CHAPTER 5 DIFFERENTIAL EXPRESSION OF THE GROUCHO RELATED GENES 4 AND 5 DURING EARLY DEVELOPMENT OF XENOPUS LAEVIS

Size: px

Start display at page:

Download "CHAPTER 5 DIFFERENTIAL EXPRESSION OF THE GROUCHO RELATED GENES 4 AND 5 DURING EARLY DEVELOPMENT OF XENOPUS LAEVIS"

Carmel Skinner
5 years ago
Views:

1 CHAPTER 5 DIFFERENTIAL EXPRESSION OF THE GROUCHO RELATED GENES 4 AND 5 DURING EARLY DEVELOPMENT OF XENOPUS LAEVIS Miranda Molenaar*, Elisabeth Brian*, Jeroen Rooseª, Hans Cleversª and Olivier Destrée Hubrecht Laboratory, Netherlands Institute for Develomental Biology, Usalalaan 8, 584 CT Utrecht, The Netherlands ªDeartment of Immunology, Utrecht Medical Center, P.O. Box 85500, 508 GA Utrecht, The Netherlands *contributed equally Mechanisms of Develoment 90, in ress (000)

3 CHAPTER 5 67 ABSTRACT Recently, we demonstrated that the Xenous Wnt effector XTcf- interacts with Groucho-related transcritional reressors. A long form of the Groucho-related genes,, was shown to reress axis formation in the Xenous embryo, whereas a short form,, acted as a otentiator. In this study, the temoral and satial exression of and is described in Xenous laevis embryos. Both genes are maternally exressed. In the gastrula, transcrits of both genes are resent in the animal as well as the vegetal region. At later stages, and show secific atterns of exression in the central nervous system (CNS), cranial ganglia, eyes, otic vesicles, stomodeal-hyohyseal anlage, cement gland, head mesenchyme, branchial arches, neural crest and derivatives, somites, ronehros, ronehric duct, heart and tailbud. Differences in the exression of and were found in the CNS, cranial ganglia, olfactory lacodes, stomodeal-haryngeal anlage, cement gland, head mesenchyme and ectoderm. INTRODUCTION Vertebrate homologues of Drosohila Groucho (Gro) 7 were isolated from human 6, mouse 8-, rat 4, Xenous, and zebrafish 7. A naturally truncated form, termed Grg-5, consists of the two amino-terminal domains of Gro, and is exressed in man mouse, rat 4 and Xenous,. The develomental exression of Gro-related genes in vertebrates was studied by Northern blot,9,,6,7 and/or in situ hybridization,8-0,7,8. Xenous Grg-4 and Grg-5 both interact with the Xenous member of the Tcf family of transcrition factors, XTcf-. reresses both Armadillo-XTcf-- driven transcritional activation of a Tcf reorter and Arm-XTcf--mediated axis dulication. In contrast, de-reresses a Tcf reorter and otentiates Arm-XTcf-- mediated secondary axis formation. To obtain a better insight in the ossible roles of different Grg family members during the earliest stages of vertebrate develoment, we studied the exression atterns of and by Northern blot and whole mount in situ hybridization.

68 DIFFERENTIAL EXPRESSION OF XGRG-4 AND -5 METHODS and cdnas and robes.

was reviously described as XAES by Choudhury et al. (997), (GenBank accession number U8776).

For Figs as shown we used a robe covering nucleotides 70 to + 650, and for, +05 to + 68.

Embryo maniulation and in situ hybridization Albino embryos were cultured and collected as described by Molenaar et al. (998).

