Identification and developmental expression of Xenopus hmga2β

BBRC Biochemical and Biophysical Research Communications 351 (2006) 392–397 www.elsevier.com/locate/ybbrc

Identification and developmental expression of Xenopus hmga2b Francesca Benini a,1, Marco Onorati a,b,1, Sandro Altamura c, Guidalberto Manfioletti c, Robert Vignali a,d,* a

d

Dipartimento di Biologia, Laboratori di Biologia Cellulare e dello Sviluppo, Universita` di Pisa, via Carducci 13, 56010 Ghezzano (Pisa), Italy b Scuola Normale Superiore, Piazza dei Cavalieri 7, 56100 Pisa, Italy c Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, via Giorgieri 1, 34127 Trieste, Italy AMBISEN Center, High Technology Center for the Study of the Environmental Damage of the Endocrine and Nervous System, Universita` di Pisa, Italy Received 22 September 2006 Available online 23 October 2006

Abstract HMGA proteins are ‘‘architectural modifiers’’ of the chromatin, characterized by three conserved ‘‘AT-hook’’ motifs, with which they bind AT-rich regions of the DNA, to assist in gene transcription. We report the identification and developmental expression of Xenopus laevis hmga2b (Xlhmga2b). We provide evidence of two forms of hmga2 (Xlhmga2a and Xlhmga2b) and of a splicing variant for Xlhmga2b with an additional AT-hook. By comparing X. laevis and X. tropicalis hmga2 DNA sequences to those of other organisms we show a high conservation of the Xlhmga2b variant. By RT-PCR, Xlhmga2b transcripts are first detected before the midblastula transition (MBT), and then become more abundant. By in situ hybridization, localized transcripts are first detected at neurula stages, in the presumptive central nervous system (CNS). At tailbud and tadpole stages, Xlhmga2b mRNA is detected in the CNS, in the otic vesicles, in neural crest cell derivatives, in the notochord, and in the medio-lateral mesoderm. Ó 2006 Elsevier Inc. All rights reserved. Keywords: HMGA; Transcription; Chromatin; Xenopus laevis; Embryogenesis; AT-hook

HMGA proteins (HMGA1 and HMGA2) are small proteins made up of about 100 amino acid (aa) residues, widely diffused among all metazoans and present also in plants [1–3]. HMGA proteins are characterized by three highly conserved ‘‘AT-hook’’ motifs, that bind cooperatively to the minor groove of AT-rich regions of DNA [4], and by an acidic C-terminal tail, whose function is not completely defined. HMGA proteins are considered important components and ‘‘architectural modifiers’’ of chromatin [1]. They have more than a mere structural role, since they participate in assembling enhanceosomes and modulate gene transcription. HMGA may either have a positive regulatory effect, as in the NF-jB/HMGA1 interaction [5] and in the CRX/HMGA1 interaction [6], or a *

1

Corresponding author. Fax: +39 050 878486 E-mail address: [email protected] (R. Vignali). These authors contributed equally to this work.

0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.10.074

negative effect, as in the interaction with some homeodomain proteins or in ERCC1 promoter regulation [7,8]. HMGA genes and proteins are mainly expressed in undifferentiated or rapidly proliferating cells. During mouse embryogenesis, Hmga1 and Hmga2 are strongly expressed in tissues derived from all three germ layers [9,10]; at later developmental stages, their expression seems progressively down-regulated to become almost null in adult tissues [11,12]. Several observations suggest their role in cell proliferation and differentiation. The functional inactivation of Hmga2 in mice leads to the pygmy phenotype, with reduced body size [13]; consistently, HMGA2 sustains expression of the cyclin A gene [14] and enhances E2F1 activity [15]. Haploinsufficiency of the Hmga1 gene causes cardiac hypertrophy and myelo-lymphoproliferative disorders [16]. Impaired spermatogenesis was observed for both Hmga1 and Hmga2 deficient mice [17,18]. A role in adipogenesis for both Hmga genes has also been demonstrated [19,20].

