Molecular Human Reproduction, Vol. 6, No. 8, 707-711,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Embryo development |
Transcription of homeobox-containing genes detected in cDNA libraries derived from human unfertilized oocytes and preimplantation embryos
Molecular Medicine Unit, Molecular Embryology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
Abstract
Genes containing the evolutionarily conserved homeodomain sequence encode a family of DNA-binding transcription factors whose functions are crucial for embryonic development in vertebrates, invertebrates and plants. We describe the detection and analysis of transcripts of homeobox-containing genes present in cDNA libraries generated from human unfertilized oocytes, single cleavage stage embryos (2-cell, 4-cell, 8-cell and blastocyst) and a 10-week old whole fetus. Using degenerate primers derived from sequences within helix 1 and helix 3 of the highly conserved region of the Antennapedia-class homeodomain, a 166 bp band was detected in all the cDNA libraries tested. Subcloning of the oocyte-derived band revealed that it contained a heterogeneous group of 166 bp fragments. Sequence analysis of 40 independent clones demonstrated the presence of HOXA7, HOXD8, and HOXD1 sequences, the ubiquitously expressed POU family member, OCT1, and HEX, a homeotic gene expressed in haematopoietic cells.
embryonic cDNA libraries/homeobox gene expression/human preimplantation development/maternal mRNA/OCT-4
Introduction
Homeotic proteins are encoded by key developmental genes conserved throughout evolution. These proteins share a highly conserved sequence of 60 amino acids known as the homeodomain. Gene expression studies and nuclear magnetic resonance investigations have demonstrated that the three dimensional structure of the homeodomain is in the form of a helix-turn-helix capable of binding promoter regions at 3'TAAT5' sequences (Gehring et al., 1994
). This implies that homeotic proteins are transcription factors controlling key developmental processes such as differentiation and patterning during early development (Favier and Dolle, 1997). It is also well established that abnormal or aberrant expression of homeobox-containing genes results in several congenital abnormalities and oncogenesis (Cillo et al., 1999).
The human and mouse HOX/Hox homeobox-containing genes are highly related to the Antennapedia (Antp) homeotic gene first cloned in the fruitfly, Drosophila melanogaster. The HOX/Hox genes are arranged in four clusters (A, B, C and D) on different chromosomes. Based on sequence similarities, the highly related genes in different clusters are designated by the same number and referred to as paralogues (for a review, see Boncinelli and Thorogood, 1997). Recent studies have shown that the specificity of binding and the activity of homeotic proteins is modulated by protein–protein interactions involving other regions of the proteins besides the homeodomain itself (Sugihara et al., 1998). Thus, several families of homeotic proteins contain a second conserved domain. Examples include: (i) the paired (prd) domain present in PAX/Pax genes, (ii) the POU domain, present in OCT/Oct genes, and (iii) the cysteine -histidine rich LIM domain (Deschamps and Meijlink, 1992).
Very little is known about the expression of homeotic genes in early development. We can assume that the regulation of human early embryonic development is initially under the control of maternally-inherited mRNA and proteins. Embryonic (zygotic) genes are activated at different times of development, starting as early as the 1-cell zygote stage (for reviews, see Daniels and Monk, 1997; Pergament and Fiddler, 1998). Maternal and/or embryonic homeobox-containing genes, and other genes whose structures imply a role in transcriptional regulation are expected to play key roles in the regulation of early human development and therefore merit investigation.
Previous studies using reverse transcription–polymerase chain reaction (RT–PCR) protocols on lysed embryos have demonstrated the transcription of known homeobox-containing genes in human unfertilized oocytes and cleavage stage embryos. Thus, HOXA4, HOXA7, HOXB4 and HOXB5 are present in oocytes and cleaving embryos (Verlinsky et al., 1995; Kuliev et al., 1996). OCT6 is expressed later than OCT4 and is detectable from the 10-cell stage. OCT4 is also continuously expressed from the unfertilized oocyte through to the blastocyst as shown by RT–PCR analysis of lysed human embryos (Abdel-Rahman et al., 1995) or by analysis of cDNA libraries derived from human oocytes and single 2-cell, 8-cell and blastocyst stage embryos (Adjaye et al., 1999). The expression of OCT4 in human oocytes and embryos supports a role of OCT4 in the maintenance of pluripotency in early human development as previously established in the mouse from studies where Oct4-deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not pluripotent (Nichols et al., 1998).
