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A POU-DOMAIN SPECIFICALLY

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GENE OF ZEBRAFISH, ZFPOUI, EXPRESSED IN THE DEVELOPING NEURAL TISSUES+

Takashi Matsuzaki*, Hiroshi Amanuma, and Hiroyuki Takeda Laboratory of Gene Technology and Safety, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba 305, Japan Received

August

19,

1992

Summary: We have isolated a POU domain-containing cDNA (ZFPOUZ) from a cDNA library of zebrafish (Bruchydanio rerio). The ZFPOUZ cDNA contained an open reading frame encoding a 425 amino acid peptide. The conserved POU domain was located near the carboxy terminus. The deduced amino acid sequence of the reading frame was most similar to that of the mouse class III POU-domain gene, Brain-Z. Northern blot analysis revealed that the ZFPOUI transcripts first appeared at the early neurula stage of embryogenesis and transiently increased thereafter. A significant level of expression, however, was not found in adult tissues except in the brain. In situ hybridization analysis demonstrated that the ZFPOUI transcripts were localized in the neural tissues of embryos, but not in mesodermal, endodermal or ectodermal tissues. In adult zebrafish, the ZFPOUI transcripts were detected in the restricted regions of the brain. Spatial and temporal expression patterns suggest that ZFPOUZ has distinct roles in the early neural development of zebrafish. o 1992~~~~~~~~ press, I,,=.

POU-domain genes were first isolated as three mammalian genes encoding the transcription factors, Pit- 1 (l), Ott- 1 (2) and Ott-2 (3), and as a Caenorhabdifis elegans gene uric-86 that is required for neuroblast differentiation (4). They have a conserved 1.50-160 amino acid region referred to as the POU domain which is responsible for highaffinity, site-specific DNA sequence recognition and protein-protein interactions (5-7). The POU domain consists of two subdomains, an N-terminal POU-specific domain and a C-terminal POU-homeo domain separated by a short variable linker region. Many POU-domain genes have been isolated from a variety of species and they show distinct expression patterns during development (for review, see Rosenfeld, ref. 7). Most of the POU-domain genes, especially those belonging to the class III, are expressed in the neural tissues throughout the course of development, indicating potential roles in the regulation of neural development (8). However, we have yet little knowledge concerning the functions of the POU-domain genes in neural development. +Sequence data Data Libraries

from this article under Accession

have been deposited No. D13045.

with

the

* To whom correspondence should be addressed. FAX: +g l-298-36-9050. 0006-291 X/92 $4.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

Inc. reserved.

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It is well established that zebrafish (Brachydunio rerio) is one of the conspicuous biological materials suitable for the study of developmental phenomena because of its brief generation time, prolific egg production, and transparent embryos (9). Since the underlying mechanisms of vertebrate development are evolutionarily

conserved, the

characterization of POU-domain genes of fish may help to elucidate the functional roles of these genes in higher vertebrates. In the present study, we report the isolation of a neural tissue-specific POU-domain gene of zebrafish, which is closely related to the mammalian POU-domain gene, Bruin-l. As a first step to demonstrate a functional role, the temporal and spatial expression of the gene was studied in embryos.