F. Benini et al. / Biochemical and Biophysical Research Communications 351 (2006) 392–397

The pronounced and complex phenotype of Hmga1 and Hmga2 knockout animals suggests their relevance in the commitment of several cellular lineages. Indeed, results obtained on embryonic stem cells reveal a function for HMGA2 in skeletal muscle differentiation [21] and for HMGA1 in lympho-haematopoietic differentiation [22]. HMGA proteins may also be involved in modulating neural cell differentiation [23], and a particularly interesting example is given by the interaction of HMGA1 with the homeodomain factor CRX to promote photoreceptor specific transcription [6]. Given the importance of HMGA in development, we decided to characterize these factors in Xenopus laevis, a model system for developmental studies. Two Hmga2-related sequences were recently cloned in X. laevis, Xlhmga2a and Xlhmga2b, but the developmental pattern of expression was only described for the first [24]. We here describe the identification and developmental expression of X. laevis hmga2b. Materials and methods Computational analysis of DNA. To identify HMGA2 cDNA sequences, the amino acid sequence corresponding to the second AT-hook of the human HMGA2 protein (PKRPRGRPKGSK) was used for extensive ESTs database search using the TBLASTN tool with an expect value of 1000. The database employed are the Xenopus EST blast server from the Sanger Institute (http://www.sanger.ac.uk) and the NCBI blast server (http://www.ncbi.nlm.nih.gov/blast/). The ESTs obtained were the following: Homo sapiens (Accession No: NP_003474); Capra hircus (Accession No: BAB64336); Macaca mulatta (Accession No: XM_001117025); Rattus norvegicus (Accession No: NP_114459); Mus musculus (Accession No: NP_835158); Gallus gallus (Accession No: NP_990332); Ambystoma mexicanum (Accession No: CO786995); X. laevis a (Accession No: AW646221); X. laevis ba (Accession No: BC082363); X. laevis bb (Accession No: BI477855); X. tropicalis (Accession No:

393

AL955898); Danio rerio 1 (Accession No: NM_212680); D. rerio 2 (Accession No: AL913631). ESTs were translated in protein using the Expasy translate tool (http://www.expasy.ch) and aligned and compared with Clustalw algorithm from EBI (http://www.ebi.ac.uk/clustalw). Cloning and RT-PCR. RT-PCR was performed as described [25]. We used the following PCR primers, derived from sequence BC082363, and containing EcoRI linkers: hmgF: 5 0 -GGGAATTCATGAGCTCAAGGGAAGGAGCG-3 0 hmgR2: 5 0 -GGGAATTCCTAGTCGTCTTCAGATTCCTGG-3 0 For amplification we used: 1 cycle at 94 °C for 5 0 ; 25 cycles at 94 °C for 3000 , 55 °C for 1 0 , 72 °C for 3000 , and 72 °C for 5 0 . The amplified HMGA2 coding region from stage 37 embryo cDNA was cloned into the pGEM7Zf(+) plasmid, to generate pGEM-Xlhmga2b. The same primers were used to analyse the temporal expression of Xlhmga2b by RT-PCR, along with ornythine decarboxylase (ODC) control primers [26]. Xenopus embryos and in situ hybridizations. X. laevis embryos were obtained and staged as described [27,28]. For whole-mount in situ hybridization (WISH), embryos were processed, bleached, and sectioned as in [29–31]. Digoxygenin (DIG)-labelled antisense and sense probes were generated by standard procedures from pGEM-Xlhmga2b template.

Results and discussion Identification of hmga2 cDNA sequences in Xenopus Extensive database search allowed us to identify several Xenopus EST sequences highly homologous to human HMGA2; two of them were recently reported [24] as XLHMGA2a and XLHMGA2b, respectively (Fig. 1). XLHMGA2b, but not XLHMGA2a, deduced peptide sequence is also present in the EST database of the close species X. tropicalis. Because X. laevis is pseudotetraploid, the Xlhmga2a sequence may represent a pseudoallelic duplicate. In fact, only the Xlhmga2b sequence was found in the X. trop-

Fig. 1. ClustalW alignment of HMGA2 amino acid sequences found in database search. Conserved AT-hooks are shaded. Amino acid identities are represented by (*), conservative amino acid substitutions by (:), and semi-conservative substitutions by (.).

