Jennifer J. Swiergiel

Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.

Lumican is one of the major keratan sulfate proteoglycans (KSPG) in vertebrate corneas. We previously cloned the murine lumican cDNA. This study determines the structure of murine lumican gene (Lum) and its expression during mouse embryonic developments. The mouse lumican gene was isolated from a bacterial artificial chromosome mouse genomic DNA library and characterized by polymerase chain reaction and Southern hybridization. The lumican gene spans 6.9 kilobase pairs of mouse genome. The gene consists of three exons and two introns. Exon 1 constitutes 88 bases (b) of untranslated sequence. Exon 2 is 883 b and contains most of the coding sequence of lumican mRNA, and exon 3 has 152 b of coding sequence and 659 b of 3′ noncoding sequence. The mouse lumican gene has a TATCA element, a presumptive TATA box, which locates 27 b 5′-upstream from the transcription initiation site. Northern hybridization and in situ hybridization indicate that in early stages of embryonic development, day 7 post coitus the embryo expresses little or no lumican. Thereafter, different levels of lumican mRNA can be detected in various organ systems, such as cornea stroma, dermis, cartilage, heart, lung, and kidney. The cornea and heart are the two tissues that have the highest expression in adult. Immunoblotting studies found that KSPG core proteins became abundant in the cornea and sclera by postnatal day 10 but that sulfated KSPG could not be detected until after the eyes open. These results indicate that lumican is widely distributed in most interstitial connective tissues. The modification of lumican with keratan sulfates in cornea is concurrent with eye opening and may contribute to corneal transparency. Lumican is one of the major keratan sulfate proteoglycans (KSPG) in vertebrate corneas. We previously cloned the murine lumican cDNA. This study determines the structure of murine lumican gene (Lum) and its expression during mouse embryonic developments. The mouse lumican gene was isolated from a bacterial artificial chromosome mouse genomic DNA library and characterized by polymerase chain reaction and Southern hybridization. The lumican gene spans 6.9 kilobase pairs of mouse genome. The gene consists of three exons and two introns. Exon 1 constitutes 88 bases (b) of untranslated sequence. Exon 2 is 883 b and contains most of the coding sequence of lumican mRNA, and exon 3 has 152 b of coding sequence and 659 b of 3′ noncoding sequence. The mouse lumican gene has a TATCA element, a presumptive TATA box, which locates 27 b 5′-upstream from the transcription initiation site. Northern hybridization and in situ hybridization indicate that in early stages of embryonic development, day 7 post coitus the embryo expresses little or no lumican. Thereafter, different levels of lumican mRNA can be detected in various organ systems, such as cornea stroma, dermis, cartilage, heart, lung, and kidney. The cornea and heart are the two tissues that have the highest expression in adult. Immunoblotting studies found that KSPG core proteins became abundant in the cornea and sclera by postnatal day 10 but that sulfated KSPG could not be detected until after the eyes open. These results indicate that lumican is widely distributed in most interstitial connective tissues. The modification of lumican with keratan sulfates in cornea is concurrent with eye opening and may contribute to corneal transparency. Corneal strength and transparency depend upon the development and maintenance of an organized extracellular matrix, including uniformly small diameter collagen fibrils with lamellae of consistent interfibrillar spacing. The collagen fibrils of adjacent lamella sheets are perpendicular to one another (1Linsenmayer T.F. Fitch J.M. Birk D.E. Ann. N. Y. Acad. Sci. 1990; 580: 143-160Crossref PubMed Scopus (72) Google Scholar, 2Hay E.D. Int. Rev. Cytol. 1980; 63: 263-322Crossref PubMed Scopus (295) Google Scholar). The mechanism that governs the formation of collagen lamellae in cornea stroma is not well understood. It has been suggested, however, that the ratios of different collagen types in making up the fibrillar corneal collagen and other extracellular specialized matrix components, e.g.proteoglycans and glycoprotein are essential for the development of a transparent cornea (1Linsenmayer T.F. Fitch J.M. Birk D.E. Ann. N. Y. Acad. Sci. 1990; 580: 143-160Crossref PubMed Scopus (72) Google Scholar, 3Hassell J.R. Cintron C. Kublin C. Newsome D.A. Arch. Biochem. Biophys. 