Qat Jlinn

MATERIALS riz

AND METHODS

‘ning CD hr . In order to isolate POU domain-containing cDNA clones of zebrafish, we employed the reverse transcriptase-polymerase chain reaction (RT-PCR) method as an initial step. Three 26-mer oligonucleotides, POUl, POU2r and POU3r, were synthesized based upon the amino acid sequences of the N-terminal and C-terminal portions of the Ott-3 POU domain (lo), LGYTQADVG, KDVVRVWFC and CNRRQKGKR, respectively. POU3r was used to prime the cDNA synthesis from 10 ug of total RNA prepared from the neurula stage embryos (9-12 hr of development at 28.5X). Thirty cycles of PCR amplification were then performed using the primers POUl and POU2r. The cycle included a 1 min denaturation step at 94’C, a 1 min annealing step at 50°C and a 2 min extension step at 72’C. After the amplification cycles, the reaction was followed by a final extension at 72’C for 7 min to complete the DNA synthesis. The PCR products of about 360 bp length were cloned and subjected to DNA sequence analysis. One clone, ZFPCRl, containing a class III POU domain was used as a probe to screen a lambda ZAP II cDNA library constructed from RNA of 9-16 hr embryos (a kind gift from Dr. D. J. Grunwald). translation and SDS-PAGE ana Iv+. RNA was synthesized in vitro 1n wtro * using mCAPTM RNA capping kit (Stratagene) from the ZFPOUl cDNA which had been inserted in the pBluescript 11 SK(+) plasmid. The ZFPOUI plasmid was linearized, and the sense RNA was made using the T7 polymerase. The RNA (1 ug) was used for in vitro translation reaction with rabbit reticulocyte lysate (Stratagene) in the presence of [35S]methionine (0.4 MBq/ul, 37.0 TBq/mmol. DuPont). The labeled products were analyzed by 10% SDS-polyacrylamide gel electrophoresis. Northern blot analvsig. Total RNA was prepared by the acid guanidinium thiocyanate-phenol-cloroform extraction method (11). Poly A+ RNA was purified from the total RNA using oligo(dT)-latex (Oligotex-dT30, Roche). Ten ug of total RNA prepared from embryos of various stages (reared at 28.5 “C) and adult ovary or 2 ug of poly A+ RNA from adult liver, intestine, and brain were separated by formaldehydecontaining 1.0% agarose gel (12), blotted onto the Hybond N+ filter and subjected to hybridization with a [32P]-labeled probe which represented the 3’ noncoding region of ZFPOUl. The hybridization buffer contained 6xSSC, SxDenhardt’s, 100 ug/ml sonicated calf thymus DNA, and 0.5% SDS. After prehybridization, the filter was incubated with new hybridization buffer containing the [32P]-labeled probe (1~10~ cpm/ml) at 57’C for 16 hr. Then, the filter was washed to a final stringency of O.lxSSC at 57 “C. The radioactive signals were detected by Fujix BAS2000 Bio-imaging analyzer (Fuji photo film). . . . m . For in situ hybridization, digoxigenin (DIG)-1 1-UTP-labeled single-stranded sense and antisense RNA probes were prepared according to the manufacturer’s instruction of the DIG RNA labelling kit (Boehringer Mannheim) using the 3’ noncoding region of ZFPOUl as a template. Embryos were fixed with 4% paraformaldehyde/PBS, frozen on dry ice and cut at 7-10 urn. Hybridization and washing were performed essentially as described (13). Immunological detection was carried out according to the manufacturer’s instruction of the DIG Detection kit (Boehringer Mannheim). Positive sites were stained in blue to brown in the sections. 1447

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RESULTS Isolation

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of a POU domain-containing

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DISCUSSION cDNA clone:

By the RT-PCR method,

several different POU domain-containing sequences were amplified and cloned using the total RNA from neurula stage zebrafish embryos. One clone of the RT-PCR products (ZFPCRI) showed more than 90% amino acid sequence similarity to the class III POUdomain genes (classification according to the proposal by He et al., ref. 8). Since most of the class III POU-domain genes are expressed mainly in the neural tissues, we attempted to isolate those of zebrafish. We screened the cDNA library made from the poly A+ RNA of neurula stage embryos using the ZFPCRl as a probe, and isolated 4 different positive clones, each having the sequence identical to that of the ZFPCRl.

we

reconstituted a composite sequence by combining them at the unique XhoI site and designated this PGU-domain cDNA as ZFPOUI. Structure of the ZFPOUI POV domain-containing cDNA: The ZFPOUI cDNA was 3171 bp long and contained a long open reading frame (nucleotides 5631894) which will produce a 425 amino acid peptide if the ATG codon at position 620 is an authentic translation initiation site (Fig. 1). This is likely because the sequence around the ATG codon at position 620 matches well with the conserved Kozak sequence for initiation of translation in eukaryotes (14). A typical POU domain was located near the C-terminus of this open reading frame, which included the POU-specific domain (69 a.a.), the linker region (15 a.a.), and the POU-homeo domain (60 a.a.) (Figs. 1 and 2). Since the sequence of the ZFPOUI POU domain was identical to that of the ZFPCRl, ZFPOUZ could be classified as a member of the class III POU-domain genes. As indicated in Fig. 2, in the linker region where the sequence is variable depending on the class, the sequence of ZFPOUI was most similar to those of the class III POU-domain genes. Among the sequences of the class III POU-domain genes, the sequence of the POU domain of ZFPOUI was almost identical to those of the mouse genes, Brain-l and

