Identification of a set of genes with developmentally down-regulated expression in the mouse brain

Vol. 185, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 30, 1992 Pages 1155-1161

IDENTIFICATION OF A SET OF GENES WITH DEVELOPMENTALLY DOWN-REGULATED EXPRESSION IN THE MOUSE BRAIN

Sharad Kumarl*, Yasuhiro Tomooka2 and Makoto Nodal

1Department of Viral Oncology, Cancer Institute, Kami-Ikebukuro, Toshima-ku, Tokyo 170, JAPAN

2 Laboratory of Cell Biology, Institute of Physical and Chemical Research, RIKEN, Tsukuba Life Science Center, Koyadai, Tsukuba 305, JAPAN

Received May 22, 1992

Summary : Using a subtraction cloning approach, we have isolated a set of cDNA clones from mouse neural precursor cells whose respective mRNA levels are down-regulated during the development of mouse brain. Single stranded DNA prepared from neuronal precursor cell cDNA library in lambda Zap vector was subtracted with poly (A)+ RNA prepared from postnatal and adult mouse brain to obtain several clones which show developmental down- regulation of expression. Their patterns of expression indicate that these genes may play important roles during the embryonic development and differentiation of central nervous system. c3 1992 Academic Press, Inc.

The development of mouse central nervous system (CNS) starts around embryonic day 8 (ES) when a neural plate is formed from the ectoderm. This neural plate then invaginates and fuses to form the neural tube around ElO. Following the closure of the neural tube, the neuroectoderm gives rise to a large number and variety of neural and glial cells, which would eventually form the CNS (l-3). Molecular events which lead to the development and differentiation of the mammalian CNS are largely unknown, although the recent advances in the culture of neural precursor cells and molecular biology techniques have resulted in the isolation of a number of genes which may play a role in the CNS development (4-6). Most of the information about CNS development at cellular level has come from the studies with Drosophila mutants exhibiting aberrant CNS development (7-9). Characterization of these mutants has led to the isolation of mutated genes and eventually some of the mammalian counterparts of these genes (lo- 12).

We are interested in identifying the genes responsible for mammalian CNS development and especially the ones which regulate the lineage determination and differentiation of the neural precursor cells (NPC). In order to study this, we had previously established a simple method

* To whom correspondence should be addressed.

Abbreviations: CNS, central nervous system; NPC, neural precursor cells.

0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc.

1155 All rights of reproduction in any form reserved.


for isolating large numbers of the mouse NPC (13). In the present communication we describe the isolation of a set of cDNA clones from a NPC derived library, whose respective mRNA am predominantly expressed in the early embryonic brain and are down-regulated during the development of the brain. To achieve this, we subtracted a cDNA library prepared from the NPC with a large excess of mRNA isolated from the postnatal and adult brain. This approach is based on the assumption that the genes required for the development and differentiation of the CNS are primarily expressed in the embryonic brain and not in the fully differentiated adult tissue. Using this strategy, we report here, the isolation of ten novel cDNA clones.

Materials and Methods

Cells and embryos NPC were isolated from the neural tube of the El0 mouse embryos as previously described (13). Approximately 24 hours post-mating was cosidered as embryonic day 1 (E 1). Embryos and postnatal (N) animals were dissected out at stages ElO, El 1, E13, E15,E17, Nl and N5 and the heads (ElO, Ell) or brain tissue (E13, E15, E17, Nl and N5) used for RNA extraction.

Preparation of NPC cDNA library Total cellular RNA was prepared from NPC according to established methods (14). Poly (A)+ RNA was prepared from total cellular RNA by absorption on Oligotex according to the instructions supplied by the manufacturer (Nippon Roche). Five pg of poy (A)+ RNA was used to synthesize cDNA primed with an oligo (dT)-adaptor primer using a cDNA synthesis kit (Suatagene) accordind to the instructions supplied with following modifications: the kit reverse uanscriptase was replaced with Superscript (BRL) and after the synthesis of double stranded cDNA, the sample was purified twice with Geneclean (Bio 101). The cDNA inserts containing the Xhol and Eco Rl likers were then ligated into Xhol/ Eco Rl sites of lambda Uni Zap vector (Stratagene). Approximately 500,000 primary clones were recovered with an average insert size of more than 1.5kb (range 0.4- 6.0 kb). The library contained a background of less than 0.1% of nonrecombinants.

