Hereditas 147: 132–135 (2010)
FISH mapping of six genes responsible for development of the nervous and skeletal systems on donkey (Equus asinus) chromosomes MONIKA BUGNO-PONIEWIERSKA1, KLAUDIA PAWLINA2, ANETA DARDZIN´ SKA2, TOMASZ ZA˛BEK1, EWA SŁOTA1 and JOLANTA KLUKOWKA-RÖTZLER3 1
Department of Immuno and Cytogenetics, National Research Institute of Animal Production, Balice, Poland Interdepartmental Biotechnology Studies Program, University of Agriculture, Kraków, Poland 3 Institute of Genetics, Vetsuisse Faculty, University of Berne, Switzerland 2
Bugno-Poniewierska, M., Pawlina, K., Dardzin´ska, A., Za˛bek, T., Słota, E. and Klukowka-Rötzler, J. 2010. FISH mapping of six genes responsible for development of the nervous and skeletal systems on donkey (Equus asinus) chromosomes. – Hereditas 147: 132–135. Lund, Sweden. eISSN 1601-5223. Received March 2, 2010. Accepted April 16, 2010. The results obtained in the present study made it possible to place selected markers responsible for development of the nervous and skeletal systems on the physical map of the donkey genome. Fluorescence in situ hybridization (FISH) was used to localize genes such as GDF5 (15q13), FRZB (4q23.1), TWIST (1q31), PAX6 (20q25), SALL1 (24q15) and SHH (1q35) on donkey chromosomes. The identification of their localization confirmed previously proposed homologies using ZOO-FISH technique, except for FRZB and SALL1 genes. This suggests that they were affected by rearrangements that changed their localization compared to horse, and in the case of the SALL1 gene also compared to human. Monika Bugno-Poniewierska, Department of Immuno- and Cytogenetics, National Research Institute of Animal Production, Krakowska 1, PL–32-083 Balice, Poland. E-mail:
[email protected]
Animals of the order Perissodactyla, family Equidae, show great diversity in chromosome morphology and number (RYDER et al. 1978; POWER 1984). Such a high degree of differentiation that occurred in short evolution time is suggestive of rapid karyotype changes (WICHMAN et al. 1991). The horse (Equus caballus) karyotype has received the most study of Equidae, followed by donkey (Equus asinus), which is mainly compared to the horse (RAUDSEPP and CHOWDHARY 2001). To detect homologies and possible rearrangements between Equus caballus and Equus asinus species, ZOO-FISH was performed using horse chromosome (ECA1-13, X and Y) probes on donkey metaphase plates (RAUDSEPP and CHOWDHARY 1999). Then, possible homologies between horse (ECA14, 16, 18, 19, 20, 21, 23, 24, 26, 28) and donkey chromosomes (EAS9, 21, 4, 5, 8, 10, 23, 7, 18, 4) were proposed based on the G-banding pattern and mapped markers (RAUDSEPP et al. 2001); in some cases, homology is only partial or requires further investigation. Homology between ECA22, 23, 25 and EAS15, 23, 10p was determined indirectly based on ZOO-FISH with human probes on horse chromosomes (ECA) (RAUDSEPP et al. 1996) and with human probes on donkey chromosomes (RAUDSEPP and CHOWDHARY 2001). In 2004, YANG et al. performed ZOO-FISH with horse probes from all autosomes and X chromosomes on donkey chromosomes, which made it possible to validate and extend previous results (YANG et al. 2004). The number of possible rearrangements between horse and donkey karyotypes was estimated by the same authors to exceed 20
© 2010 The Authors. This is an Open Access article.