1 MATAASNPYL?&SSILSSGS 61 AMAASNGGHMLSHAHQWVTS 121 NSGRDDLHSGTALHNRAPHL 181 VNGMHSPPGSQSLVHPGLVR 241 PTSPDLEHFAKOFKORRIKJ, 301 KPLLNKWLEEADSSTGSPTS 361 TSLADNLOLEKEVVRVWFCN 421 MFSET* 425

IVHSDSbGGMQQGSAAVTSV LPk@iAAAAAAAVAAAEAjX GPHQTYAGAWGSTTAAHIPS GDTPELaHSSHHHHHHHQHQ GFKOADVCTALGTLYGNVFS IDKIAAQGPKRKKRTSEVS RROKEKRMTPPGVPQTPEDV

SGGYRGDPTVKMVQSDFMQG PWSSSPVGITGSPQQQDVKN LTG-LIYFAPGGFT HHQQAH&VNSHDPHSDEDT OTTICRFEALOLSFKNMCKL VKGALESHFLKCPKPSAOEI YSQVGNVSADTPPPSMDCKR

Fig. 1, The deduced amino acid sequence encoded by the zebrafish POU domaincontaining cDNA, ZFPOUI. ZFPOUI was 3171 bp long and contained one long open reading frame which will produce a 425 amino acid peptide. The POU-specific domain and the POU-homeo domain are indicated by underlines. These domains are separated by the linker region. Boxes upstream of the POU domain indicate several characteristic regions rich in particular amino acids, such as serine (residues 12-26), alanine (residues 84-98), glutamine (residues 165-169), and histidine/glutamine (residues 208-227). Amino acids are shown in one-letter codes,

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180 240 3oo 360 420

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B sub-domain

SFPDDl

DD~~~~~I~~~~~Y~...SPTTICRPEALQLS~L

III

BrdIl-1 Brain-2 Brdn-4 OCt-6 XI2001 xLPon2 Cfl-a Cab-6

----9--------------T----------------...------------------------------------------------......... ----*--------------=----------------...-----------------------------------s------------.......,. ----9--------------T----------------...------------------------------------------------......... ----9--------------T----------------...-------------------------------*---s----~------.,,..,,., ----P--------------T----------------...-------------------------------T--T-----~------.,,...,.. -=--q--------------*----------------...-------------------------------------w----------......... ----A--------------T---------------. ..-------------------------Q---------T-------------.....,.,, ----G--------------T--------------I-. ..-------------------------p---------T--N...,.,,

I

pit-1

Lg--*--NB--"------YT-~--E--~~-sE-...---------N-------*----*~-s------B~~*-........,.

II

act-1 act-2

IV v

BP--T--EB---------VT-----K--~-XMPGVCSt--S-----S-T-I-.....

I-POD

------VT-----S--BN-KIP~GSL--S------S-T--~--IA---I-~-----E~~~~E~G......... RE--A--ER---------~T-----K--~-~~~~--S------S-T--~--~---~-~---K-

Ore-3

K&--*---LL--K--T--YT------T--“-F-K--...--------------L------B---E----NNWLQEI.....,,

POU-HOME0

LINKER

-

SSTGSPTSIDIUAAQ.........

one-06

Em-3

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DOMAIN -

-

RESEARCH

DOMAIN

BPDAPSWA..

Similarity

Identity

91.9% 91.9 95.0 96.5 95.0 95.1 94.4 00.9

97.2% 91.2 94.4 93.1 93.1 93.0 94.4 83.3

SPPOUl

Ill

act-6

KLPOUl XLPOD2 Cfl-a Cmh-6

----------------------------------N---S----------------------------------------------------------s------------------------------------"--T--------A----S----S--------------------------------G-----------------B---G---S--------------------------------G------N----------B-------S------------------------------------"--T--------A-L-------S---------------------------------------p--B-*-------------S--------------------------------N--SR--F--*SN~--N-----*"-~---------------------

I

pit-1

.----K--T-SULA-D---B---S---MRH-EE---

76.0

61.7

II

act-1 Ott-2

.-R--------TN~RV---~--ENP--TSB---H---*-~---I--------------R--------~---~--~~--TSB--u--BQ-----I-------------

81.3 82.6

68.1 68.1

IV

Dnc-86 Bm-3 I-PW

.D-~R------B-RB--~-K*~-N----A-~--N-D-K-N--------*---~--B-XR-----AAPB-RS--Ay-A”*-N--S~-~-~-D-K-N--------B-K..----~B-RS--AY-A”~K----~-~-D-K-N--------*---*--