394

F. Benini et al. / Biochemical and Biophysical Research Communications 351 (2006) 392–397

icalis genome database. Interestingly, the Xlhmga2 gene structure organization is similar to that of the human and mouse genes. It consists of five exons, similar in size to those of the human and mouse genes (data not shown). Besides Xlhmga2a and Xlhmga2b, we also found a splicing variant of Xlhmga2b (referred to as Xlhmga2bb) that encodes an additional 17-aa sequence, including an extra AT-hook (Fig. 1). XLHMGA2bb therefore is peculiar since it contains four AT-hooks instead of the usual three. At a close inspection, the additional aa sequence of XLHMGA2bb is identical to aa 66–83 of the third AThook. This sequence is encoded by a single exon of the known hmga2 genes and therefore may result from an internal duplication of this exon. Significantly, it is not present in X. tropicalis. Only two ESTs from unfertilized eggs were found for the Xlhma2bb variant, possibly representing maternal transcripts, while eight sequences were found for Xlhmga2ba, deriving from different developmental stages. From the above, reported considerations, and from aa sequence comparison, showing that the XLHMGA2b variant is more similar than XLHMGA2a to HMGA2 sequences from other species, we decided to concentrate on XLHMGA2b. Developmental expression of Xlhmga2b The use of a variant-specific set of primers allowed us to amplify Xlhmga2b from stage 37 embryos. No Xlhmg2a

sequences were found in the amplification products. With this primer set, we studied the developmental expression of Xlhmga2b by RT-PCR (Fig. 2A). A low level of maternal transcripts seems to be present at the 2-cell stage, but after mid-blastula Xlhmga2b expression becomes progressively up-regulated, especially after neurula stage, to decline at the swimming tadpole stage (stage 42). These results are consistent with those of Hock et al. [24], reporting that Xlhmga2b transcripts become the most abundant form beyond tailbud stage. Interestingly, only PCR amplification products of the size corresponding to the Xlhmga2ba isoform were detected in all the stages, suggesting that Xlhmga2bb splicing variant expression is very low and stage specific. In whole-mount in situ hybridization (WISH) experiments, we used a probe consisting of the coding region of Xlhmga2b. Because the Xlhmga2a and Xlhmga2b sequences are about 83% identical at the nucleotide level, in principle we are not sure to discriminate between the two transcripts; however, since we were not able to see the early expression pattern described for Xlhmga2a [24], we presume that our probe does not cross-hybridize under the conditions used. Therefore, we will refer to the following results as to Xlhmga2b expression; this assumption is reinforced by the fact that Xlhmga2a transcripts level off after gastrulation [24]. In WISH, we did not detect localized Xlhmga2b transcripts before neurula stage. At stage 15, Xlhmga2b

Fig. 2. (A) RT-PCR analysis of Xlhmga2b and ODC transcription during development. Developmental stages numbered as in [28]. (B–K) Expression of Xlhmga2b transcripts as detected by WISH during neurula stages. (B) stage 15, dorsal view: arrows show faint labelling in the anterior region of the neural plate; arrowheads show staining around the blastopore; (C) stage 15, posterior view; (D) stage 18, dorsal–lateral view; (E) stage 18, posterior view; (F) stage 18, anterior view; (G) stage 18 embryo hybridized with a sense probe, in dorsal view; (H) stage 20, dorsal view; (I) stage 20, posterior view; (J) stage 20, anterior view: arrowheads show more intensely labelled stripes in the presumptive brain region; (K) stage 20, sense probe, dorsal–lateral view.

F. Benini et al. / Biochemical and Biophysical Research Communications 351 (2006) 392–397

transcripts are detectable around the blastopore (Fig. 2B, arrowheads; C) and, with very faint intensity, in the anterior neuroectoderm (Fig. 2B, arrows). The entire neuroectoderm is strongly labelled at a slightly later stage (stage 18) (Fig. 2D–G), just before neural tube closure. Probe staining is detectable in the anterior neural plate, most probably embracing also the presumptive eye field (Fig. 2F). A strong labelling is still detectable around the blastopore (Fig. 2E). Use of a sense probe shows that this pattern is specific, both for this and the previously described stage (Fig. 2G and data not shown). At neural plate closure (stage 20), Xlhmga2b expression is maintained in the most anterior neural plate region and the eye field (Fig. 2H and J); more intensely labelled transversal stripes are visible within the presumptive brain (Fig. 2J, arrowheads). Xlhmga2b expression is also detected in the posterior neural ectoderm, as a thin stained line posterior to the hindbrain (Fig. 2H), and around the blastopore (Fig. 2I). No staining was observed with the sense probe (Fig. 2K). At tailbud stage (stage 25, stage 29, and stage 33/34), Xlhmga2b transcripts are detected in the brain region and, at lower level, in the spinal cord. Expression in the proencephalic, mesencephalic, and rhombencephalic regions has a striped pattern reflecting different intensities of labelling (Fig. 3A–D). Transcripts are also detected in