Gene knockout studies in mice have shown that blastocysts in females homozygous for the loss of Hoxa10 and Hoxa11 genes die by 3.5 days post-coitum, suggesting that these genes and their corresponding proteins are required for embryonic development to implantation (Hsieh-Li et al., 1995; Satokata et al., 1995). Further work is required to establish precise functions for these and other key developmental homeotic genes in early mammalian embryos, both mouse and human.
In this paper, rather than analysing expression of specific known homeobox genes, we analyse cDNA libraries from human unfertilized oocytes and single 2-cell, 4-cell, 8-cell and blastocyst stage embryos (Adjaye et al., 1997, 1999) for the complete spectrum of expressed homeobox-containing genes, including the possibility of novel genes transcribed in early human development. So far, we have shown transcription of HOXA7, HOXD8, HOXD1 sequences, of the ubiquitously expressed POU family member, OCT1, and of HEX, a homeotic gene expressed in haematopoiesis.
Materials and methods
PCR amplification of homeodomain sequences
PCR amplifications were carried out in a total volume of 25 µl consisting of 1x PCR buffer, 1 IU Amplitaq DNA polymerase (Perkin Elmer, UK), 200 µmol/l of each dNTP (Pharmacia, UK), 10 ng of cDNA sample from each library and 25 pmoles of each primer. The libraries were prepared as described previously (Adjaye et al., 1999) from four human unfertilized oocytes, and single preimplantation embryos at the 2-cell, 4-cell, 8-cell and blastocyst stage of development and a whole 10-week old fetus to serve as a somatic control. The degenerate primer sequences directed at helix 1 and 3 of the Antennapedia homeodomain (Murtha et al., 1991) are as follows:
Sense: 5'-TACCAGAC(C/G)(C/T)TGGA(A/G)CTGGAGAA(A/G) GA(A/G)TT(C/T)C-3'
Antisense: 5'-C/T)TTCCA(C/T)TTCAT(C/G)C(G/T)(A/C/G/T)CG (A/G)TTCTG(A/G)AACCAGAT-3'
Primers were synthesized by Genosys Biotechnologies (Europe) Ltd, Cambs, UK.
Cycling parameters consisted of an initial denaturation step at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 40°C for 1 min, elongation at 72°C for 30 s, then a final elongation step at 72°C for 10 min. The PCR amplification was carried out using a Perkin Elmer thermal cycler.
Gel electrophoresis, and subcloning of fragments.
After PCR amplification, 5 µl of the reaction products were resolved on 3% agarose gels containing 2 µg/ml ethidium bromide for 60 min at 100 V. The products were visualized and photographed under short wavelength UV light. For subcloning of the PCR fragments in the 166 bp band from the oocyte cDNA library, the remaining PCR mixture for the oocyte library was resolved and the 166 bp band (Figure 1) excised and eluted using Qiagen elution columns (Qiagen, Crawley, UK). The fragments were ligated into a TA-cloning vector using 100 ng of cDNA and 50 ng of pGEM-TEasy (Promega, Southampton, UK). The ligated mixture was transformated into Escherichia coli strain XL-10 (Stratagene, Amsterdam, The Netherlands).
|
Plasmid DNA isolation and nucleotide sequencing
Isolation and restriction enzyme analysis of plasmid DNA and all other standard molecular biology procedures followed established protocols (Sambrook et al., 1989
). cDNA inserts were sequenced in both orientations employing a Sequenase cycle sequencing kit (Amersham Pharmacia Biotech). Sequence analyses were performed with computer programs implemented within the University of Wisconsin Genetics Computer Group (GCG) software on the HGMP network. Results
Amplification of homeodomains by the polymerase chain reaction
To identify homeobox-containing genes involved in the regulation of preimplantation development in humans, we employed degenerate primers for the Antennapedia-homeo- domain (Murtha et al., 1991) for PCR amplification of homeobox sequences present in cDNA libraries derived from human unfertilized oocytes (n = 4) and single 2-cell, 4-cell, 8-cell and blastocyst stage embryos. A library from a 10-week gestation whole fetus served as a somatic control for comparisons of gene expression in embryonic and somatic cells (Adjaye et al., 1999). The degenerate primers (see Materials and methods) are directed at the homeodomain sequences YQTLELEKEF (helix 1, nucleotide positions 31–61) and KIWFQNRR (helix 3, nucleotide positions 139-172) respectively with position 1 defined as the first nucleotide of the first codon of the Antennapedia-homeodomain. These sequences represent regions common to a large number of homeotic genes with Antennapedia-like homeodomains (Affolter et al., 1990). The amplification product of 166 bp was detected in human oocytes and throughout preimplantation development (Figure 1
).