67.1 61.6 60.8

53.8 50.4 52.4

v

act-3

..-------NR-RWS--1W--------I-9--BI-NP-G---D------------G--

76.4

63.2

Brain-l Brain-2 Brain-4

&& Comparisons of the deduced amino acid sequence of the ZFPOUl POU domain with those of other POU-domain genes. The sequence of ZFPOUI is presented at the top. A horizontal bar represents an identical residue, and a dot indicates a gap. Roman numerals on the left show the classes of the POU-domain genes (8). References for the sequences of the POWdomain genes are as follows: Brain-l, Brain-2, and Brain-4 (15); Ott-6 (25); XLPOUl and XLPOU2 (26); Cfl-a (27); CA-6 (28); Pit-l (1); Ott-1 (2); Oct.2 (3 ); Uric-86 (4); Brn-3 (8): I-POU (29); Ocr-3 (10).

Bruin-2, isolation and the sequences of which were recently reported (15) (Fig. 2). In the region outside the POU domain, the ZFPOUI cDNA showed a significantly high sequence homology with the Brain-l gene (75% identity at the amino acid level), and a moderate sequence homology with the Brain-2 and Brain-4 genes (15). Thus, the ZFPOUI gene could be a zebrafish homologue of the mouse Brain-l gene. The deduced amino acid sequence of ZFPOUl indicated several regions rich in specific amino acids; namely, serine-rich (residues 12-26), alanine-rich (residues 84-98), glutamine-rich (residues 165169), and histidine-rich (residues 208-227) regions (Fig. 1). Similar characteristic sequences have been found in various transcription regulators (7). 1449

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F&&. Northern blot analysis of the ZFPOLJI transcripts in embryos and adult tissues. Ten pg of total RNA (adult ovary and embryos) or 2 pg of poly A+ RNA (adult liver, intestine, and brain) was analyzed using the 3’ noncoding region of the ZFPOUl cDNA as a probe. The lower panel shows the ethidium bromide staining of the filter.

The ZFPOUI

cDNA was in vitro transcribed and the resultant RNA was used for

in vitro translation. The synthesized ZFPOUl protein product appeared as a 49 kDa molecule in the SDS-polyacrylamide gel electrophoresis (not shown). This apparent molecular weight was only slightly different from the molecular mass of 45.6 kDa calculated from the deduced amino acid sequence of ZFPOUZ. Expression

of the ZFPOUI

gene in embryos and adult tissues: Northern

blot

analysis was carried out using the 3’ noncoding region of the ZFPOUl cDNA as a probe, which revealed that the ZFPOUI transcripts of about 4.2 kb size were present in embryos of various stages (Fig. 3). The ZFPOUI

transcripts were first detected at the early

neurula stage (9-12 hr of development at 28.YC) where the neural tube begins to be formed. The expression increased greatly at the 16-somite stage, reached the maximum level at 36 hr of development, and thereafter decreased. In adult tissues, the ZFPOUI transcripts were not detected in ovary, liver, or intestine, but the distinct signals were obtained with the poly A+ RNA fraction from brain (Fig. 3). In addition to the 4.2-kb transcripts, slightly larger transcripts (5.2 kb) were also detected in the brain RNA sample. In order to determine the tissue distribution of the ZFPOUI transcripts, embryos and adult tissues of zebrafish were subjected to in situ hybridization analysis using the digoxigenin-labeled RNA probes. Consistent with the results of the Northern blot 1450

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Fig. In situ hybridization analysis of the ZFPOUZ transcripts in embryos and adult brain. In situ hybridization was performed using the DIG-labeled antisense RNA probe. (A) A horizontal section of the i6-somite stageembryo (16 hr of development). (B)(C) Frontal sections of the 24-hr embryo made either at midbrain (B) or at the boundary between hindbrain and spinal cord (C). Positive signals were specifically detected in neural tissues. Yolk granules were nonspecifically stained. Numerous small spots in (B) are also nonspecific staining. Dark cells found in the somite region in (C) are not positively stained but contain melanin particles. (D) Schematic presentation of the results with the parasagitally sectioned adult zebrafish brain. Positive signals are drawn by dots. ce, cerebellum; d, diencephalon; e, eye; fp, floor plate; h, hindbrain; ht. hypothalamus; il, inferior lobe of the hypothalamus; ni, nucleus isthmi; m, midbrain; nt, notochord, OS,optic stalk; rp, roof plate; s, somite; SC,spinalcord; spv, stratum periventxicularof the optic tectum; t, telencephalon; tc, tectum; VI, ventro-lateral portion of the neural tube; y, yolk granule. Scale bar, 100 pm.

analysis, the ZFPOUl transcripts were obviously detected in the 6-somite stage embryos (not shown). In this stage embryos, positive signals were exclusively localized in the neural tube. A distinct pattern of localization of the transcripts was observed in embryos of the 16-somite stage.