395

the otic vesicles (Fig. 3D and G). Only limited, diffused expression is observed in the eye vesicle by stage 29 and 33/34 (Figs. 3F and 4F; data not shown). Xlhmga2b expression is strong in the neural crest-derived material that will form the pharyngeal arches (Fig. 3B, C, E, and F). In general, the endodermal layer did not show strong labelling by the probe, close to the visceral arches (Fig. 3E). This is at variance with what was described for both Hmga1 and Hmga2 in the mouse, where they are transcribed in numerous endodermal derivatives [9,10]; however, it must be observed that endodermal derivatives are somehow refractory to probe penetration in Xenopus [29]. Instead, Xlhmga2b expression is readily detected in the splanchnic layer of the intermediate mesoderm (Fig. 3B, C, H, and K), just ventral to the somites, that will give rise to the vascular structures of the excretory system [32]. In strongly stained embryos, the Xlhmga2b probe labelled the whole lateral and ventral aspects, suggesting that it may be expressed at a lower level also in this part of the mesoderm (data not shown). Moreover, Xlhmga2b is expressed in the heart-forming region, in the developing myocardium and endocardium (Fig. 3G). In many sections, labelling was observed dorsal to the neural tube and adjacent to the dorsal tip of the somites, as well as in the medial aspect of somites (Fig. 3H and data not shown); it is possible that this labelling is due, in part, to migrating neural crest cells

Fig. 3. Expression of Xlhmga2b transcripts at tailbud stages, as detected by WISH. (A) Expression at stage 25; labelling is clearly visible in the head region, and in the dorsal trunk region. (B) Expression at stage 29: strong labelling is detected in the neural tube (nt), especially in transversal stripes (black arrowheads), in the intermediate mesoderm (im, black arrowhead), and in the pharyngeal arches (pa, white arrowheads). (C) Expression at stage 33/34: arrowheads indicate expression in the pharyngeal arches, intermediate mesoderm, and in the notochord (no). (D) Detail of the head region of a stage 25 Xenopus embryo showing distribution of Xlhmga2b transcripts in the neural tube (arrowheads), and in the otic vesicle (arrows). (E) Horizontal section of a stage 29 embryo following in situ hybridization with a Xlhmga2b probe, showing intense labelling in the neural crest derivatives of the pharyngeal arches (from I to IV), but not in the endoderm. (F) Transversal section at an anterior level of a stage 33/34 embryo showing Xlhmga2b expression in neural crest cells (ncc) and in the pharyngeal arches (pa). (G) Transversal section of a stage 33/34 embryo at the level of the otic vesicles, showing Xlhmga2b transcript localization in the otic vesicles (ov), as well as in the heart-forming region (my, myocardium; ec, endocardium). (H) Transversal section of a stage 33/34 embryo at the trunk level, showing Xlhmga2b transcript localization in the neural crest cells (ncc) close to the dorsal neural tube and in the splanchnic layer of the intermediate mesoderm (im); the notochord is only slightly positive. (K) Transversal section of a stage 33/34 embryo at the posterior trunk level, showing Xlhmga2b transcript localization in the notochord (no) and intermediate mesoderm (im).