Amino acid sequence analysis of oocyte-derived fragments
Subcloning of the oocyte derived band and sequence analysis of 40 independent clones provides an indication of the relative abundance of different homeobox gene transcripts. For the amino acid sequence alignments of the various homeodomains and their comparison with the archetypal Antennapedia-class homeodomain see Figure 2.
|
Of the 40 clones, 35 sequences were represented by OCHD-1 (87.5%). Two of the 35 clones had amino acid sequence identities of 98%, and the remaining 33 clones had 100% identity with HOXA7 respectively (Boncinelli et al., 1989
). The observed low frequency of amino acid substitutions (2%) may be due to base changes resulting from PCR-induced misincorporation or allele-specific polymorphisms. More clones will have to be sequenced to validate allele-specific polymorphisms.
Two of the 40 clones (OCHD-26) showed 100% identity with HOXD8 (Oliver et al., 1989), and one of the 40 clones (OCHD-25) showed 100% identity with human HEX, (aematopoietically xpressed homeobo) (Crompton et al., 1992). Clone OCHD-37, which appeared once, showed 100% identity with HOXD1, a human homeodomain sequence derived from the embryonic carcinoma cell line NTERA-2 (Stornaiuolo et al., 1990) and 99% identity (sequence not shown) with mouse Hoxd1 (Frohman and Martin, 1992). Another clone OCHD-3, appeared once and shared 99% identity with OCT1 (Sturm et al., 1988). There is a conserved amino acid substitution–valine (V) to isoleucine (I) in helix 3 of OCHD-3 (I) compared to the published OCT1(V) sequence (highlighted in bold, Figure 2).
Discussion
As an initial step towards identifying regulatory genes that might be involved in the control of human preimplantation development, we surveyed for the presence of homeobox-containing transcripts at early stages of human development using cDNA libraries (Adjaye et al., 1999) prepared from four human unfertilized oocytes, and single preimplantation embryos at the 2-cell, 4-cell, 8-cell and blastocyst stages of development. A heterogenous 166 bp band representing various homeodomains was detected in all the stages analysed by PCR amplification. Sequence analysis of 40 independent clones derived from the unfertilized oocyte library revealed the presence of transcripts of HOXA7, HOXD8, HEX, HOXD1 and OCT1. Interestingly, HOXA7 has previously been shown to be transcribed in human oocytes and cleavage stage embryos (Verlinsky et al., 1995; Kuliev et al., 1996). However, the detection of HOXD8, HEX, HOXD1 and OCT1 at these early stages of human development is reported here for the first time.
The high level of expression of a member of the HOXA cluster relative to expression within clusters B, C and D, in human unfertilized oocytes has not been previously demonstrated and the significance of this is not known at present. Employing degenerate primers based on the archetypal Antennapedia-class homeodomain (Murtha et al., 1991), which is highly conserved within the HOX genes, results in a bias towards detection of the HOX family genes compared to the other homeodomain-containing genes. Despite the bias towards the HOX family (38/40, 95%), transcription of HEX and OCT1 genes was also detectable. This suggests that the primers are capable of amplifying other homeobox-containing genes but at a reduced efficiency. As shown in Figure 2, amino acid sequence conservation is highest within the helix-turn-helix motif especially, as expected, within helix 1 (YQTLELEKEF) and helix 3 (KIWFQNRR) where the primers are located. This increase in conservation is also manifested by the observed higher amino acid sequence identity of the HOX homeodomains relative to OCT1 and HEX at these regions.
The haematopoetic gene, HEX, is expressed in multipotential progenitor cells and in B-lymphocytes and myeloid lineages, but is not expressed in T-lymphocytes nor erythroid cells. This pattern of expression suggests a role in haematopoietic differentiation (Bedford et al., 1993). OCT1 is a ubiquitously expressed member of the family of octamer-binding proteins containing the POU-homeodomain (Sturm et al., 1988). The conserved valine to isoleucine substitution in helix 3 of OCT1 (V) and OCHD-3 (I) could be an allele-specific polymorphism, or a result of nucleotide misincorporation by the Taq polymerase.
For additional, and a more extensive analysis of the complete spectrum of known and novel homeobox-containing genes, PCR screens will have to be carried out on the original pool of uncloned cDNAs rather than the cloned (cDNAs inserted into a vector) libraries. This strategy should overcome the unavoidable phenomenon of reduced mRNA representation associated with library constructions.
