Embryonic expression patterns of the mouse and chick Gas1 genes

Mechanisms of Development 101 (2001) 293±297

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Gene expression pattern

Embryonic expression patterns of the mouse and chick Gas1 genes Catherine S. Lee, Chen-Ming Fan* Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210, USA Received 8 November 2000; received in revised form 19 December 2000; accepted 20 December 2000

Abstract Control of cell proliferation is essential to generate the de®ned form of a multi-cellular organism. While much is known about the regulators for cell cycle progression, relatively little is known about the state of growth arrest. Growth arrest (G0) is de®ned as a cell in a metabolically active but proliferation-quiescent state (reviewed in Baserga (1985) The Biology of Cell Reproduction), typically induced by serum starvation in vitro. Using subtractive hybridization, Schneider et al. (Cell 54 (1988) 787) identi®ed six genes (Gas1 through Gas6) whose expressions are upregulated in serum-deprived NIH3T3 cells. Among the Gas genes, Gas1 is the only one that can cause growth arrest when expressed in cultured cell (Cell 70 (1995) 595; Int. J. Cancer 9 (1998) 569). Here, we describe for the ®rst time the expression pattern of Gas1 during mouse embryogenesis. Our data reveal that Gas1 is expressed in many regions that the cells are actively proliferating and suggest that it may have other roles during development than negatively regulating cell proliferation. Furthermore, we have cloned the chick GAS1 gene and documented the similarity and divergence of Gas1 gene expression patterns between the two species. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gas1; Proliferation; Growth arrest; Patterning; Embryogenesis; Somite; Dermomyotome; Sclerotome; Myotome; Neural tube; Notochord; Lateral plate mesoderm; Intermediate mesoderm; Endoderm; Heart; Limb bud; Cartilage; Digits; Neural retina; Retinal pigmented epithelium; Brain; Cerebral cortex; Dentate gyrus; Ventricular zone; Chick

1. Results and discussion While many growth factors have been identi®ed to govern cell proliferation and differentiation during development (Gilbert, 1994), little is known about the G0 growtharrested state (Baserga, 1985) and whether this state exists during embryogenesis. In surveying genes expressed in the mouse somites, we isolated the Gas1 cDNA. Although well known as a marker of G0 and for its anti-tumor growth function (Del Sal et al., 1995; Evdokiou and Cowled, 1998), Gas1 expression during embryogenesis is not known. We therefore investigated whether Gas1 marks a unique population of G0 cells during development. Using RT-PCR to detect Gas1 transcripts during mouse embryogenesis, we found them to be present from E6.5 and onwards (not shown). To de®ne the patterns of expression, we performed whole mount and sectioned in situ hybridization (ISH) using DIG or 35S-labeled probes. While we present the whole mount data for 3-D visualization, our descriptions are based on sectioned ISH data, some of which are highlighted here. Gas1 expression is detected in the primitive streak at E6.5 (Fig. 1A) and in the somites as * Corresponding author. Tel.: 11-410-554-1222; fax: 11-410-243-6311. E-mail address: [email protected] (C.-M. Fan).

soon as they appear at E7.0 (Fig. 1B). The somitic expression persists until E12.5 (Fig. 1B±G). The presomitic region is negative for Gas1 expression (Fig. 1C±E). Sectioned ISH indicates that Gas1 is strongly expressed in the dermomyotome and much weaker in the myotome and sclerotome (Fig. 1H,I). It is also detected in the surface ectoderm (Fig. 1I) and the lateral plate mesoderm (Fig. 1D,E,H,I), but not the endoderm (e.g. gut and heart) and splanchnic mesoderm (Fig. 1H,I, and not shown). Gas1 expression follows the trunk dermatome cells as they disperse to populate the dermal layer of the skin (Fig. 1F,G). There is expression in the branchial arches (Fig. 1D,E) and Gas1 expression is noticeably missing in the whisker follicles (Fig. 1G). In the limb, it displays an anterior-speci®c expression as soon as the limb bud forms at E9.0 (Fig. 1J±L). At the onset of chondrogenesis in the limb, Gas1 is expressed in cells next to the condensing cartilage (Fig. 1O,P). At E14.5, it is expressed in the mesenchymes surrounding and between digits and metacarpals (Fig. 1Q). Gas1 is expressed in the spinal cord at E8.0±E11.5, extending down just prior to the presomitic level and absent from the tail bud (Fig. 1C±F). This expression is dorsally restricted and no longer observable at E12.5 (Fig. 1D±I, and not shown). Weak expression is also detected in the dorsal diencephalon, midbrain, and hindbrain (Figs. 1D±F,R and

