarried out in 50% formamide, 0.5% sodium dodecyl sub-fate, 5 X SSC (1 x SSC = 1 50 mM NaCI and 15 ms.i sodiumcitrate), and 5X Denhardt’s solution at 42#{176}Cfor 48 h. Filterswere extensively washed in 0.1 x SSC containing 0.5% so-dium dodecyl sulfate at 60#{176}C.

Tissue Preparation

For in Situ Hybridization. After dissection, the embryosor tissues were transferred to O.C.T. embedding medium(Miles Laboratories) and then mounted on a cryostat speci-men holder. The embedded samples were frozen by immers-ing them in liquid nitrogen. Samples were kept in the cryostat

at -18#{176}Cfor at least 1 h to equilibrate to the appropriatetemperature before sectioning. Cryostat sections of 8- to1 Ojjm thickness were collected on polyionic slides (Super-frost plus), air dried at room temperature for 30 mm, and thenstored at -70#{176}Cuntil used.

For Immunocytochemistry. Following d issection, tissueswerefixed overnight in modified Bouin’s solution (0.9% pic-nc acid, 5% acetic acid, and 9% v/v formaldehyde), dehy-drated in increasing concentrations of ethanol, and embed-ded in paraffin wax (Fisher Scientific Co.). Floating sectionsof 5 to 7 pm were air dried for 30 mm on polyionic slidesat room temperature and then stored at 4#{176}Cprior to use.

In Situ Hybridization

A murine cDNA fragment of erp, positions 1 to 390, wascloned into Bluescript KS+. For in situ hybridization, senseand antisense [35S]UTP labeled probes were prepared fromlinearized template DNAs using T3 or T7 RNA polymerases(Promega). Probes were degraded to an average size of 200nucleotides by limited alkaline hydrolysis. After removal ofunincorporated nucleotides with a Sephadex G-50 column(Pharmacia), the labeled probes were stored at -80#{176}Cin asolution containing i 0 mwi DTT and 1 �ig/ml yeast tRNA untilused. in situ hybridization was performed essentially as pre-viously described (43) with minor modifications. Briefly, thetissue sections were fixed in freshly prepared 4% paraform-aldehyde at 4#{176}Cfor i h and then treated with Proteinase K(0.1 �jWml). After washing in PBS, the sections were fixedagain in 4% paraformaldehyde, acetylated, dehydrated, andair dried. Just prior to hybridization, the labeled probes werediluted with hybridization buffer containing 50% deionizedformamide, 1 0% dextran sulfate, i 0 mt�i DTT, 0.3 M NaCI, 20mM Tris-HCI (pH 7.4), 10 m�i NaH2PO4 (pH 8.0), 2 mt�iEDTA, 1 x Denhardt’s solution, and 0.5 mg/mI yeast tRNA,to approximately 4 X 10� cpm/pl. After heating at 80#{176}Cfor2 mm, 25 to 50 p1 of the solution were applied to eachsection and covered with a siliconized coverslip. Hybrid-

ization was performed in a humid chamber at 52#{176}Cfor 16h. Sections were then washed in 5X SSC containing 10 ms�DII for 30 mm at 52#{176}Cfollowed by an additional 30-mmwash at 65#{176}Cin 50% formamide, 2 x SSC, and 1 0 m� DTT.The slides were then treated with RNase A (20 �jg/ml) andRNase Ti (2 �iWml) for 1 h at 37#{176}C,followed by a 3-h in-cubation in 50% formamide, 2 x SSC, and 10 m�i DTT, andthen washed for 1 5 mm in 0.1 x SSC at 37#{176}C.After dehy-dration in graded (35 to 1 00%) ethanol solutions containing0.3 M ammonium acetate, the slides were air dried, dippedinto NTB-2 nuclear track emulsion (IBI; Kodak) and exposedfor 3 to 4 weeks at 4#{176}C.After developing, the samples weredehydrated, mounted with coverslips, and microphoto-graphed under dark-field. For bright-field photography, sec-tions were stained with hematoxylmn.

Immunohistochemistry

The procedure used was a modified version of the methodreported by Heine et a!. (44). Prior to immunostaining, sec-tions were deparaffinized and then submerged in a solutionofmethanol:hydrogen peroxide (9:1) for 10 mm. After wash-ing the slides with PBS for 1 0 mm, they were treated withsaponin (0.5 mg/mI in PBS) for 30 mm and subsequentlywashed three times with PBS for 5 mm. Tissue sections wereincubated overnight at 4#{176}Cwith a rabbit polyclonal antibodyraised against a peptide containing amino acids 219 to 230of ERP at a concentration of 5 pg/mI (diluted in 1 0% non-immune goat serum). Control slides were incubated witheither an IgG fraction of preimmune rabbit serum at 5 pg/mI(diluted in 1 0% nonimmune goat serum) or without primaryantibody. Slides were then extensively washed in PBS andincubated for 1 h at room temperature with biotinylated goatanti-rabbit antibody (1 :50; DAKO). After washing in PBS, thesections were exposed to streptavidin-peroxidase complex(Zymed Laboratories) for 1 0 mm at room temperature andwashed again in PBS. Samples were then treated with ami-noethyl carbazole with 0.03% v/v hydrogen peroxide for 5to 10 mm and counterstained with hematoxylmn.

Acknowledgments

We thank R. Metz, H. Macdonald-Bravo, R. Smeyne, S. Lira, and M. Grudafor their valuable comments.

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