(RAUDSEPP et al. 2001). The standard donkey karyotype was proposed based on G-banding (GTG) chromosome analysis (RAUDSEPP et al. 2000). Research is also underway to compare donkey and human karyotypes (RAUDSEPP et al. 1999; RAUDSEPP and CHOWDHARY 2001). In the present study six genes were localized with FISH technique on donkey chromosomes. GDF5 (Growth/Differentiation Factor 5), also known as CDMP1 (Cartilage-Derived Morphogenetic Protein 1) or LAP4 (Lipopolysaccharide-Associated Protein 4), is a member of the TGF-beta superfamily (STORM et al. 1994). GDF5 is one of the factors required for the proper formation of bones and joints in the skull, limbs and axial skeleton (SETTLE et al. 2003). FRZB (Frizzled-Related Protein), also known as SFRP3 (Secreted Frizzled-Related Protein 3), is expressed in the developing skeletal structures such as the appendicular skeleton, several craniofacial bones and epiphyseal ends of the rib cage (HOANG et al. 1996). FRZB is also suggested as a tumor suppressor gene (LEYNS et al. 1997). TWIST1 (TWIST, Drosophila, Homolog Of, 1), also known as TWIST, belongs to the basic helix-loop-helix (bHLH) class of transcriptional regulators (PAN et al. 2009). According to SHISHIDO et al. (1993) Twist might affect the transcription of FGFRs (Fibroblast Growth Factor Receptors) being an upstream regulator of FGFRs. PAX6 (Paired Box Gene 6) is a member of the paired box gene family and encodes a transcriptional regulator involved in oculogenesis and other developmental processes. PAX6
DOI: 10.1111/j.1601-5223.2010.02178.x
Hereditas 147 (2010) affects the eye development, including the neuroectoderm, the eye surface ectoderm as well as related tissues (ASHERY-PADAN and GRUSS 2001). PAX6 is involved in the development of the Rathke pouch and early anterior pituitary gland, and its expression controls the established boundaries of somatotrope, lactotrope and thyrotrope cell types (KIOUSSI et al. 1999). SALL1 (SAL-Like 1) encodes a protein of 1306 amino acids. The SALL1 gene is expressed in a wide range of human tissues, with highest expression in kidney, brain and liver (KOHLHASE et al. 1996). SHH (Sonic Hedgehog) is a homolog of the Drosophila gene HH (Hedgehog) that encodes inductive signals during embryogenesis. SHH plays a vital role during early embryogenesis in vertebrates. It is expressed in the Hensen node, the floorplate of the neural tube, the early gut endoderm, the posterior of the limb buds, and throughout the notochord (ECHELARD et al. 1993; ROELINK et al. 1994). MATERIAL AND METHODS Chromosome preparation and G-banding Pokeweed-stimulated peripheral blood lymphocyte cultures from donkey were used to obtain metaphase preparations. For FISH and karyotyping the donkey cell suspensions were dropped onto microscope slides. The G-banding of donkey chromosomes was performed by trypsin treatment according to WANG and FEDOROFF (1974). FISH mapping The INRA equine BAC library (MILENKOVIC et al. 2002) was screened by polymerase chain reaction (PCR) using primers designed to amplify parts of the equine GDF5, FRZB, TWIST, PAX6, SALL1 and SHH genes. Primers were first tested for PCR amplification on whole genomic equine DNA before screening the library. DNA of positive clones (GDF5: INRA0190A08, FRZB: INRA0035B12 TWIST: INRA0038D02, PAX6: EBAA30E6, SALL1: EBAA90A4, SHH: INRA0522B12) was prepared from 500 ml overnight cultures of the positive BAC clone using the Qiagen Midi plasmid kit (Qiagen AG, Basel, Switzerland) according to the alkaline lysis protocol for BACs. Direct sequencing of BACs was performed on an ABI 3730 sequencer using BigDye ver. 3.1 chemistry (Applied Biosystems, Rotkreuz, Switzerland). One hundred ng BAC DNA was labelled with Biotin – 16-dUTP and used for FISH on photographed G-banded donkey metaphase. Donkey chromosomes were identified according to the GTG-banded chromosome nomenclature (RAUDSEPP et al. 2001) using 10–20 mataphase spreads. Hybridization followed the standard protocol (PINKEL et al. 1986). The analysis of FITC fluorescence signals was done on propidium iodide-stained slides.
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RESULTS The FISH experiments led to the chromosomal assignment of predicted donkey genes as follows: GDF5 on EAS15q13, FRZB on EAS4q23.1, TWIST on EAS1q31, PAX6 on EAS20q25, SALL1 on EAS24q15, SHH on EAS1q35 (Fig. 1, Table 1).