1983; 222: 362-369Crossref PubMed Scopus (176) Google Scholar, 4Linsenmayer T.F. Gibney E. Igoe F. Gordon M.K. Fitch J.M. Fessler L.I. Birk D.E. J. Cell Biol. 1993; 121: 1181-1189Crossref PubMed Scopus (247) Google Scholar, 5Hahn R.A. Birk D.E. Development. 1992; 115: 383-393PubMed Google Scholar, 6McLaughlin J.S. Linsenmayer T.F. Birk D.E. J. Cell Sci. 1989; 94: 371-379PubMed Google Scholar, 7Marchant J.K. Hahn R.A. Linsenmayer T.F. Birk D.E. J. Cell Biol. 1996; 135: 1415-1426Crossref PubMed Scopus (101) Google Scholar, 8Ruggiero F. Burillon C. Garrone R. Invest. Ophthalmol. Vis. Sci. 1996; 37: 1749-1760PubMed Google Scholar). In addition to interaction with collagen fibrils, proteoglycans in stroma also play a role in corneal hydration due to their high negative charge of sulfated carbohydrate moieties (9Rawe I.M. Tuft S.J. Meek K.M. Histochem. J. 1992; 24: 311-318Crossref PubMed Scopus (30) Google Scholar, 10Bettelheim F.A. Plessy B. Biochim. Biophys. Acta. 1975; 381: 203-214Crossref PubMed Scopus (92) Google Scholar, 11Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. Biochem. Soc. Trans. 1991; 19: 871-876Crossref PubMed Scopus (48) Google Scholar). The hydrophilic properties of the stroma result from stromal proteoglycans that constitute the second most abundant biological materials in stroma, after collagen (12Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. J. Biol. Chem. 1991; 266: 14226-14231Abstract Full Text PDF PubMed Google Scholar, 13Funderburgh J.L. Conrad G.W. J. Biol. Chem. 1990; 265: 8297-8303Abstract Full Text PDF PubMed Google Scholar). The keratan sulfate proteoglycans (KSPGs) 1The abbreviations used are: KSPG, keratan sulfate proteoglycan; KS, keratan sulfate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PC, post coitus; SLRP, small leucine-rich proteoglycan; kb, kilobase pair(s); b, base(s); bp, base pair. are uniquely abundant in the cornea, constituting the major proteoglycans of the corneal stroma. Currently, three corneal KSPG core proteins have been identified, i.e. keratocan, lumican, and mimican (osteoglycin), which were previously designated 37A, 37B, and 25, respectively (13Funderburgh J.L. Conrad G.W. J. Biol. Chem. 1990; 265: 8297-8303Abstract Full Text PDF PubMed Google Scholar, 14Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 15Funderburgh J.L. Corpuz L.M. Roty M.R. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1996; 37 (abstr.): S645PubMed Google Scholar, 16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar, 17Grover J. Chen X.-N. Korenberg J.R. Roughley P.J. J. Biol. Chem. 1995; 270: 21942-21949Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 18Funderburgh J.L. Funderburgh M.L. Brown S.J. Vergnes J.P. Hassell J.R. Mann M.M. Conrad G.W. J. Biol. Chem. 1993; 268: 11874-11880Abstract Full Text PDF PubMed Google Scholar). These proteins are structurally and antigenically related, and each bears from one to threeN-linked keratan sulfate chains in addition to several nonsulfated oligosaccharides (14Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 15Funderburgh J.L. Corpuz L.M. Roty M.R. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1996; 37 (abstr.): S645PubMed Google Scholar, 18Funderburgh J.L. Funderburgh M.L. Brown S.J. Vergnes J.P. Hassell J.R. Mann M.M. Conrad G.W. J. Biol. Chem. 1993; 268: 11874-11880Abstract Full Text PDF PubMed Google Scholar). Lumican belongs to the family of small leucine-rich proteoglycans (SLRPs) that includes decorin, biglycan, fibromodulin, keratocan, epiphycan, and osteoglycin (20Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar). Each of these proteoglycans possesses 6–10 leucine-rich repeating units between the flanking cysteine-rich disulfide-bonded domains at the N and C termini of the core protein. The presence of a common structural motif implies that these proteoglycans may share common functional properties. Such a common function is thought to be the interaction with fibrillar collagen. The tissue distributions of each proteoglycan are distinct; therefore, it is likely that each family member fulfills a different role in connective tissues (20Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar, 21Kresse H. Hausser H. Schonherr E. Exper. Suppl. (Basel). 1994; 70: 73-100PubMed Google Scholar, 22Scott J.E. Biochemistry. 1996; 35: 8795-8799Crossref PubMed Scopus (215) Google Scholar). For example, lumican only exists as a proteoglycan in cornea, it is a glycoprotein in the rest of connective tissues (11Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. Biochem. Soc. Trans. 1991; 19: 871-876Crossref PubMed Scopus (48) Google Scholar, 14Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 15Funderburgh J.L. Corpuz L.M. Roty M.R. