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779 Development 105, 779-786 (1989) Printed in Great Britain © T h e Company of Biologists Limited 1989 Differential gene expression in the anterior neural plate during gastrulation of Xenopus laevis MILAN JAMRICH and SHERYL SATO Laboratory of Molecular Genetics, N1CHD, National Institutes of Health, Bethesda, Maryland 20892, USA Summary We have isolated three cDNA clones that are preferentially expressed in the cement gland of early Xenopus laevis embryos. These clones were used to study processes involved in the induction of this secretory organ. Results obtained show that the induction of this gland coincides with the process of neural induction. Genes specific for the cement gland are expressed very early in the anterior neural plate of stage-12 embryos. This suggests that the anteroposterior polarity of the neural plate is already established during gastrulation. At later stages of development, two of the three genes have secondary sites of expression. The expression of these genes can be induced in isolated animal caps by incubation in lOmM-NHtCl, a treatment that is known to induce cement glands. Introduction dorsal mesodermal cells form the chordamesoderm (notochord and somites). This is called the primary or mesodermal induction, though it is possible that other inductive processes precede this step. In the process of gastrulation, the involuting chordamesoderm induces the overlying ectoderm to form the neural plate. This is called secondary or neural induction. As O. Mangold noticed in 1933, neural induction is not a uniform process, because different regions of the archenteron roof induce different structures in the overlying ectoderm. These structures are formed in a predetermined order (head structures, forebrain, midbrain, hindbrain, spinal cord), although it is not known at what stage of development they are specified. The first morphological signs of differentiation of the neural plate into subregions are observable during neurulation. At this time, as the embryo begins to elongate, the neural folds increase in elevation and subdivide the originally uniform plate into a sense plate, neural plate and gill plates. In order to establish how early in development the different regions of neural plate are specified, we attempted to isolate molecular markers that would allow a study of this question. In this paper, we describe three cDNA clones that are suitable for the study of cement gland development from the earliest stage of formation of this organ. The cement gland (also called adhesive organ, mucous gland, or sucker) is derived from the outside ectodermal cells of the lower sensory plate. It is an induced structure (Spemann & Schotte, 1932; Picard, 1975a,b), consisting of elongated columnar cells which secrete adhesive substances such as The development of the metazoan organism is a continuous process involving the generation of new cell types and their subsequent differentiation until the final body plan is realized. In amphibians, the formation of new cell types depends on the utilization of localized maternal factors and on inductive interactions between neighbouring cells. Although most of the developmentally important maternal components are likely to be uniformly distributed in the egg, there is good evidence that at least some of them are localized (Bonoure, 1934; Spemann, 1938). More recently, Melton (1987) demonstrated the presence of localized maternal RNA in the vegetal pole of developing Xenopus oocytes. This RNA is transcribed from a gene which shows similarity to the TGF-beta gene family (Weeks & Melton, 1987). Members of this gene family are likely to play an important role in mesoderm formation (Kimmelman & Kirschner, 1987; Rosa et al. 1988). The data of Sargent et al. (1986) and Jamrich etal. (1987) suggest the presence of a localized maternal component in the animal hemisphere of Xenopus embryos which regulates the synthesis of keratin genes in the embryonic ectoderm. Embryonic induction has been one of the most popular topics for study in the amphibian embryo since its discovery by Spemann & Mangold in 1924. Embryonic induction can be divided into at least two major phenomena. The first is the induction of mesodermal cells by vegetal cells, primarily in the dorsal but also including the lateral and ventral marginal zone. The Key words: cement gland, gastrulation, in situ hybridization, neural induction, NH4CI induction, Xenopus laevis. 