396

F. Benini et al. / Biochemical and Biophysical Research Communications 351 (2006) 392–397

Fig. 4. Expression of Xlhmga2b transcripts at tadpole stage 39 as detected by WISH. (A) Distribution of Xlhmga2b transcripts at stage 39. (B,C,D) Sections of stage 39 embryos following WISH to Xlhmga2b probe show transcript distribution in the head region, in the ventricular zone (vz, arrowheads) of the neural tube, in the condensing cartilaginous cells of the first pharyngeal (maxillary) arch (m), and in the condensing ceratohyal originating from the second pharyngeal arch (ch, arrowheads). (E) Horizontal section of a stage 39 embryo reveals strong staining of the notochord by the Xlhmga2b probe; somites (so) are only faintly stained. (F,G) Sections of the developing eye at stage 29 and 33/34, respectively, may be compared to those in section (D), at stage 39, showing initial limited expression of Xlhmga2b in the developing lens and in the surrounding neural retina territory (F, stage 29), that increases later at stage 33/34 to decline by stage 40. Picture shown in (D) was obtained by juxtaposition of two separate pictures of the same section.

[33,34]. At stage 25 and 29 faint labelling was observed on the notochord (data not shown), but at stage 33/34 Xlhmga2b transcripts are clearly detected in the middle part of the notochord (Fig. 3K). This pattern of expression tends to be maintained in the following tadpole stages up to stage 39 with an apparently increased level of expression, consistent with RT-PCR results. At stage 39, the anterior region of the embryo stains very strongly with Xlhmga2b probe (Fig. 4A), especially in the pharyngeal region. In the anterior central nervous system, Xlhmga2b expression is restricted to the inner part of the neural tube, possibly corresponding to the ventricular region (Fig. 4B–D). The rest of the neural tube is now only faintly labelled. The notochord, instead, is intensely labelled along most, if not all, of its length (Fig. 4E). Consistent with RT-PCR, Xlhmga2b expression at later stages (stage 42) is strongly down-regulated in the embryo tissues. Transcripts are detected only in the pharyngeal arches, and, at low levels, in restricted parts of the CNS (data not shown). Xlhmga2b expression pattern shows important similarities, as well as differences, with that of mouse. Similarly to mouse Hmga2 and Hmga1, it is expressed during embryogenesis in several tissues and organs of diverse embryological origin. Although RT-PCR analysis detected Xlhmga2b transcripts before MBT ([24] and present data) our WISH failed to reveal localized mRNA before neurula stage, when transcripts are detected in the CNS. Expression in the CNS is intense and widespread during tailbud stages, but then becomes down-regulated at later stages, to persist, in the ventricular region of the brain; this is similar to what was observed in the mouse [10,35] and is consistent with the idea that HMGA2 plays a role in proliferation of neural precursors [36]. While mouse Hmga2 expression is mainly limited to the telencephalic region [10,35], Xlhmga2b is also

transcribed in the spinal cord, recalling some aspects of mammalian Hmga1 expression [9]. Expression in the eye is rather limited, and may better correspond to the pattern observed for Hmga2 in mouse [35] than to the strong expression seen in the eye for Hmga1 [9]. Xlhmga2b is also expressed in neural crest cell derivatives in the anterior region, such as the visceral skeleton of the pharyngeal region; this expression is shared with mouse Hmga1 [9], while it is not clear if murine Hmga2 is expressed in these neural crest derivatives [10]. A new site of expression for Xlhmga2b, not previously described for mouse Hmga genes, is here found in the notochord. In conclusion, we have characterized the expression pattern of Xlhmga2b during Xenopus early development. This expression may complement, in time, that of Xlhmga2a [24], whose transcripts were found in the blastula stage embryo. It is possible that the two resulting proteins have similar biochemical properties (being 90% identical) but perform their actions at different developmental times due to differential transcriptional control of their genes. Alternatively, they could perform different biochemical functions because of minor, but important, differences in their peptide sequences. Future gain- and loss-of function experiments need to be performed to address the developmental role of these genes in Xenopus. Our present data provide the necessary expression knowledge to interpret the phenotypic effects that may result from such experiments.

Acknowledgments We thank Donatella De Matienzo, Marzia Fabbri, and Salvatore Di Maria for technical assistance and frog care. This work was supported by PRIN 2005–2007 contribution to G.M. and to R.V., and by the ‘‘Giovani Ricercatori