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1996; 37 (abstr.): S645PubMed Google Scholar, 23Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. J. Biol. Chem. 1991; 266: 24773-24777Abstract Full Text PDF PubMed Google Scholar, 24Funderburgh J.L. Conrad G.W. Greiling H. Scott J.E. Keratan Sulphate: Chemistry, Biology, and Chemical Pathology. Biochemical Society, London1989: 39-52Google Scholar, 25Funderburgh J.L. Caterson B. Conrad G.W. J. Biol. Chem. 1987; 262: 11634-11640Abstract Full Text PDF PubMed Google Scholar). The presence of sulfated lumican molecules in cornea suggests that in this tissue lumican may have unique functions, e.g. maintaining corneal transparency; however, its role serving in other noncorneal tissues remains elusive. Mouse lumican is a 338-amino acid protein with high sequence homology to bovine, human, and chicken lumican (16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar, 17Grover J. Chen X.-N. Korenberg J.R. Roughley P.J. J. Biol. Chem. 1995; 270: 21942-21949Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 18Funderburgh J.L. Funderburgh M.L. Brown S.J. Vergnes J.P. Hassell J.R. Mann M.M. Conrad G.W. J. Biol. Chem. 1993; 268: 11874-11880Abstract Full Text PDF PubMed Google Scholar, 26Blochberger T.C. Vergnes J.P. Hempel J. Hassell J.R. J. Biol. Chem. 1992; 267: 347-352Abstract Full Text PDF PubMed Google Scholar). To examine the structure and function relationship of mouse lumican gene using transgenic mice and site-directed mutagenesis techniques; it is imperative to isolate and characterize the mouse lumican cDNA and genomic DNA and to determine the spatial-temporal expression of lumican gene during mouse development. In the present studies, we have cloned and determined the primary structure of mouse lumican gene (Lum). In situ and Northern hybridization were used to determine the temporospatial expression of Lum. Lumican isolated from eye shells (cornea plus sclera) at various developmental stages were also biochemically characterized. Our results indicate that lumican is widely expressed in a variety of connective tissues. Sulfation of the lumican in cornea occurs concomitantly with eye opening and therefore may be an essential step in providing corneal transparency. A pair of primers, sense 5′-CATGTATGGGCAAATATC and antisense 5′-TGTAGAAGGTTGTGGTCA (16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar), derived from mouse lumican cDNA was used in polymerase chain reaction to screen a mouse bacterial artificial chromosome-genomic DNA library (Research Genetics, Inc., Huntsville, AL). A positive clone of 200 kb was isolated. The clone was characterized with restriction enzyme digestion and Southern blot hybridization with 32P-labeled lumican cDNA. A 6-kbSalI-XbaI fragment and an 8-kbXbaI-XbaI fragment together encoding the full-length cDNA were subcloned into pBSSK vector (Stratagene, La sequence of the lumican gene was determined with by the DNA core in the of at of A antisense to exon 1 and a sequence were used to a DNA fragment by polymerase chain reaction using a fragment of mouse lumican genomic DNA clone as the sense was with using a by the The antisense was with by Inc., The DNA was to of mouse mRNA from 1 day post This reaction was with units of R. Kao H. J. Justice M.J. Stech M.E. Kao W.W.-Y. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The was used with the as a The was an To the transcription initiation a sequence reaction using the antisense and mouse lumican genomic DNA fragment as was with S. Biochemical A antisense to exon 1 was with by and to of mouse was as previously R. Kao H. J. Justice M.J. Stech M.E. Kao W.W.-Y. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The reaction were the as the one used in the For of lumican mRNA in a blot 2 of from different developmental by a was from The blot was with 32P-labeled mouse lumican cDNA as previously (14Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, R. Kao H. J. Justice M.J. Stech M.E. Kao W.W.-Y. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). For tissue were from mouse tissues using as previously (16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar). 10 of were in with The were to and with 32P-labeled lumican and mouse 3-phosphate cDNA in a hybridization at as previously (16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar, R. Kao H. J. Justice M.J. Stech M.E. Kao W.W.-Y. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The were by three with and at for The hybridization were detected with a The of lumican mRNA were with the mRNA in To the types that lumican, the mouse tissues were with and in as previously Kao Kao W.W.