780 M. Jamrich and S. Sato B Q Q O z Z z o o t- o 1- Ill Q < UJ z X \- Z) Q < UJ z X 1- D z z LU < 2 UJ < 2 ui LU >• CC LU ^ O LU m O w m z z < 2 LU UJ 01 O UJ UJ 28S' 18S XCG2 XCG7 XCG13 XCG2 XCG7 XCG13 Fig. 1. (A) Localization of XCG 2, XCG 7, and XCG 13 RNA in dissected stage-24 Xenopus embryos. Northern blot analysis of equal amounts of RNA (1 jig per lane) using nick-translated XCG 2, XCG 7, and XCG 13 probes. In every case, the corresponding RNA is present in the head region only. (B) Localization of XCG 2, XCG 7, and XCG 13 in dissected regions of stage-24 Xenopus heads. Northern blot analysis of equal amounts of RNA (1 jig per lane) using nick-translated probes. RNA corresponding to the XCG 2, XCG 7, and XCG 13 is present in the cement glands. mucoproteins (Eakin, 1963). It is homologous to the balancers of urodeles and it is the first organ in Xenopus embryos to become functionally differentiated. Knowing the exact timing of cement gland induction would help to establish when in development the regional identity of the original neural plate is specified. Since the cement gland is not morphologically noticeable before stage 15, we monitored the expression of cement-gland-specific RNAs as an indicator of tissue differentiation. The study of cement gland induction offers additional advantages. Although inductive phenomena are frequently studied, only a few experimental models of induction exist that deal with a homogeneous cell population and the cement gland is one of them. In contrast to mesodermal and neural induction, which result in the formation of a variety of tissues, cement gland induction represents a homogeneous transition from one cell type into another. Furthermore, this transition can be achieved by treatment of the isolated animal caps of gastrulae in 10 mM-NtLjCl, which causes the outside ectodermal cells to differentiate into cement gland cells (Picard, 1975a,b). The ability to study this inductive process in explants makes this system especially suitable for biochemical analysis. Materials and methods Preparation of RNA RNA was prepared as previously described by Sargent et al. (1986). Poly(A)+ RNA was purified according to Aviv & Leder (1972). In the plus-minus screening, single-stranded 32 P-cDNA probes were prepared by using Moloney murine leukemia virus (Mo-MLV) reverse transcriptase (Sargent, 1988). Filter blotting and hybridization RNA electrophoresis and Northern blot hybridization was performed as previously described (Bailey & Davidson, 1976; Church & Gilbert, 1984). Filters were washed as described in LaFlamme et al. (1988). Gene expression in the neural plate during gastrulation 781 B CG Fig. 2. (A) In situ hybridization of XCG 2 antisense transcripts to a stage-34 embryo viewed with dark-field optics. Hybridization is to the cement gland. The plane of section is indicated in B. (C) A phase-contrast view of the same section; B, brain; CG, cement gland; E, eye. (D) In situ hybridization of XCG 7 probe to a sagittal section of a stage-25 embryo. Though most of the hybridization is to the cement gland, secondary hybridization to the olfactory pit can be observed (arrow). (E) A phase-contrast view of the section in Figure D; CG, cement gland; O, olfactory placode; P, pharynx. (F) In situ hybridization of XCG 13 probe to a cross section of a stage-34 embryo. The plane of section is indicated in B. Hybridization is to the cement gland. Arrows indicate the concentration of pigment in the embryonic eyes. These areas diffract light in darkfield, but do not show any real hybridization. (G) Phase-contrast picture of Figure F; CG, cement gland; E, eye; N, notochord; P, pharynx. 782 M. Jamrich and S. Sato Fig. 3. (A) In situ hybridization of a clone XCG 7 probe to a section of a stage-12 embryo. This dark-field view shows the hybridization to the anterior portion of the neural plate (arrow). (B) Phase-contrast view of the section of stage 12. Boxed area shows the region enlarged in A; A, archenteron; BL, blastocoel; Y, yolk plug. (C) A higher magnification of the neural plate to show the position of the future brain; B, brain. The apparent signal in the archenteron floor is due to trapped air bubbles and not to silver grains. In situ hybridization The hybridization procedure used is a modification of existing protocols (Angerer & Angerer, 1981; Akam, 1982; Jamrich etal. 1984; Ingham etal. 1985; Kintner & Melton, 1987). Albino embryos were dejellied in 2% cysteine pH7-8 and fixed in 4 % paraformaldehyde in phosphate-buffered saline (PBS) for 30min. They were dehydrated in an ethanol series (70%, 95%, 100%), cleared in xylene and embedded in paraffin (Paraplast-Plus with DMSO). 