4 68 DIFFERENTIAL EXPRESSION OF XGRG-4 AND -5 METHODS and cdnas and robes. A Xenous brain cdna library in λgt 5 was screened at low stringency with murine Grg-4 and Grg-5 cdna robes as described by Roose et al., 998. was reviously described as XAES by Choudhury et al. (997), (GenBank accession number U8776). encodes full length and is identical to the artial cdna described as XESG by Choudhury et al. (997). The accession number for is AJ4945. For Figs as shown we used a robe covering nucleotides 70 to + 650, and for, +05 to Northern blotting RNA isolation and Northern blot hybridization were erformed as described by Destrée et al. (99) 4. Embryo maniulation and in situ hybridization Albino embryos were cultured and collected as described by Molenaar et al. (998). The and cdna s were used to generate digoxigenin labeled antisense RNA robes. The whole mount ISH rocedure was adated and modified from Harland (99) 6. RESULTS AND DISCUSSION Both and are maternally exressed, as shown before by Choudhury et al. (997), and transcrits of both genes are resent throughout early A Egg S. 8 S. 9 A S. 9 V S. 0 S. S. 4 S. 7 S. 5 S. 40 B Egg S. 8 S. 9 S. 9 A S. 9 V S. 0 S. S. 4 S. 7 S. 5 S. 40 8S 8S 8S 8S H actin Fig. Northern blot analysis of and X-Grg-5 transcrits in Xenous laevis embryos at different stages. (A) and histone H control, (B) and actin control. Exression of and is found both in the animal (A) and the vegetal (V) arts of stage 9 embryos.

5 CHAPTER 5 69 develoment (figure ). Whole mount in situ hybridization shows that at the end of gastrulation, relatively high levels of and are resent in the rosective neural late region (figure ). Posteriorly, exression is restricted to two shar S S Fig. Whole mount in situ hybridization of gastrula stage embryos with antisense and robes. Dorsal views of stage embryos (A, B). exression along the midline is indicated by arrows (B). lines along the midline (figure ). In neurula stages, and transcrit levels remain highest in the sensorial layer of the neurectoderm (figure A-E and sections, not shown), decreasing from anterior to osterior. A broad transverse band of high exression is seen just anterior to the border of the neural groove (figure A, B). Sections demonstrate that this staining is localized in the anterior region of the entoderm, in a thin layer of adjacent mesenchyme and in the ectoderm; an area corresonding to the stomodeal-hyohyseal anlage (figure C). Within the anterior neural late relatively high exression of is detected in localized areas, i.e. anteriorly in the floor of the neural groove (figure A, B), at the level of the resumtive ros-mesencehalic boundary and in the anterior 4 C. 4 5 S4 S6 D. S6 E. F. XEn- 4 S4 S6 S8 Fig. and exression in neurula stages. A, B, D, E and F: antero-dorsal views. C: Sagittal section of a stage 6 embryo. F: XEn- marks the mid-hindbrain border region., cement gland, () stomato-hyohyseal anlage, () anterior neural late, () rosective ros-mesencehalic boundary, (4) rim along the neural groove, (5) rosective anterior rhombencehalon.

6 70 DIFFERENTIAL EXPRESSION OF XGRG-4 AND -5 rosective rhombencehalon (figure A, 5 B; cf Eagleson and Harris (990) ). A seckled attern of exression in the eidermal ectoderm, aears in stage S5 Fig 4. Exression of in the ectoderm. Seckled exression of in the ectoderm in stage 5 (B) and transverse section of a stage embryo (A). 4 embryos (figurea and figure 4) and disaears at stage 5. This might reresent exression in recursors of ciliated eidermal cells. and are exressed in the anlage of the cement gland at stage 4 (figure A, B). transcrits are mainly located in the area of the rosective telencehalon and between the eye anlagen. At stage, and transcrits are abundantly resent in the eye vesicles, the ear lacodes and the rosective branchial arches (figure 5). In sections, intensive staining is also observed in cells between the olfactory lacodes, in the lateral mesenchyme of the branchial arches and around the otic vesicles and cement gland (not shown). and transcrits are resent in the ronehric anlage, the rosective ronehric duct region, the resomitic mesoderm and the somites (figure 5). Pronounced differences between and exression are found in the cement ov C. ov ba * ov ba D. ov ov E. F. ba sha ov sha * Fig 5. Localization of and transcrits in early tailbud stage (). A, B, C: High exression at the ros-mesencehalic boundary (), at the mes-rhombencehalic boundary () and in the rhombencehalon (). D, E, F: High exression in the rosencehalon (), in the anterior rhombencehalon () and in the osterior rhombencehalon (). Arrows in A and D: somites. Asterisks in C and F: mesenchyme between the rosective olfactory lacodes. ba, branchial arches; ov, otic vesicle; ov, otic vesicle;, ronehros;, ronehric duct; sha, stomodeal-hyohyseal anlage. Embryos are viewed from lateral (A, D), dorsal (B, E) or anterior (C, F).