DISCUSSION Six new genes related to development of the nervous and skeletal systems were placed on the physical map of Equus asinus. The loci of these genes were first identified in humans and then in horses and donkeys (Table 1). Comparison of their localization in these species makes it possible to detect similarities and differences, and thus to infer possible rearrangements that might have occurred during evolution. Chromosome 15 of the donkey (EAS15) shows homology to the entire horse chromosome 22 (ECA22) (RAUDSEPP et al. 1996; RAUDSEPP and CHOWDHARY 2001; YANG et al. 2004), and both of them show complete homology to human chromosome 20 (HSA20) (YANG et al. 2004). Chromosome 20 (HSA20) is highly conservative and its homologies were found in all hitherto investigated mammals (WIENBERG and STANYON 1997; CHOWDHARY et al. 1998; HAIG 1998) except the gibbon (KOEHLER et al. 1995). Even in mice, whose karyotype underwent rapid evolution, synteny has been preserved on a single MMU2 chromosome (CHOWDHARY et al. 1998). However, despite the conservation, no similarity is observed in the G-banding pattern of chromosomes homologous to HSA20 (RAUDSEPP and CHOWDHARY 2001). The p arm of donkey chromosome 4 (EAS4p) shows homology to the entire chromosome ECA28, and the q arm (EAS4q) to ECA18. EAS4 is homologous to HSA22, 12 and 2 fragments; ECA28 to HSA22 and 12 fragments; and ECA18 to a HSA2 fragment (YANG et al. 2004). However, our results differ from the data presented above. The FRZB gene was localized on the q arm of donkey chromosome 4 (EAS4q), and on the q arm of horse chromosome 15 (ECA15q) rather than chromosome 18 (ECA18), as suggested by ZOO-FISH results. Homology is shown for the localization in the human and the donkey; in humans, the FRZB gene is located on the q arm of chromosome 2 (HSA2q), which is also confirmed by ZOO-FISH, because homology is shown between HSA2q and EAS4q. Differences in our and ZOO-FISH results may be due to the fact that ZOO-FISH shows homology of whole segments or chromosomes but provides no details on the organization and arrangement of single genes that may differ despite the homology shown; therefore, EAS4 and ECA18 chromosomes can be homologous except the fragment in which the FRZB gene is localized on EAS4, which ZOOFISH is unable to demonstrate.
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Hereditas 147 (2010) Table 1. Comparison of the results of physical mapping. HSA-Homo sapiens chromosomes, ECA-Equus caballus chromosomes, EAS-Equus asinus chromosomes. Gene name GDF5 FRZB TWIST SHH PAX6 SALL1
Fig. 1. Chromosomal assignment of the equine GDF5, FRZB, TWIST, PAX6, SALL and SHH genes by FISH. Chromosomes with signals are in the left panels. GTGbanding patterns of the corresponding metaphase spreads are in the right panels. A comparison of the G-banded ideograms and FISH-hybridized chromosomes are shown in the central panels.
A rearrangement – translocation or deletion and insertion of that chromosome fragment in which the FRZB gene is located – could have occurred during karyotype evolution, as a result of which it is located on different chromosomes in the donkey and horse. Further investigations are necessary to confirm this.
HSA
ECA
EAS
20q11.2 2q31-q33 7p21 7q36 11p13 16q12.1
22q16 (Zabek et al. 2007) 15q24 (Zabek et al. 2007) 4q14 (Zabek et al. 2007) 4q26 (Zabek et al. 2007) 7q19 (Zabek et al. 2007) 3p16 (Zabek et al. 2009)
15q13 4q23.1 1q31 1q35 20q25 24q15
The situation is analogous for the TWIST and SHH genes, because both occur on the same chromosome. An EAS1p fragment shows homology to ECA31 (YANG et al. 2004), and EAS1q to ECA4 and ECA31 (RAUDSEPP and CHOWDHARY 1999; YANG et al. 2004). EAS1p is also homologous to an HSA6 fragment, and EAS1q to HSA7 and a fragment of HSA6, while ECA4 is homologous to HSA7 fragments (YANG et al. 2004). Thus, the results obtained confirm the results of ZOO-FISH. Another mapped gene – PAX6 – was localized on the q arm of donkey chromosome 20 (EAS20q). This chromosome is homologous to horse chromosome 7 (ECA7), and both are homologous to fragments of human chromosomes 11 and 19 (HSA11 and 19) (RAUDSEPP and CHOWDHARY 1999; YANG et al. 2004), which is consistent with the results obtained. Donkey SALL1 gene is localized on chromosome 24 (EAS24), which is homologous to the q arm of horse chromosome 10 (ECA10q), and both are homologous to human chromosome 6 (HSA6) (RAUDSEPP and CHOWDHARY 1999; YANG et al. 2004). However, the localization of the SALL1 gene differs from the homology shown. This gene is localized on chromosome 24 in the donkey (EAS24) and on chromosome 3 (ECA3) rather than chromosome 10 (ECA10) in the horse, which would have been expected based on ZOO-FISH. In humans, the SALL1 gene is localized on chromosome 16 (HSA16) rather than chromosome 6 (HSA6), as also indicated by ZOO-FISH results. Conformity with ZOO-FISH only occurs when we compare the localization of this gene in horse and in human, because the p arm of horse chromosome 3 (ECA3p) is homologous to the q arm of human chromosome 16 (HSA16q) (RAUDSEPP et al. 1999; YANG et al. 2004). As for the FRZB gene, the discrepancy between the results obtained and ZOO-FISH when comparing the donkey with horse and human may be due to the fact that the ZOO-FISH technique shows homology of larger segments without providing information on the exact organization, which means that small rearrangements may go undetected. Presumably, an event (translocation or deletion-insertion) that occurred during karyotype evolution of the donkey
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