In terms of function, it is known that the anterior HOX/Hox genes regulate specific hindbrain segments and neural crest derivatives, while the posterior genes regulate growth and morphogenesis of skeletal structures along the proximo-distal axis of developing limbs (for reviews, see Favier and Dolle, 1997; Boncinelli and Thorogood, 1997). The function of the HOX/Hox cluster genes in oogenesis and preimplantation development is unknown at present. It is possible that in human oocytes, transcripts of HOXA7, HOXD8, HEX and HOXD1 may perform non-essential roles. Alternatively, these transcripts may be present as `masked' maternal mRNAs maintained in a translationally repressed state during oogenesis by their association with specific subsets of mRNA-binding proteins (Lieb et al., 1998; Paynton, 1997) until such a time when the functional proteins are required. A precedent for this level of translational control is observed in Drosophila bicoid and caudal, which are homeotic genes expressed both maternally and embryonically. Both mRNAs are expressed in the oocyte but are translationally repressed (Dworkin and Dworkin-Rastl, 1990; Niessing et al., 1999).
Our high quality human preimplantation embryo cDNA libraries (Adjaye et al., 1997, 1999), have enabled the demonstration of transcription of homeobox-containing genes by PCR. However, the functional role in proliferation and differentiation during human early development remains to be determined. In addition, PCR analyses in the blastocyst library has been used to demonstrate the expression of arylamine N-acetyltransferase (NAT1), an enzyme which catalyses the acetylation of the folate catabolite p-aminobenzoyl-L-glutamate (pABGlu) to produce the major urinary product, N-acetyl-p-ABGlu (Smelt et al., 2000). Such analyses of expression of key developmental genes, as well as the isolation and characterization of novel embryonic genes, will provide greater insight into the control of early human development at the molecular level.
Acknowledgments
This work was supported by the Medical Research Council.
Notes
1 To whom correspondence should be addressed at: Molecular Medicine Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. E-mail: J.Adjaye{at}ich.ucl.ac.uk 
References
Abdel-Rahman, B., Fiddler, M., Rappolee, D. and Pergament, E. (1995) Expression of transcription regulating genes in human preimplantation embryos. Mol. Hum Reprod., 1, see Hum Reprod., 10, 2787–2792
Adjaye, J., Daniels, R., Bolton, V. and Monk, M. (1997) cDNA libraries from single human preimplantation embryos. Genomics, 46, 337–344.[Web of Science][Medline]
Adjaye, J., Bolton, V. and Monk, M. (1999) Developmental expression of specific genes detected in high quality cDNA libraries from single human preimplantation embryos. Gene, 237, 373–383.[Web of Science][Medline]
Affolter, M., Schier, A. and Gehring, W. (1990) Homeodomain proteins and the regulation of gene expression. Curr. Opin. Cell Biol., 2, 485–495.[Medline]
Bedford, F.K., Ashworth, A., Enver, T. et al. (1993) HEX: a novel homeobox gene expressed during haematopoiesis and conserved between mouse and human. Nucleic Acid Res., 21, 1245–1249.
Boncinelli, E. and Thorogood, P. (1997) The expression of Hox genes in postimplantation human embryos. In Strachan, T., Lindsay, S. and Wilson, D.I. (eds), Molecular Genetics of Early Human Development. Bios Scientific Publishers, Oxford, UK, pp. 171–189.
Boncinelli, E., Acampora, D., Pannese, M. et al. (1989) Organization of human class I homeobox genes. Genomics, 31, 745–756.
Cillo, C., Faiella, A., Cantile M. et al. (1999) Homeobox genes and cancer. Exp. Cell Res., 248, 1–9.[Web of Science][Medline]
Crompton, M.R, Bartlett, T.J, MacGregor, A.D. et al. (1992) Identification of a novel vertebrate homeobox gene expressed in haematopoietic cells. Nucleic Acid Res., 20, 5661–5667.
Daniels, R. and Monk, M. (1997) Gene expression in human preimplantation development. In Strachan, T., Lindsay, S. and Wilson, D.I. (eds), Molecular Genetics of Early Human Development. Bios Scientific Publishers, Oxford, UK, pp. 155–166.
Deschamps, J. and Meijlink, F. (1992) Mammalian homeobox genes in normal development and Neoplasia. Crit. Rev. Oncogenesis, 3, 117–173.[Medline]
Dworkin, M.B. and Dworkin-Rastl, E. (1990) Functions of maternal mRNA in early development. Mol. Reprod. Dev., 26, 261–297.[Web of Science][Medline]
Favier, B. and Dolle, P. (1997) Developmental functions of mammalian Hox genes. Mol. Hum. Reprod., 3, 115–131.