7 CHAPTER 5 7 gland, the head mesenchyme, the brain, the ov S5 C. D. V VII ov ov sha ba S5 boundaries (figure 5B, C). In the sinal sha S5 and and two distinctive bands in the mesencehalic and mes-rhombencehalic o is more ronounced in the rosencehalon exression is detected at the ros- IX X ba osterior rhombencehalon, while rhombencehalon (figure 5B-F). High ov o develoing brain, and is most abundant in the mesencehalon and V VII sinal cord and the ectoderm. In the transcrits are resent throughout; S5 Fig 6. Localization of and exression at tailbud stage 5. A, B:. C, D:. () Pros-mesencehalic boundary, () mes-rhombencehalic boundary. V-X: cehalic ganglia V-X. Embryos are viewed from lateral (A, C) or anterior (B, D). cord, both and are exressed in an anterior to osterior decreasing gradient with in an asymmetric, scattered attern (figure 5B, E). At stage 5, and exression atterns are similar to those at stage. In the cement gland, exression of is high, while RNA is detected only in the basal region (not shown). A strong signal of exression is observed at the ros-mesencehalic boundary. At later tailbud stages, is strongly exressed in the keel of the foregut (sections, not shown). In the brain, exression of and is very dynamic. Both and are weakly exressed in the eihysis (sections not shown) and in the infundibulum (not shown). At stage 5, and exression is high in the future choroid lexus of the telencehalon and in the eendymal layer (sections, not shown). In the brain, both and exression is highest laterally. Throughout the neural tube, and are exressed in a dorsal to ventral gradient (sections not shown). In the canalis neurentericus, has a scattered exression, while RNA is evenly distributed. and dislay a similar exression attern in the develoing eye: in the rosective ganglion cell layer, the ciliary marginal zone and the lens (sections, not shown). At stage 0, exression is found in cehalic ganglia V and VII (not shown) and in stage 5, also IX and X (figure 6A). is only weakly exressed in ganglia V and VII (figure 6C). Both and are exressed in