F. Benini et al. / Biochemical and Biophysical Research Communications 351 (2006) 392–397

2004’’ contribution from Scuola Normale Superiore di Pisa to M.O. References [1] R. Reeves, Molecular biology of HMGA protein: hubs of nuclear function, Gene 277 (2001) 63–81. [2] K.D. Grasser, Chromatin-associated HMGA and HMGB proteins: versatile co-regulators of DNA-dependent processes, Plant Mol. Biol. 53 (2003) 281–295. [3] R. Sgarra, A. Rustighi, M.A. Tessari, J. Di Bernardo, S. Altamura, A. Fusco, G. Manfioletti, V. Giancotti, Nuclear phosphoproteins HMGA and their relationship with chromatin structure and cancer, FEBS lett. 574 (2004) 1–8. [4] R. Reeves, M.S. Nissen, The AT-DNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure, J. Biol. Chem. 2651 (1990) 8573–8582. [5] J. Yie, M. Merika, N. Munshi, G. Chen, D. Thanos, The role of HMG I(Y) in the assembly and function of the IFN-beta enhanceosome, EMBO J. 18 (1999) 3074–3089. [6] K.Y. Chau, N. Munshi, A. Keane-Myers, K.W. Cheung-Chau, A. Kwong-Fuk Tai, G. Manfioletti, K. Dorey, D. Thanos, D.J. Zack, S.J. Ono, The architectural transcription factor high mobility group I(Y) participates in photoreceptor-specific gene expression, J. Neurosci. 20 (2000) 7317–7324. [7] P. Arlotta, A. Rustighi, F. Mantovani, G. Manfioletti, V. Giancotti, G. Tell, G. Damante, High mobility group I proteins interfere with the homeodomains binding to DNA, J. Biol. Chem. 272 (1997) 29904–29910. [8] L. Borrmann, R. Schwanbeck, T. Heyduk, B. Seebeck, P. Rogalla, J. Bullerdiek, J.R. Wisniewski, High mobility group A2 protein and its derivatives bind a specific region of the promoter of the DNA repair gene ERCC1 and modulates its activity, Nucleic Acids Res. 31 (2003) 6841–6851. [9] G. Chiappetta, V. Avantaggiato, R. Visconti, M. Fedele, S. Battista, F. Trapasso, B.M. Merciai, F. Fidanza, V. Giancotti, M. Santoro, A. Simeone, A. Fusco, High level expression of the HMGI(Y) gene during embryonic development, Oncogene 13 (1996) 2439–2446. [10] U. Hirning-Foltz, W.M. Rippe, J. Bullerdiek, H. Hameister, The expression pattern of Hmgic gene during development, Genes Chromosomes Cancer 23 (1998) 350–357. [11] G. Manfioletti, V. Giancotti, A. Bandiera, E. Buratti, P. Sautiere, P. Cary, C. Crane-Robinson, B. Coles, G.H. Goodwin, cDNA cloning of the HMGI-C phosphoprotein, a nuclear protein associated with neoplastic and undifferentiated phenotypes, Nucleic Acids Res. 19 (1991) 6793–6797. [12] J. Liu, J.F. Schiltz, P.C. Shah, K.F. Benson, K.K. Chada, Genomic structure and expression of the murine Hmgi(y) gene, Gene 246 (2000) 197–207. [13] X. Zhou, K.L. Benson, H.R. Ashar, K. Chada, Mutation responsible for the mouse pygmy phenotype in the developmentally regulated factor HMGI-C, Nature 376 (1995) 771–774. [14] M. Tessari, M. Gostissa, S. Altamura, R. Sgarra, A. Rustighi, C. Salvano, G. Caretti, C. Imbriano, R. Mantovani, G. Del Sal, V. Giancotti, G. Manfioletti, Transcriptional activation of the cyclin A gene by the architectural transcription factor HMGA2, Mol. Cell. Biol. 23 (2003) 9104–9116. [15] M. Fedele, R. Visone, I. De Martino, G. Troncone, D. Calmieri, S. Battista, A. Ciarmiello, P. Pallante, C. Arra, R.M. Melillo, K. Helin, C.M. Croce, A. Fusco, HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity, Cancer Cell 9 (2006) 459–471. [16] M. Fedele, V. Fidanza, S. Battista, F. Pentimalli, A.J. Klein-Szanto, R. Visone, I. De Martino, A. Curcio, C. Morisco, L. Del Vecchio, G.