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C.S. Lee, C.-M. Fan / Mechanisms of Development 101 (2001) 293±297

2O 0 , and not shown). The forebrain expression is detectable at E7.0 (Fig. 1B), primarily localized to the alar region and devoid from the ventral midline (also at E8.0, Fig. 1C and not shown). In the telencephalon, Gas1 is expressed in the

dorsomedial region corresponding to the neocortex and hippocampus (Fig. 1D±G,U±V). We are intrigued by its restricted expression in the proliferative zone (the ventricular zone) of the brain. At P0, Gas1 is still expressed in the

Fig. 1.

C.S. Lee, C.-M. Fan / Mechanisms of Development 101 (2001) 293±297

cells lining the ventricle of the neocortex, the dentate gyrus (both the granule cells and the neuroepithelium), the epithalamic ventricle (the dorsal 3rd ventricle), the aqueduct, the forth ventricle, the olfactory and the striatum neuroepithelium, and the lateral migratory stream (Fig. 1W±b). We did not ®nd any differentiated neuronal ®eld expressing Gas1 in the brain ± except for the granule cells of the dentate gyrus. Gas1 is also expressed in the optic and otic vesicle at E9.5 (Fig. 1D,R±T). Its expression in the otic vesicle is transient (Fig. 2O 0 ). The eye expression is ®rst detected in the optic vesicle and the overlying ectoderm. As the lens evaginates, its expression is absent from the central region of the lens epithelium and the neural retina. It is expressed in the presumptive RPE (Fig. 1S), the mature RPE, the ciliary body, and the adjacent periorbital mesenchyme (Fig. 1T). We next sought to document the conserved expression patterns in other vertebrate species. To this end, we isolated a chick homolog of Gas1. One gene in the chick genome was found and it is contained in a single exon as the mammalian counterparts. The sequence alignment is in Fig. 2A. There are 20 cysteins and one N-glycosylation site conserved in the chick GAS1, which (when applicable) are also conserved in the frog, zebra ®sh and worm genes identi®ed in the database. A ¯y homolog has not yet been identi®ed. Importantly, the chick gene functions similarly to the mouse gene as it can suppress proliferation when overexpressed in the NIH3T3 cells (not shown). Evolutionarily conserved expression sites are found in the chick. It is transiently detected in the primitive streak (st. 6) before somitogenesis. Chick GAS1 expression in the somite is delayed until st.10 (Fig. 2B±H). Once commenced, the somitic expression is similar to that of the mouse: absent from the presomitic mesoderm, enriched in the dermomyotome, and weak in the myotome and sclerotome (Fig. 2E±G, and not shown). Chick GAS1 is expressed in the anterior neural plate starting from st. 4, but excluded from the midline region (Fig. 2B±D). The spinal cord and cortex expression is localized dorsally (Figs. 2L, P), but the midbrain and hindbrain expression extends ventrally (Fig. 2M,O). Its expression in the developing eye (mesenchymes, optic vesicle, RPE, and the rim of neural retina) and in the