-Y. 1996; PubMed Scopus Google Scholar). and sense of lumican were and used in in situ hybridization To were to a in at and with of at for 1 by with at as previously Kao Kao W.W.-Y. 1996; PubMed Scopus Google Scholar). The hybridization were with using by were isolated from eye shells (cornea plus sclera) of day postnatal and and 1 of mice in of a and as by Funderburgh J.L. Corpuz L.M. Roty M.R. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1996; 37 (abstr.): S645PubMed Google Scholar). The tissue was by and for at The were to were a of in the The was with in the and proteoglycans were with proteoglycans were in for at and keratan proteoglycans were by the addition of to the to This isolated sulfated and of lumican J.L. Funderburgh M.L. Mann M.M. Conrad G.W. J. Biol. Chem. 1991; 266: 24773-24777Abstract Full Text PDF PubMed Google Scholar). The proteoglycans were by in in and in of 10 of protein of the corneal KSPG or with each of and for 2 at 37 was a 3 to The was in for 2 and in in acid and in the KSPG core proteins were by digestion of of KSPG protein with as and a The proteins were to and KSPG proteins were detected using KSPG as previously (14Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The genomic DNA clone isolated from a mouse bacterial artificial chromosome genomic DNA library was characterized by Southern hybridization and 3′ of mouse lumican cDNA. The full-length mouse lumican gene spans 6.9 kb in mouse genome. 1 that gene has three exons and two introns. The genomic and were and the were determined in by the primary structure of lumican gene and the acid sequence from The not have a TATA box, it has a TATCA element, a presumptive TATA box, that is 27 bases 5′-upstream from the transcription initiation site. is a of at b to the transcription initiation site. The initiation is at the base from the of exon exon 1 the untranslated of lumican Exon 2 most of in lumican, and exon 3 acid and a 3′ untranslated 1 and 2 are and bp, of the and The is found from the in exon derived from and the and that the transcription initiation is the at b to the initiation and 27 b 3′ of the TATCA as with the sequence of lumican genomic DNA was determined by using the used in of the of 2 and To the of lumican it is to determine the and expression of mouse lumican during development. Northern hybridization and in indicate that in early stages of embryonic development day 7 post coitus the embryo not lumican or expresses only not A that the isolated from contains little lumican mRNA The levels of lumican mRNA in the at and at a high To the expression of lumican mRNA by various tissues during mouse development, Northern hybridization was with isolated from cornea, heart, lung, and kidney. that levels of lumican in to that of in cornea and are in embryonic and of postnatal and in heart levels of lumican expression are The lumican mRNA at a but and have of lumican expression the not To determine the types that lumican mRNA, tissue from mouse and mice were with as and The in situ hybridization that the stroma in mouse cornea to lumican at embryo day the lumican expression in cornea stroma at postnatal day 1 and situ hybridization also detected the expression of lumican mRNA in several other such as heart, lung, and kidney. of these to lumican mRNA at embryo day that the lumican mRNA is expressed by the and in the situ hybridization of mouse lumican mRNA in various tissues at antisense sense and C and and the expression of lumican in various tissues during mouse development. day 10 PC, the to mRNA in and day PC, the expresses lumican mRNA, but the the early development and also expresses lumican mRNA and It is of to that interstitial of various tissues lumican mRNA after of expression of lumican during mouse and of tissues from mouse at different developmental stages were to in situ hybridization with cDNA as and in a of tissues from mouse at different developmental stages were to in situ hybridization with cDNA as and KSPG core proteins were by with from of cornea and sclera and detected by using in at postnatal day little of KSPG core proteins was detected with this day 10 and were of the KSPG core proteoglycans were detected with a that the sulfated chains of the KSPG postnatal day 1 sulfated KSPG could not be detected but by day 10 of KSPG in that of mice was A in the and of KSPG between 10 and with the at day that of of the KSPG with an enzyme for sulfated moieties in the results in the of in These indicate that cornea and sclera not of sulfated KSPG to 10 after a in which the eyes are in The mouse lumican belongs to family and has the of a of leucine-rich