SHORT COMMUNICATION ______www.paper.edu.cn Lhx4, a LIM Tsuyoshi Yamashita, Kenji Moriyama,* Hui Z. Sheng, and Heiner Westphal1 Laboratory of Mammalian and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2790; and *Department of Anatomy, Shimane Medical University, 89-1 Enya-cho, Izumo, Shimane 693, Japan

Received February 19, 1997; accepted June 10, 1997

closely related gene Lhx4 (originally termed Gsh-4)is LIM homeobox genes are well conserved in evolution expressed in specific fields of the brain and spinal cord, and play important roles as transcriptional regulators and mutant embryos that lack Lhx4 function die at of embryonic development. Here we report on the birth (4). Lhx4 is located on 1, approxi- structure of LIM domains of the mouse Lhx4 (Gsh4) mately 3.3 cM proximal to the Pmx paired mesoderm gene. The cDNA was generated by modified reverse homeobox locus (3). Screening of a variety of cDNA transcription-PCR from midgestation embryo tem- libraries to determine the structure of Lhx4, however, -plates, using a degenerate consensus primer. The de- failed to produce the 5؅ end of these transcripts, pre duced amino acid sequence of the first LIM domain dicted to encode the LIM motifs (Ref. 4, and our own reveals 77% identity and that of the second domain observations). reveals 86% identity with the corresponding sequences of the closely related Lhx3 gene. In addition, there is To establish Lhx4 as a member of the Lhx gene fam- 38–56% similarity to other members of the Lhx gene ily, we applied a modified reverse-transcription (RT)- family. The LIM consensus sequence is well conserved PCR method to generate cDNA that contains LIM do- in Lhx4. ᭧ 1997 Academic Press mains of Lhx4. Thirty micrograms of total RNA isolated with the help of RNAzol (Tel-Test, Inc.) from E12.5 FVB/N embryos was used as a template to generate A recent review by Dawid et al. (1) summarizes the first-strand cDNA, using reverse primer A (Table 1) initial experiments that identified the family of LIM and the superscript II RT kit (GIBCO, BRL) for the RT homeodomain and shed light on their function reaction as described by the manufacturer. Subse- in cell determination and specification in the devel- quently, seminested PCR was performed using modi- oping organism, from Caenorhabditis elegans to verte- fied primer sets. The degenerate forward primer B in- brates. The characteristic feature of these proteins is cluded an XbaI restriction site within stabilizer se- a double zinc-finger LIM motif located N-terminally of quences, and the reverse primer D contained two an adjacent homeodomain. A rapidly growing number nucleotide substitutions that enabled us to create SalI of LIM homeobox genes are being isolated from worm, restriction sites for further subcloning. The PCR mix- fly, frog, rodent, chicken, and human genomes, and a ture consisted of one-fourth of the RT product in a 50- strong evolutionary conservation of sequence and func- ml reaction containing 0.25 mM concentrations of prim- tion among homologs is suggested by all available data. ers B and C (Table 1), 0.25 mM dNTP, 1.5 mM MgCl2 , The goal of our own research is to study the Lhx genes, 1 ml of DNA polymerase mixture (Taq/Pwo polymer- mouse equivalents of known vertebrate LIM homeobox ase), and 5 ml of reaction buffer provided by the Expand genes. High Fidelity PCR System (Boehringer-Mannheim). Lhx3 (9) was cloned as the apparent ortholog of the After denaturation for 5 min at 94ЊC, the first 30 cycles Xenopus Xlim-3 gene (7). Expression is highly re- of PCR were carried out at 94ЊC for 1.5 min, 42ЊC for stricted to specific fields of the developing brain, the 1 min, and 72ЊC for 3 min. Subsequently, a second retina, the pituitary, and the spinal cord. Lhx3 null round of 35 cycles (equal conditions) was performed mutant embryos lack the anterior and intermediate with primers B and D, which were added to a 100- lobes of the pituitary, indicating an important role of fold dilution of the first-round products (Table 1). The this gene in the development of this organ (5). The identity of the 0.6-kb amplification product was con- firmed by Southern hybridization with a specific probe Sequence data from this article have been deposited with the corresponding to the C-terminal region of Lhx4. The EMBL/GenBank Data Libraries under Accession No. U89343. fragment was digested with XbaI and SalI restriction 1 To whom correspondence should be addressed at Laboratory of enzymes and subcloned into pBluescript KS (Stra- Mammalian Genes and Development, National Institutes of Child Health and Human Development, Building 6B, Room 413, National tagene). Sequencing was carried out by dideoxy chain Institutes of Health, Bethesda, MD 20892-2790. Telephone: (301) termination using the Sequenase Version 2.0 Sequenc- 496-1855. Fax: (301) 402-0543. E-mail: [email protected]. ing kit (U.S. Biochemicals). To verify the sequence data

GENOMICS 44, 144–146 (1997) 144 ARTICLE NO. GE974852 0888-7543/97 $25.00 Copyright ᭧ 1997 by Academic Press All rights of reproduction in any form reserved.