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otic vesicle is also conserved (Fig. 2G,M±O 0 ). Its expression in the limb follows the mouse pattern: initially localized anteriorly and then in the mesenchyme between the condensing cartilage of the digits and metacarpals (Fig. 2H±J,Q). There is also a weak expression in the mesenchyme surrounding the mesonephros (Figs. 1H and 2L). Interestingly, the notochord expression is unique to the chick (Fig. 2L). Embryonic Gas1 expression pattern as documented here does not necessarily correlate with regions where the cells are not proliferating. This is particularly noticeable in the developing cortex where the expression is localized to the ventricular zone. Thus, it is likely that Gas1 functions differently during embryonic development from the previously de®ned growth arrest function. 2. Materials and methods 2.1. Mouse and chick stages The vaginal plug day is assigned as day 0.5 (E0.5) for the mouse. The chick embryos were staged according to Hamberger and Hamilton, 1951. The nomenclatures and the identi®cation of speci®c embryonic structures are primarily based on Altman and Bayer, 1995; Franklin and Paxinos, 1996; Hamberger and Hamilton, 1951; Kauffman, 1992. 2.2. Low stringency hybridization The conditions used for the low stringency hybridization to obtain the chick GAS1 gene is described by Echelard et al., 1993, using the coding region of mouse Gas1 as a probe. The chick genomic library was from the ATCC and the chick cDNA library was a generous gift from Dr Susan Mackem. For comparison, the sequences of the GAS1 homologs of each species are derived from the ESTs or full-length cDNAs in the Genbank: human (NM002048), mouse (X65128), rat (AW533982), worm (Z48582), zebra ®sh (AI558523), and frog (AW 158169).

Fig. 1. Mouse Gas1 expression pattern: Whole mount in situ hybridization were performed on E6.5 (A, lateral view), E7.5 (B, front-ventral view), E8.0 (C, dorsal view), E9.5 (D), E10.5 (E), E11.5 (F), and E12.5 (G) (lateral view for D±G) mouse embryos. Ps, primitive streak; anp, anterior neural plate; s, somite; rp, roofplate; t, telencephalon; ba, branchial arches; lp, lateral plate mesoderm; e, eye; ot, otic vesicle; ¯, forelimb; hl, hindlimb; w, whisker. (H, I) Transverse/ horizontal oblique sections of the E9.5 forelimb region by whole mount using DIG probe and the hindlimb region by sectioned ISH using 35S-labeled probe, respectively. mn, mesonephros. en, endoderm, h, heart; spm, splanchnic mesoderm. The limb orientation: A, anterior; P, posterior. The arrowheads point to the mid-hindbrain junction lack of Gas1 expression. (J±L) Dorsal view of the forelimbs of E9.0, E9.5, and E10.5; (M, N) Dorsal view of forelimb and hindlimb of E12.5. The white lines indicate the section planes presented in (O, P). (Q) A horizontal section of E14.5 forelimb hybridized with Gas1 DIG probe. M, mesenchyme; c condensing cartilage. Developing eye at E9.5 (R), E10.5 (S), E14.5 (T) in coronal sections. Lenp, lens pit; ov, optic vesicle; rpe, retinal pigmented epithelium; nr, neural retina; le, lens epithelium; cor, cornea; lens, l. (U, V) Cortext (ctx) expression at E10.5 and E14.5 by coronal sections. hip, hippocampus; BG, basal ganglia. (W-b) Coronal sections of newborn brain. At the most anterior level (a), the expression is in the neuroepithelium of the olfactory bulb (onp). Slightly posterior, expression in the lateral migratory stream (lms) and the striatum neural epithelium (snp) is found (b). Throughout different levels of the cortext (W, and not shown), expression is found in the neuroepithelium of the cortext and the dentate gyrus (dg). The expression in the dentate gyrus includes the granule cells and the neural epithelial cells. amg, amygdala. (X±Z) Expression of Gas1 in the cells lining the 3rd ventricle of epithalamus (V3e), aqueduct (Va), and the forth ventricle, V4i and V4l (intermediate and lateral 4th ventricles). Cerebellum, cer. Note that the sectioned ISH images are presented as overlays of a dark ®eld and a bright ®eld images. The positive signals are the bright silver granules.