Preparation of subtracted library The general strategy for subtracting the NPC cDNA library with adult brain mRNA is shown in Figure 1 and is similar to that described recently (15). The complete cDNA library was rescued as phagemid clones by in vivo excision of lamda Zap library according to the protocols supplied by the manufacturer (Stratagene). Highly purified single stranded DNA, essentially free from contaminating double stranded DNA, was prepared from this library according to the protocol of Rubenstein et al. (15). This single stranded DNA, in addition to the library derived DNA, contained a large amount (approximately 70%) of DNA from R408 helper phage which was used for the rescue of the Bluescript phagemid. This contaminating DNA does not interfere with the subsequent steps in the subtraction process and infact acts as a carrier and internal control, and thus was not removed.

Total RNA was isolated from postnatal day 5 (N5) and adult mouse brain (14) and poly (A)+ RNA prepared by Oligotex. Twenty pg of this poly (A)+ RNA (10 pg each of N5 and the adult brain ) were labeled with photobiotin and precipitated with one pg of single stranded DNA prepared above. Dried precipitates were dissolved in 50 ~1 of Berk-Sharp hybridisation buffer (16), heated at 65’ C for 10 minutes and incubated at 37’ C for 48 hours. Then 401tl of a HEPES (lOmM), EDTA (1mM) solution and 12 pl of 2M sodium acetate solution were added and nucleic acids precipitated with 2.5 volumes of ethanol. Nucleic acid pellet was dissolved in 50 ~1 of HEPES-EDTA solution and biotin-labeled nucleic acids (RNA and RNA/ DNA hybrids) were removed by two cycles of streptavidin-agarose binding (15). Remaining DNA was ethanol precipitated and rehybridised with 1Opg of biotin-labeled adult brain poly (A)+ RNA as described above. After two cycles of subtraction, the single stranded DNA was converted to double stranded form by the use of Ml3 reverse sequencing primer and Vent polymerase (NEB) at 70’ C for 20 min, extracted once with phenol/ chloroform, purified on a

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Nensorb column (NEN),and vacuum dried. Nucleic acids were dissolved in 10 ~1 of water and 1~1 aliquots were introduced into E. coli (Electromax DH IOB, BRL) by electroporation using a Biorad apparatus.

Screening of subtracted clones The bacterial colonies obtained after electroporation were directly picked into 200 l.tl of medium in 96- well plates and grown overnight at 37’C. One fourth of each stock culture was made 15% in glycerol and frozen at -70’ C while 3x 50~1 aliquots were blotted onto nitrocellulose filters using a Biorad apparatus. Filters were processed according to the standard protocols. [32P]-labeled cDNA probes from mRNA were prepared using 5 Itg of poly (A)+ RNA transcribed with random hexamers plus oligo (dT) and Superscript reverse transcriptase, followed by removal of RNA template by alkali hydrolysis using standard protocols (14). Probes were further purified by Nensorb columns.

Other methods Poly (A)+ RNA for northern blot analysis were isolated using a single cycle of oligo (dT) cellulose absorption by Fast Tract kit (Invitrogen ). Northern analysis of RNA , cloning and sequencing of DNA and other related methods were according to standard protocols (14). An oligonucleotide, 5’-ATACGGCCAAATCCGTCACACCGACCTICACCAT, derived from the cDNA sequence of glyceraldehyde-3-phosphate dehydrogenase (GA-3-PDH) (17) was used as a control in northern blot analyses.

Results and Discussion

As the starting material for the subtraction cDNA library we prepared a unidirectional cDNA library in lambda Zap vector from the mRNA isolated from NPC. The use of isolated NPC

instead of total embryos or the dissected heads eliminates the cDNA clones originating from tissues other than of those of CNS lineage. The unidirectional library in the lambda Zap vector

enables the rescue of the negative strand of the cloned cDNA which could be directly hybridised to mRNA to be subtracted. Using this subtraction cloning approach, a total of 348

clones were obtained. Rescreening of these clones further, using the cDNA probes derived