AID GENO 4852 / 6r3f$$$181 07-22-97 03:02:28 gnmxa SHORT COMMUNICATION 145 ______中国科技论文在线 www.paper.edu.cn TABLE 1 Primers for Reverse Transcription Reaction and PCR

Reverse transcription (Primer A 5؅-AGTGTGGTCTCCGCCTCCTGGGA-3؅ (Reverse Nested PCR 1st PCR (Primer B 5؅-TGCTCTAGAATTCTGTGCNGGCTG-3؅ (Forward XbaI (Primer C 5؅-CCTCAAGTCCTGATAGGATTG-3؅ (Reverse 2nd PCR (Primer B 5؅-TGCTCTAGAATTCTGTGCNGGCTG-3؅ (Forward XbaI (Primer D 5؅-CTCCTG* TC*GACACTCTTGTAGAACTG-3؅ (Reverse SalI

Note. Primer A is located in the 3؅-UTR; Primers C and D correspond to the coding region between homeobox and 3؅ UTR. Primer D contains two nucleotide substitutions (*) for creating a SalI restriction site. The degenerate primer B contains sequence (Boldface) that was predicted from related sequences of LIM domain 1 of Lhx1, 2, 3, and 5. obtained from cDNA generated by PCR, BAC genomic amino acid sequence of the Lhx4 LIM domains is com- clones (Genome Systems, Inc.) screened with the above- pared with those of the other known members of the mentioned Lhx4-specific probe were subcloned and se- Lhx gene family, Lhx3 is clearly identified as the closest quenced as well (data not shown). relative (Fig. 1b). This close structural relationship be- We found that the LIM consensus sequence (1) was tween the Lhx4 and the Lhx3 genes had been deduced perfectly conserved (Fig. 1a). The two LIM domains earlier from a comparison of their homeodomains (4). are encoded by two separate exons. When the deduced Taken together, these observations invite a close exam-

FIG. 1. (a) The nucleotide sequence of the Lhx4 LIM domains. Amino acids that identify the LIM motifs are underlined. Arrows indicate exon–intron boundaries. (b) Comparison of the deduced amino acid sequence of various Lhx4, Lhx3 (9), Lhx1 (2), Lhx2 (8), and Lhx5 sequences (6). Dots mark identity with the Lhx4 sequence. The degree of identity is indicated to the right.

AID GENO 4852 / 6r3f$$$182 07-22-97 03:02:28 gnmxa 146 SHORT COMMUNICATION ______中国科技论文在线 www.paper.edu.cn ination of potential functional synergy between these Scott, W. J., and Potter, S. S. (1994). Gsh-4 encodes a LIM-type two genes, which is a focus of our current efforts. homeodomain, is expressed in the developing nervous system, and is required for early postnatal survival. EMBO J. 13: 2876– 2885. ACKNOWLEDGMENTS 5. Sheng, H. Z., Zhadanov, A. B., Mosinger, B., Jr., Fujii, T., Ber- tuzzi, S., Grinberg, A., Lee, E. J., Huang, S. P., Mahon, K. A., We thank S. Steven Potter and Hung Li for providing the Lhx4- and Westphal, H. (1996). Specification of pituitary cell lineages specific probe for Southern hybridization and genomic screening. by the LIM homeobox gene Lhx3. Science 272: 1004–1007. 6. Sheng, H. Z., Bertuzzi, S., Chiang, C., Shawlot, W., Taira, M., Dawid, I. B., and Westphal, H. (1997). Expression of murine REFERENCES Lhx5 suggests a role in specifying the forebrain. Dev. Dyn. 208: 1–12. [In press] 1. Dawid, I. B., Toyama, R., and Taira, M. (1995). LIM domain 7. Taira, M., Hayes, W. P., Otani, H., and Dawid, I. B. (1993). . C.R. Acad. Sci. Paris 318: 295–306. Expression of LIM class homeobox gene Xlim-3 in Xenopus de- 2. Fujii, T., Pichel, J. G., Taira, M., Toyama, R., Dawid, I. B., and velopment is limited to neural and neuroendocrine tissues. Dev. Westphal, H. (1994). Expression patterns of the murine LIM Biol. 159: 245–256. class homeobox gene lim-1 in the developing brain and excretory 8. Xu, Y., Baldassare, M., Fisher, P., Rathbun, G., Oltz, E. M., system. Dev. Dyn. 199: 73–83. Yancopoulos, G. D., Jessel, T. M., and Alt, F. W. (1993). LH-2: 3. Kern, M. J., Argao, E. A., Birkenmeier, E. H., Rowe, L. B., and A LIM/homeodomain gene expressed in developing lymphocytes Potter, S. S. (1994). Genomic organization and chromosome lo- and neural cells. Proc. Natl. Acad. Sci. USA 90: 227–231. calization of the murine homeobox gene Pmx. Genomics 19: 9. Zhadanov, A. B., Bertuzzi, S., Taira, M., David, I. B., and West- 334–340. phal, H. (1995). Expression pattern of the murine LIM class 4. Li, H., Witte, D. P., Banford, W. W., Aronov, B. J., Weistein, M., homeobox gene Lhx3 in subsets of neural and neuroendocrine Kaur, S., Wert, S., Singh, G., Schriener, C. M., Whitsett, J. A., tissues. Dev. Dyn. 202: 354–364.

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