by and domains with (16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar, 18Funderburgh J.L. Funderburgh M.L. Brown S.J. Vergnes J.P. Hassell J.R. Mann M.M. Conrad G.W. J. Biol. Chem. 1993; 268: 11874-11880Abstract Full Text PDF PubMed Google Scholar, R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar, 26Blochberger T.C. Vergnes J.P. Hempel J. Hassell J.R. J. Biol. Chem. 1992; 267: 347-352Abstract Full Text PDF PubMed Google S. N. Hassell J.R. 1995; PubMed Scopus (101) Google Scholar). The structure of mouse gene is to that of fibromodulin, which has three with the second exon encoding leucine-rich Biochim. Biophys. Acta. 1993; PubMed Scopus Google Scholar). are different from the other two in another of i.e. and biglycan, which are of exons and the sulfate chains (20Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar, S. R. J.L. R.V. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, Y. J. S. R.V. 1995; PubMed Scopus Google Scholar). The mouse lumican gene not a TATA an TATCA is present at 27 b 5′-upstream to the transcription initiation site. of that have been characterized this TATA for of transcription initiation N. J.P. Mol. 1993; Google Scholar). In a is at b from the transcription It has been that the presence of the may the of a TATA box, by transcription For example, the and the TATA may these and the proteins to to transcription J. PubMed Scopus Google Scholar). It has been that are of tissue and in their to the of extracellular matrix in connective tissues (20Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar, S. E.D. Biol. 35: PubMed Scopus Google Scholar, S. E.D. Acad. Sci. S. PubMed Scopus Google Scholar). The of a matrix with small and and interfibrillar is essential for the development and maintenance of transparent cornea R.A. J. Scopus Google Scholar). corneal and the of corneal it has been that are in cornea stromal proteoglycan that may for the formation of N. M.M. 1995; PubMed Scopus Google Scholar, J.R. Cintron C. Kublin C. Newsome D.A. Arch. Biochem. Biophys. 1983; 222: 362-369Crossref PubMed Google Scholar, J.L. Cintron C. Conrad G.W. Invest. Ophthalmol. Visual Sci. Google Scholar, J.R. Newsome D.A. M.M. Acad. Sci. S. 1980; PubMed Scopus Google Scholar, M.E. Biophys. J. 1996; 70: Full Text PDF PubMed Scopus Google Scholar). In the corneal the of KSPG is and the of sulfate proteoglycan A to KSPG is upon of corneal transparency J.R. Cintron C. Kublin C. Newsome D.A. Arch. Biochem. Biophys. 1983; 222: 362-369Crossref PubMed Google Scholar). The corneal is characterized by the of the of sulfated due to a in the sulfate proteoglycan or the J.R. Newsome D.A. M.M. Acad. Sci. S. 1980; PubMed Scopus Google Scholar, Hassell J.R. Newsome D.A. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. N. Arch. Ophthalmol. PubMed Scopus Google Scholar). Our that is a of KSPG core proteins in the mouse cornea at postnatal day 10 with that of day Lumican with keratan sulfate chains at postnatal day This is consistent with that of studies that in a and sulfated keratan sulfate proteoglycans during the embryonic development of the cornea at day the cornea to transparent T.C. Hassell J.R. Invest. Ophthalmol. Visual Sci. 1994; 35: Google Scholar, J.L. Caterson B. Conrad G.W. Biol. PubMed Scopus (92) Google Scholar). of in KSPG during corneal development that the core protein of lumican and its keratan sulfate chains are to the development of corneal transparency T.C. Hassell J.R. Invest. Ophthalmol. Visual Sci. 1994; 35: Google Scholar, J.L. Caterson B. Conrad G.W. Biol. PubMed Scopus (92) Google Scholar, G.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Sulfation of the chain the core protein by 10 KSPG not to until day levels at day this the mouse eyes open. This is consistent with the study by Gibney E. Gordon M.K. J.K. Birk D.E. Linsenmayer T.F. 1996; 63: PubMed Scopus Google Scholar), a of mRNA of chicken an enzyme in the of the chain during embryonic the extracellular is detected and an in the stroma J.L. Caterson B. Conrad G.W. Biol. PubMed Scopus (92) Google Scholar). The chicken transparency during this and are consistent with the that lumican and other proteoglycans may play an role in the development and maintenance of corneal transparency. It is of to that the levels of lumican mRNA in in the mice and a in to as with of embryonic at day and the of lumican protein in the tissue not with the of lumican the and of lumican may be at to has been in collagen W.W.-Y. R.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, W.W.