Frohman, M.A. and Martin, G.R. (1992) Isolation and analysis of embryonic expression of Hox-4.9, a member of the murine labial-like gene family. Mech. Dev., 38, 55–67.[Web of Science][Medline]
Gehring, W.J., Qian, Y.Q., Bileter, M. et al. (1994) Homeodomain-DNA recognition. Cell, 78, 211–223.[Web of Science][Medline]
Hseih-Li, H.M., Witte, D.P., Weinstein, M. et al (1995) Hoxa11 structure, extensive antisense transcription, and function in male and female fertility. Development, 121, 1373–1385.[Abstract]
Kuliev, A., Kukharenko, V., Morozov, G. et al. (1996) Expression of homeobox-containing genes in human preimplantation development and in embryos with chromosomal aneuploidies. J. Assist. Reprod. Genet., 13, 177–181.[Web of Science][Medline]
Lieb, B., Carl, M., Hock, R. et al. (1998) Identification of a novel mRNA-associated protein Exp. Cell, Res., 245, 272–281.
Murtha, M.T., Leckman, J.F., and Ruddle, F.H. (1991) Detection of homeobox genes in development and evolution. Proc. Natl Acad.. Sci. USA, 88, 10711–10715.
Nichols, J., Zevnik, B., Anastassiadis, K. et al. (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor OCT4. Cell, 59, 379–391.
Niessing, D., Dostatni, N., Jackle, H. et al. (1999) Sequence interval within the PEST motif of Bicoid is important for translation repression of caudal mRNA in the anterior region of the Drosophila embryo. EMBO J., 18, 1966–1973.[Web of Science][Medline]
Oliver, G., Sidell, N., Fiske, N. et al. (1989) Complementary homeoprotein gradients in developing limb buds. Genes Dev., 3, 641–650.
Paynton, B.V. (1998) RNA-binding proteins in mouse oocytes and embryos: expression of genes encoding Ybox, DEAD box RNA helicases, and polyA binding proteins. Dev. Genet., 23, 285–298.[Web of Science][Medline]
Pergament, E. and Fiddler, M. (1998) The expression of genes in human preimplantation embryos. Prenat. Diagn., 18, 1366–1373.[Web of Science][Medline]
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratories Press, Cold Spring Harbor, New York, USA.
Satokata, I., Benson, G. and Maas, R. (1995) Sexually dimorphic sterility phenotypes in Hoxa10-deficient mice. Nature, 374, 460–463.[Medline]
Smelt, V.A., Upton, J., Adjaye, J. et al. (2000) Expression of arylamine N-acetyltransferases in pre-term placentas and in human pre-implantation embryos. Hum. Mol. Genet., 9, 1101–1107.
Stornaiuolo, A., Acampora, D., Pannese, M. et al. (1990) Human HOX genes are differentially activated by retinoic acid in embryonal carcinoma cells according to their position within the four loci. Cell Diff. Dev., 31, 119–127.[Web of Science][Medline]
Sturm, R.A., Das, G. and Herr, W. (1988) The ubiquitous octamer-binding protein Oct-1 contains a POU domain with a homeobox subdomain. Genes Dev., 12, 1582–1599.
Sugihara, T.M., Bach, I., Kioussi, C. et al. (1998) Mouse Deformed epidermal autoregulatory factor 1 recruits a LIM domain factor, LMO-4 and CLIM coregulators. Proc. Natl Acad. Sci. USA, 95, 15418–15423.
Verlinsky, Y., Morozov, G., Gindilis, V. et al. (1995) Homeobox gene expression in human oocytes and pre-embryos. Mol. Reprod. Dev., 41, 127–132.[Web of Science][Medline]
Submitted on January 7, 2000; accepted on May 12, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. M. Kocabas, J. Crosby, P. J. Ross, H. H. Otu, Z. Beyhan, H. Can, W.-L. Tam, G. J. M. Rosa, R. G. Halgren, B. Lim, et al. The transcriptome of human oocytes PNAS, September 19, 2006; 103(38): 14027 - 14032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Huntriss, M. Hinkins, and H.M. Picton cDNA cloning and expression of the human NOBOX gene in oocytes and ovarian follicles Mol. Hum. Reprod., May 1, 2006; 12(5): 283 - 289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Serafica, T. Goto, and A. O. Trounson Transcripts from a human primordial follicle cDNA library Hum. Reprod., August 1, 2005; 20(8): 2074 - 2091. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Holding, V. Bolton, and M. Monk Detection of human novel developmental genes in cDNA derived from replicate individual preimplantation embryos Mol. Hum. Reprod., September 1, 2000; 6(9): 801 - 809. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||