8 7 DIFFERENTIAL EXPRESSION OF XGRG-4 AND -5 the future endocardium and ericardium, while is additionally exressed in the future myocardium (sections not shown). Both in mouse and Xenous, Grg-5 0 is exressed in the heart and foregut. While mgrg-5 is exressed in the liver rimordium and the ventral sinal cord floor of the develoing brain, no exression of is detected at these ositions at the stages analyzed in Xenous. The exression attern of is strikingly similar to that of mgrg-4 8, suggesting functional conservation. REFERENCES. Choudhury, K., Kim, J., Kung, H.F. & Li, S.S. (997). Cloning and develomental exression of Xenous cdnas encoding the Enhancer of slit groucho and related roteins. Gene 95: Chu, D.T. & Klymkowsky, M.W. (989). The aearance of acetylated alha-tubulin during early develoment and cellular differentiation in Xenous. Dev Biol 6: Dehni, G., Liu, Y., Husain, J. & Stifani, S. (995). TLE exression correlates with mouse embryonic segmentation, neurogenesis, and eithelial determination. Mech.Dev. 5: Destrée, O.H.J., Lam, K.T., Peterson-Maduro, L.J., Eizema, K., Diller, L., Gryka, M., Frebourg, T., Shibuya, E. & Friend, S.H. (99). Structure and exression of the Xenous retinoblastoma gene. Dev.Biol. 5: Eagleson, G.W. & Harris, W. (990). Maing of the resumtive brain regions in the neural late of Xenous laevis. J Neurobiol : Harland, R.M. (99). In situ hybridization: an imroved whole mount method for Xenous laevis. Meth.Enzymol. 6: Hartley, D., Preiss, & Artavanis Tsakonas, S. (988). A deduced gene roduct from the Drosohila neurogenic locus, enhancer of slit, shows homology to mammalian G-rotein beta subunit. Cell 55: Koo, K.E., MacDonald, L.M. & Lobe, C.G. (996). Transcrits of Grg4, a murine groucho-related gene, are detected in adjacent tissues to other murine neurogenic gene homologues during embryonic develoment. Mech.Dev. 59: Leon, C. & Lobe, C.G. (997). Grg, a murine Groucho-related gene, is exressed in the develoing nervous system and in mesenchyme-induced eithelial structures. Dev.Dyn. 08: Mallo, M., Franco del Amo, F. & Gridley, T. (99). Cloning and develomental exression of Grg, a mouse gene related to the groucho transcrit of the Drosohila Enhancer of slit comlex. Mech.Dev. 4: Miyasaka, H., Choudhury, K., Hou, E.W. & Li, S.S. (99). Molecular cloning and exression of mouse and human cdna encoding AES and ESG roteins with strong similarity to Drosohila enhancer of slit groucho rotein. Eur.J.Biochem. 6: 4-5. Molenaar, M., Roose, J., Peterson, J., Venanzi, S., Clevers, H. & Destrée, O. (998). Differential exression of the HMG box transcrition factors XTcf- and XLef- during early Xenous develoment. Mech.Dev. 75: Roose, J., Molenaar, M., Peterson, J., Hurenkam, J., Brantjes, H., Moerer, P., van de Wetering, M., Destrée, O. & Clevers, H. (998). The Xenous Wnt effector XTcf- interacts with Groucho-related transcritional reressors. Nature 95: Schmidt, C.J. & Sladek, T.E. (99). A rat homolog of the Drosohila enhancer of slit (groucho) locus lacking WD-40 reeats. J Biol Chem 68: Schrama, L.H., Leerdinger, G., Moritz,, van den Engel, N.K., Marquart,, Oestreicher,, Eggen, J., Hage, W.J., Richter, K. & Destree, O.H. (997). B-50/growth-associated rotein-4, a marker of neural develoment in Xenous laevis. Neuroscience 76: 65-65

9 CHAPTER Stifani, S., Blaumueller, C.M., Redhead, N.J., Hill, R.E. & Artavanis-Tsakonas, S. (99). Human homologs of a Drosohila Enhancer of Slit gene roduct define a novel family of nuclear roteins. nature genetics : Wülbeck, C. & Camos-Ortega, J. (997). Two zebrafish homologues of the Drosohila neurogenic gene groucho and their attern of transcrition during early embryogenesis. Dev.Genes Evol. 07: Yao, J., Liu, Y., Husain, J., Lo, R., Henderson, J. & Stifani, S. (998). Combinatorial exression atterns of individual TLE roteins during cell determination and differentiation suggest nonredundant functions for mammalian homologs of Drosohila Groucho. Dev.Growth Diff. 40: - 46

CHAPTER 3 DIFFERENTIAL EXPRESSION OF THE HMG BOX TRANSCRIPTION FACTORS XTCF-3 AND XLEF-1 DURING EARLY XENOPUS DEVELOPMENT.

CHAPTER 3 DIFFERENTIAL EXPRESSION OF THE HMG BOX TRANSCRIPTION FACTORS XTCF-3 AND XLEF-1 DURING EARLY XENOPUS DEVELOPMENT. CHAPTER 3 DFFERENTAL EXPRESSON OF THE HMG BOX TRANSCRPTON FACTORS XTCF-3 AND XLEF-1 DURNG EARLY XENOPUS DEVELOPMENT. Miranda Molenaar, Jeroen Roose #, Josi Peterson, Serenella Venanzi*, Hans Clevers #