Intensive staining was restricted to the neural tissues.

Diencephalon, midbrain, cerebellum, hindbrain, and spinal cord were positive for the staining, but telencephalon, optic stalk, and the posterior portion of midbrain were negative (Fig. 4A). Non-neural tissues such as notochord, somite, eye, and skin were also negative. In the 24-hr embryos, the staining pattern was almost identical to that in the 16-somite stage embryos (Fig. 4B,C). Trigeminal ganglion and the inner part of otic vesicles also showed the ZFPOUl transcripts (not shown). On the other hand, no or very weak signals were observed in the dorsal and ventral regions of both hindbrain and 1451

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spinal cord. These regions include the roof plate and the floor plate cells which are thought to be important for neurogenesis (Fig. 4C; ref. 16,17). In addition, we noticed that the ZFPOUl transcripts were not present in the ventro-lateral portions of hindbrain and spinal cord, which correspond to the areas where motor neurons will differentiate in (Fig. 4C). The spatial expression pattern of the ZFPOUZ gene was similar to that of the zebrafish paired-box containing gene, pax[zfu]

(18). The expression pattern of the

second zebrafish paired-box containing gene, par[zf-b],

was almost complementary to

that of the pax[zf-a] gene (19). The regions where the ZFPOUI transcripts were not detected corresponded well to the regions where the pax[zf-b] transcripts were detected; namely, optic stalk, the ventro-lateral portions of hindbrain and spinal cord, and the posterior portion of midbrain. Transcripts of two other zebrafish genes, a homeoboxcontaining gene en-2 (20) and a proto-oncogene writ-l (21), were also localized in the posterior portion of midbrain. The localized expression of the zebrafish genespar, en-2, and writ-Z suggests that segmentation or regionalization of developing neural tissues is essential for differentiation of the central nervous system, and these genes are thought to be involved in determining

the specialization.

Similarly, ZFPOUZ,

a putative

transcription regulator gene showing the confined pattern of expression in the neural tissues during neurogenesis, seems to perform an indispensable function in the specification of neural cell fates as well as in the segmentation process of brain at early embryonic stages. The ZFPOUI transcripts were detected in restricted areas of adult brain, such as the inferior lobe of hypothalamus, the stratum periventricular of optic tectum, the nucleus isthmi, and a part of area dorsalis of telencephalon (according to ref. 22,23; Fig. 4D). The nucleus isthmi receives a substantial projection from the optic tectum, and in turn projects mainly to the upper part of the stratum griseum centrale of the optic tectum in teleosts (24). The nucleus isthmi is thought to be homologous to the parabigeminal nucleus in mammals and to modify both visual and nonvisual input which is transmitted in optic tectum from sensory organs (24). In conclusion, the zebrafish POU-domain gene, ZFPOUI, is a neural tissue specific POU-domain gene which is closely related to the mammalian gene Bruin-l. Its spatial and temporal patterns of expression suggest that the ZFPOUl

gene could be

involved in early neurogenesis and also in some specific function of adult brain.

ACKNOWLEDGMENTS

We would like to thank Dr. C. B. Kimmel for providing the AB-camp strain of zebrafish and Dr. D. J. Grunwald, Mrs. R. Riggleman and K. Helde for providing the cDNA library. We greatly appreciate Dr. Y. Oka for help in identifying the brain structure of zebrafish. and Mr. T. Oki for his excellent technical assistance.

1.

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MOLVEN, A., NJ~LSTAD, P.R. AND FJOSE, A. (1991) EMBO J. 10,799-807. NIEUWENHUYS R. (1982) Amer. Zool. 22, 287-310. VANEGAS, H., EBBENSSON, S.O.E. AND LAUFER, M. (1984) In Comparative neurology the optic tectum, pp 93-120. Plenum, New York. ITO, H., SAKAMOTO, N. AND TAKATSUJI, K. (1982) J. Comp. Neurol. 205,299-311. SUZUKI,N., ROHDEWOHLD, H., NEUMAN, T., GRUSS, P. AND SCHOLER, H.R. (1990)

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