[17]

[18] [19] [20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28] [29]

[30] [31]

[32] [33]

[34] [35]

[36]

397

Baldassarre, C. Arra, G. Viglietto, C. Indolfi, C.M. Croce, A. Fusco, Haploinsufficiency of the Hmga1 gene causes cardiac hypertrophy and myelo-lymphoproliferative disorders in mice, Cancer Res. 66 (2006) 2536–2543. P. Chieffi, S. Battista, M. Barchi, S. Di Agostino, G.M. Pierantoni, M. Fedele, L. Chiarotti, D. Tramontano, A. Fusco, HMGA1 and HMGA2 protein expression in mouse spermatogenesis, Oncogene 21 (2002) 3644–3650. J. Liu, J.F. Schiltz, H.R. Ashar, K.K. Chada, HmgA1 is required for normal sperm development, Mol. Reprod. Dev. 66 (2003) 81–89. A. Anand, K. Chada, In vivo modulation of Hmgic reduces obesity, Nat. Genet. 24 (2000) 377–380. M. Fedele, S. Battista, G. Manfioletti, C.M. Croce, V. Giancotti, A. Fusco, Role of the high mobility group A proteins in human lipomas, Carcinogenesis 22 (2001) 1583–1591. L. Caron, F. Bost, M. Prot, P. Hofman, B. Binetruy, A new role for the oncogenic high-mobility group A2 transcription factor in myogenesis of embryonic stem cells, Oncogene 24 (2005) 6281–6291. S. Battista, F. Pentimalli, G. Baldassarre, M. Fedele, V. Fidanza, C.M. Croce, A. Fusco, Loss of Hmga1 gene function affects embryonic stem cell lympho-hematopoietic differentiation, FASEB J. 17 (2003) 1496–1498. F. Cerignoli, C. Ambrosi, M. Mellone, I. Assimi, L. di Marcotullio, A. Gulino, G. Giannini, HMGA molecules in neuroblastic tumors, Ann. N.Y. Acad. Sci. 1028 (2004) 122–132. R. Hock, F. Witte, J. Brocher, M. Schutz, U. Scheer, Expression of HMGA2 variants during oogenesis and early embryogenesis of Xenopus laevis, Eur. J. Cell Biol. 85 (2006) 519–528. L. Poggi, T. Vottari, G. Barsacchi, J. Wittbrodt, R. Vignali, The homeobox gene Xbh1 cooperates with proneural genes to specify ganglion cell fate within the Xenopus neural retina, Development 131 (2004) 2305–2315. T. Bouwmeester, S. Kim, Y. Sasai, B. Lu, E.M. De Robertis, Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann’s organizer, Nature 382 (1996) 595–601. J. Newport, M. Kirschner, A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription, Cell 30 (1982) 687–696. P.D. Nieuwkoop, J. Faber, Normal Table of Xenopus laevis (Daudin), second ed., North-Holland, Amsterdam, 1967. R.M. Harland, In situ hybridization: an improved wholemount method for Xenopus embryos, Methods Cell Biol. 36 (1991) 675– 685. R. Mayor, R. Morgan, M.G. Sargent, Induction of the prospective neural crest of Xenopus, Development 121 (1995) 767–777. M. Pannese, G. Lupo, B. Kablar, E. Boncinelli, G. Barsacchi, R. Vignali, The Xenopus Emx genes identify presumptive dorsal telencephalon and are induced by head orgnaizer signals, Mech. Dev. 73 (1998) 73–83. A. Brandli, Towards a molecular anatomy of the Xenopus pronephric kidney, Int. J. Dev. Biol. 43 (1999) 381–395. B. Sadaghiani, C.H. Thiebaud, Related Articles, Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy, Dev. Biol. 124 (1987) 91–110. P. Hausen, M. Riesebell, The early development of Xenopus laevis, an atlas of the histology, Springer Verlag, Berlin, 1991. J. Lord-Grignon, M. Abdouh, G. Bernier, Identification of genes expressed in retinal progenitor/stem cell colonies isolated from the ocular ciliary body of adult mice, Gene Expr. Patterns 6 (2006) 992–999. K.Y. Chau, G. Manfioletti, K.W. Cheung-Chau, A. Fusco, N. Dhomen, J.C. Sowden, T. Sasabe, S. Mukai, S. Ono, Derepression of HMGA2 gene expression in retinoblastoma is associated with cell proliferation, Mol. Med. 9 (2003) 154–165.