Table 1. Summary of the NEDD clones

clone name

insert gene size (kb) name

transcript size (kb)

relative mRNA levels El0 El4 adult

2C3 0.80 NEDD-I Z:::

tt t+ + 2C8 0.80 NEDD-2 it ++ 2A7 0.45 NEDD-3

A:;, 4.3 +tt ii+ +

4Cl 0.39 NEDD-4 +++ +++ + 4D12 0.24 NEDD-5 it tt t 4A7 0.43 NEDD-6 i:; t+t ttt t lC4 0.20 NEDD-7 tt++ ++++ ++ 2D1 0.53 NEDD-8 A:775 ii+++ ii+++ t+ 1C6 1.10 NEDD-9 if if 2H2 0.60 NEDD-IO ;:; tt t

The cloned inserts were released as EcoRl/ Xhol fragments and used as probes for hybridisation to poly (A)+ RNA from the NPC or El4 brain to determine the sizes of corresponding transcripts. To determine the relative levels of mRNA during the CNS development, poly (A)+ RNA isolated at the indicated stages were reverse transcribed in the presence of [s2P] UTP and used as probes to hybridise to plasmid DNA from various clones immobilised on nylon membrane. Number of positives (+) represent the relative intensity of hybridisation, while negative (-) indicates that no signal was detected under the conditions of hybridisation.

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from total poly (A)+ RNA from NPC and adult brain indicated that 195 of these clones hybridised strongly to both pools of cDNA probes and were omitted from further screening. Among the remaining 153 clones, 73 were found to contain no cDNA insert at all. Since the background of nonrecombinants in the original NPC library, used as the starting material, was less than O.l%, the presence of approximately 21% nonrecombinants in the subtracted population would indicate that an enrichment of at least 200 fold was achieved using the directional, single-stranded library subtraction strategy. The plasmid DNA from the remaining 80 clones was further analysed by hybridisation to [32P]-labeled cDNA probes from El0 head and El4 and adult brain. After two cycles of hybridisation, a total of 10 clones were selected which showed positive hybridisation to cDNA probes from El0 and E14, and negative or reduced hybridisation to cDNA probes derived from the adult brain (Table 1). We have named the genes corresponding to these cDNA clones 'NEDD' : NPC-Expressed, Developmentally Down- regulated (genes) (Fig. 1). The cDNA inserts from these clones released by digestion with EcoRl plus Xhol were used as probes to hybridise to poly (A)+ RNA from El0 heads and adult brain, immobilised on nylon membrane using a slot blot apparatus (Figure 2). It is clear from the data that the relative abundance of NEDD -derived mRNA is much lower in the adult brain than the El0 head. The intensities of hybridisation signals of NEDD probes to the RNA samples were quite different from one another indicating the different relative abundances of the steady state levels of the respective messages (Figure 2 and Table 1). In El0 heads, the relative expression of NEDD-8 was the highest followed by (in decreasing order) NEDD-4, NEDD-7, NEDD-4, NEDD-3,

NPC cDNA library poslnatcll +adult brain mRNA

1 1 ssDNA (-ve strand) label with photobiofin

\ /

hybfidise 4

renwe unhybridised mRNA + hybrids by streptavidin agarose

NPC enriched ssDNA population

ds- DNA conversion

1 E.coli transformation

hybridise to kttxled adult brain cDNA

d&d p&mid DNA minipreps

without ins,,/ ‘with inserts

1 1 discard differential hybridisation

screeninf

/ select differentially ca.prcssed cDNA clones (NEDD-cDNA)

Figure 1. Flow diagram of the strategy used to isolate the NEDD cDNA clones.

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1 2 3 4 5 6

7 8 9 10 a b

Figure 2. Slot blot analysis of NEDD mRNA expression. 0.25 ug of poly (A)+ RNA applied to nylon membrane using a Biorad apparatus, fixed by UV crosslinking, and hybridised to random primed [3*P]-labelled NEDD probes. After washing, the filters were exposed for varying lengths of time and analysed by BAS 2000 Imageanalyser (Fuji). Numbers l- 10 represent the corresponding NEDD probes while a and b are controls. In a, end-labelled GA-3-PDH probe was used The probe in b was derived from a randomly picked cDNA clone from NPC library which shows relatively invariable mRNA expression during the CNS development.

NEDD-5, NEDD-1, NEDD-2, NEDD-9, and NEDD-10. The expression of some of these

genes (NEDD-2, NEDD-9 and NEDD-IO) was not detectable in the adult brain under these

conditions, although the presence of very low levels of transcripts in the adult brain remains a

possibility.