-Y. J. J. Biol. Chem. 1983; Full Text PDF PubMed Google Scholar). the of lumican in tissues may be due to an by the of chains to the core protein. studies are to examine the Lumican the keratan sulfate chains may be only to the it is that it is also present in a variety of noncorneal e.g. cartilage, heart, lung, as a sulfated or nonsulfated glycoprotein (11Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. Biochem. Soc. Trans. 1991; 19: 871-876Crossref PubMed Scopus (48) Google Scholar, 23Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. J. Biol. Chem. 1991; 266: 24773-24777Abstract Full Text PDF PubMed Google Scholar, 24Funderburgh J.L. Conrad G.W. Greiling H. Scott J.E. Keratan Sulphate: Chemistry, Biology, and Chemical Pathology. Biochemical Society, London1989: 39-52Google Scholar, 25Funderburgh J.L. Caterson B. Conrad G.W. J. Biol. Chem. 1987; 262: 11634-11640Abstract Full Text PDF PubMed Google Scholar). This may play that are to be in the maintenance of tissue It likely that lumican in noncorneal tissues is a of collagen it to be in in addition to cornea the of lumican gene may have organ are in

We have characterized an mRNA that increases in abundance after serum stimulation of quiescent mouse fibroblasts. This mRNA, designated 18A2, encodes a predicted polypeptide of 101 amino acids with homology to known calcium binding proteins. A variety of mouse tissues express the 18A2 mRNA, with the highest levels detected in the non-pregnant uterus and in the placenta. The concentration of 18A2 mRNA in total placental RNA decreases from day 8 to day 10 of pregnancy, and is below detection throughout the latter half of gestation. In serum-stimulated fibroblasts, the increase in 18A2 mRNA is dependent on protein synthesis. The 18A2 mRNA is similar in size, serum-inducibility, and sequence to the 2A9 mRNA (1), but these mRNAs are derived from distinct genes. This suggests that the mouse genome harbors a family of serum-inducible genes encoding proteins predicted to bind calcium.

Proliferin-related protein (mPRP) is a member of the PRL/GH family in the mouse. We have generated an antiserum against mPRP expressed as a bacterial fusion protein; this antiserum detects mPRP in the conditioned media of placental tissue cultures as a heterogeneous population of glycoproteins. We have also expressed mPRP in mammalian tissue culture cells and purified the secreted protein. N-terminal sequence analysis of the purified protein reveals that it is secreted as a 214 amino acid protein after removal of a 30 amino acid signal polypeptide. An antiserum raised against the purified protein detects high levels of mPRP in maternal serum during gestation. The site of synthesis of this protein has been localized by in situ hybridization to the basal zone of the day-10 mouse placenta, which is distinct from the site of synthesis of other placental proteins in this family.

In Xenopus laevis, the formation of the adult olfactory epithelium involves embryonic, larval and metamorphic phases. The olfactory epithelium in the principal cavity (PC) develops during embryogenesis from the olfactory placode and is thought to respond to water-borne odorants throughout larval life. During metamorphosis, the PC undergoes major transformations and is exposed to air-borne odorants. Also during metamorphosis, the middle cavity (MC) develops de novo. The olfactory epithelium in the MC has the same characteristics as that in the larval PC and is thought to respond to water-borne odorants. Using in situ hybridization, we analyzed the expression pattern of the homeobox genes X-dll3 and Pax-6 within the developing olfactory system. Early in development, X-dll3 is expressed in both the neuronal and non-neuronal ectoderm of the sense plate and in all cell layers of the olfactory placode and larval PC. Expression becomes restricted to the neurons and basal cells of the PC by mid-metamorphosis. During metamorphosis, X-dll3 is also expressed throughout the developing MC epithelium and becomes restricted to neurons and basal cells at metamorphic climax. This expression pattern suggests that X-dll3 is first involved in the patterning and genesis of all cells forming the olfactory tissue and is then involved in neurogenesis or neuronal maturation in putative water- and air-sensing epithelia. In contrast, Pax-6 expression is restricted to the olfactory placode, larval PC and metamorphic MC, suggesting that Pax-6 is specifically involved in the formation of water-sensing epithelium. The expression patterns suggest that X-dll3 and Pax-6 are both involved in establishing the olfactory placode during embryonic development, but subtle differences in cellular and temporal expression patterns suggest that these genes have distinct functions.