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C.S. Lee, C.-M. Fan / Mechanisms of Development 101 (2001) 293±297

Fig. 2. Chick Gas1 gene and its expression pattern: (A) Sequence comparison between the mouse, human, rat, chick, frog, zebra ®sh, and worm Gas1 homologs. The conserved cysteins are in red and the GPI-signal sequence, green. (B±R) Expression patterns of the chick GAS1. (B-G) St. 4, st. 6, st. 8, st. 10 (dorsal views), st. 15 and st. 18 (lateral views) chick embryos viewed by whole mount in situ hybridization. (H, I) Dorsal view of the forelimb at st. 15 and 20. (J) Horizontal section of the limb hybridized with 35S-UTP labeled probe. (K, L) Transverse sections of the presomitic level (or segmental plate level) and the trunk level at st. 15. (M, N) Eye expression at st. 18 and st. 22 in coronal sections. (O) Transient otic vesicle (ot) expression conserved in chick (st.11) and mouse (O 0 ). (P) Cortex expression (st.28). (Q) Expression in the mesenchyme between the digits and the metacarpals (st. 34). ps, primitive streak; np, neural plate; hn, hensen's node; nc, notochord; nt, neural tube; rp, roof plate; mb, midbrain; ctx, cortex; s, somite; dm, dermomyotome; h, heart; ba, branchial arches; lb, limb; AER, apical ectodermal ridge; en, endoderm; lp, lateral plate mesoderm; eye, e; se, surface ectoderm; le, lens; M, mesenchyme; c, condensing cartilage. Note that the sectioned-35S ISH photos were overlays of a bright ®eld image with blue ®lter and a dark ®eld image with red ®lter and the positive signals are in red.

C.S. Lee, C.-M. Fan / Mechanisms of Development 101 (2001) 293±297

2.3. Northern analysis Multi-tissue bot was purchased from Clonetech and hybridized with Gas1 probe following the instruction of the manufacturer. 2.4. In situ hybridization For whole mount in situ hybridization, the Wilkinson protocol was used for the mouse embryos and the Belmonte protocol was used for chick embryos (Wilkinson, 1992). For the radioactive in situ, we followed the protocol outlined by Frohman et al., 1990. Both the mouse and chick probes were synthezised by T7 polymerase using the pSK-Gas13IÂRI and pCGas13IÂNotI plasmids. Sense probes gave no speci®c signal under the same conditions. References Altman, J., Bayer, S.A., 1995. Atlas of Prenatal Rat Brain Development, CRC press. Inc, Boca Raton, FL. USA. Baserga, R., 1985. The Biology of Cell Reproduction, Harvard University, Cambridge, MA, USA.

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Del Sal, G., Ruaro, E.M., Philipson, L., Schneider, C., 1995. The growth arrest-speci®c gene. Gas1 is involved in growth suppression. Cell 70, 595±607. Echelard, Y., Epstein, D.J., St. Jacques, B., Shen, L., Mohler, J., McMahon, J.A., McMahon, A., 1993. Sonic Hedgehog, a member of a family of putative signaling molecules, is implicated in regulating the CNS polarity. Cell 75, 1417±1430. Evdokiou, A., Cowled, P.A., 1998. Tumor-suppressive activity of the growth arrest-speci®c gene GAS1 in human tumor cell lines. Int. J. Cancer. 9, 569±577. Franklin, K.B.J., Paxinos, G., 1996. The Mouse Brain in Stereotaxic Coordinates, Academic Press, Inc, San Diego, CA, USA. Frohman, M.A., Boyle, M., Martin, G., 1990. Isolation of the mouse hox2.9 gene: analysis of embryonic expression suggests that positional information along the anterior-posterior axis is speci®ed by mesoderm. Development 110, 589±607. Gilbert, S.F., 1994. Developmental Biology, . 4th EditionSinauer Associates, Inc, Sunderland, MA. USA. Hamberger, W., Hamilton, H.L., 1951. A series of normal stages of development of the chick embryo. J. Morphol. 88, 49±92. Kauffman, M.H., 1992. The Atlas of Mouse Development, Academic Press, Inc, San Diego, CA, USA. Wilkinson, D.G., 1992. Whole Mount In Situ Hybridization of Vertebrate Embryos, , In Situ Hybridization: A Practical Approach, IRL Press, Oxford University Press, England.