We further analysed the expression of some of the NEDD genes by northern blot analysis of

poly (A)+ RNA isolated from brain (or head for El0 and El 1) at several stages of embryonic

development (Figure 3). To facilitate the detection of genes expressed at low levels, we have used 5 pg of poly (A)+ RNA at every stage. The results indicate that in majority of the cases, a

gradual decrese in the steady state levels of the respective mRNA during the embryonic

development was observed (for example, NEDD-1,2,3 and 4), while NEDDd and NEDD-8 mRNA levels have little changes during the embryonic development but show decresed

expression during postnatal development of brain ( Figure 3). Since we have used complete brain tissues (heads in the case of El0 and El 1 embryos), it is conceivable that the decrease in

the levels of particular NEDD mRNA reflect more limited expression in some cell types in the

adult brain than overall down-regulation in the brain. This possibility is being checked by in situ hybridisation of NEDD probes to the embryonic and the adult brain sections.

So far we have cloned the near full length cDNA from NEDD-I, NEDD-2 ( S.K. et al., unpublished) and NEDD-3 (T. Sazuka, M. Kinoshita, Y.T., T. Matsuzaki, T. Sakakura, Y. lkawa, M.N. and S.K., manuscript in preparation). Partial cDNA sequences from the rest of

the clones were also obtained. All these sequences have been deposited in the nucleotide sequence database and will appear in the DDBJ, EMBL and GenBank databases under

following accession numbers: D10712 (NEDD-I), D10713 (NEDD-2), D10715 (NEDD-3), D10714 (NEDD-4), D10915 (NEDD5), D10916 (NEDD-6), D10917 (NEDD-7), D10918

(NEDD-8), D10919 (NEDD-9) and D10920 (NEDD-20). A search for related seqences in the

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0.9, 6

GA-3-PDH

Figure 3. Northern blot analyses of NEDD mRNA expression during the development of mouse brain. Five pg of poly (A)+ RNA isolated from the heads (El0 and El 1) or brain tissue (E13- adult) were electrophoresed on 1.2% agarose/ 2.2M formaldehyde gel, transferred to Biodyne A membrane (Pall), fixed by UV-crosslinking and hybridised to random primed [32P]-labeled DNA probes. Numbers on the right hand side correspond to the respective NEDD probes. GA-3-PDH and mouse tenascin, a developmentally regulated gene (19, 20), were used as controls. Only relevant portions of the blots are shown. The respective sizes of the detected transcripts are shown on the left hand side.

databases using FASTA program (18) showed that NEDD-I predicted gene product shares partial homology with R-subunit of GTP binding proteins (S.K. et al., unpublished), NEDD-3 encodes a novel GTP-binding protein (T. Sazuka et al., manuscript in preparation ), while NEDD-8 shows strong homology with ubiquitin both at nucleotide and protein levels (S.K. et

al., unpublished). The nucleotide sequences of all the other clones do not show any significant identities with the sequences in the databases however as most of these partial sequences probably represent the 3’-noncoding regions of the respective cDNA, the possibility remains that some of them represent mouse homologues of certain known genes in other species. In summary, we have identified a set of genes which show developmental down- regulation of their expression in the mouse brain. As these genes are expressed at much higher levels in

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embryonic brain than the adult brain, they are likely to play some roles in the development and differentiation of the CNS. Cloning of the full length cDNA and determination of the structure and the in vivo expression patterns of their respective proteins will provide the way to determine the biological roles of these genes.

Acknowledgments: We thank Y. Ikawa for providing encouragement and support and Y. Saga for providing the mouse tenascin probe.This project was supported by grants from RIKEN and Science and Technology Agency of Japan (STA). S.K. was supported by a visiting fellowship from STA while in the Laboratory of Molecular Oncology at RIKEN.

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M.H., and Usdin, T.B. (1990) Nucleic Acids Res. 18, 4833-4842. 16. Berk, A.J., and Sharp, P.A. (1977) Cell 12, 721-732. 17. Sabath, D., Broome, H.E., and Prystowsky, M.B. (1990) Gene 91, 185-191. 18. Pearson, W.R., and Lipman, D.J. (1988) Proc. Natl. Acad. Sci., U.S.A. 85, 2444-2448. 19. Saga, Y.,Tsukamoto, T., Jing, N., Kusakabe, M., and Sakakura, T. (1991) Gene 104,

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Identification of a set of genes with developmentally down-regulated expression in the mouse brain

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