5/an sections were cut and transferred to a drop of water on a polylysine-coated slide. After sections had dried down and attached to the slide, they were incubated in xylene to remove paraffin and rehydrated through an ethanol series. Slides were incubated in 2xSSC for 30min at 65 °C, treated with Proteinase K (2//gmr 1 in 10mM-Tris-HCl pH7-5, 5mM-EDTA) for 15 min at 37°C. Digestion was terminated by placed slides in 2mgml~1 glycine in PBS for lmin. Slides were washed in PBS and acetylated (Hayashi et al. 1978). They were washed in 2 X SSC, dipped in water and hybridized overnight with 35Slabelled transcripts (Green et al. 1983). Hybridization was carried out in 50 % formamide, 5 x SSC, 0-1 M-sodium-potassium phosphate pH7-0, 1 x Denhardt's solution, 5 % dextran sulphate, 100mM-dithiothreitol (DTT), lOOfigmr1 E. coli tRNA at 50°C under glass coverslips. Typically 7/il hybridization solution was used per slide containing 300000 cts min"1. Coverslips were sealed with rubber cement. After hybridization, rubber cement was manually removed and the coverslips were floated off in 2 x SSC and 10mM-DTT. Slides were incubated in this solution for 2 h and then in 50 % formamide, 1 x SSC, lOmM-DTT for lh at 50°C. Sections were treated with RNase A (2/igmP 1 in 2 x SSC for 30min at 37°C), washed in 2 x SSC for 2h, dipped in water and covered with autoradiographic emulsion (Kodak NTB-2 diluted 2:1 with water). Slides were typically exposed for 2-10 days, developed in D-19 developer for 60 s at 18°C,fixedin Rapid Fix for 2 min and rinsed in water. They were dried and viewed in a microscope using dark-field optics or stained with Giemsa using standard procedures. NH4Cl induction Animal caps of stage-10 embryos were dissected and incubated for 6h in lOmM-NKtCl. They were transferred into 67 % L15 medium (GIBCO) and collected 12 h later for RNA preparation and Northern blot analysis. Staging of embryos was done according to Nieuwkoop & Faber (1967). Results Cement-gland-specific cDNA clones were obtained as Gene expression in the neural plate during gastruladon neural plate 783 notochord i B endoderm Fig. 4. In situ hybridization of XCG 13 probe to a section of a stage-17 embryo. The plane of section is indicated in B; A, anterior; P, posterior. part of an experiment designed to isolate cDNA clones specifically expressed in the head region of Xenopus embryos. Heads were separated from the trunks of stage-24 embryos, and RNA was prepared from both fractions. 32P-labelled cDNA was prepared from both RNA preparations and duplicate filters of a Xenopus neurula cDNA library (Richter et al. 1988) were screened using these probes. Plaques hybridizing with head cDNA but not trunk cDNA were isolated and purified. Head region specificity was confirmed by hybridizing the isolated clones to Northern blots of head and trunk RNA. Fig. 1A shows three clones preferentially hybridizing to head RNA. Clone XCG 13 hybridizes to a very large RNA. The hybridization signal appears smeared in all our RNA preparations. We presume that this is due to the partial degradation of this RNA; however, it is possible that this clone hybridizes to multiple transcripts. Further analysis of the expression of these clones by microdissection of the head region into brain, eyes and cement gland revealed that they are preferentially expressed in the cement gland (Fig. IB). More detailed analysis by in situ hybridization to sections of embryos confirmed that all three clones are predominantly expressed in the cement gland (Fig. 2). We hybridized these clones to embryos of progressively earlier stages of development to determine when and in what region these clones are first activated. The earliest detectable hybridization was limited to the anterior neural plate of stage-12 embryos (Fig. 3). This is an important result as it shows that the different regions of the neural plate are already specified during gastrulation, well before the different regions can be recognized as morphological entities. A few hours later, at stage 17, the hybridization is restricted to the anterior ventral region of the embryo in what can now be recognized as the lower part of the sense plate (Fig. 4). Whereas clone XCG 13 appears to be expressed exclusively in the cement gland throughout development, secondary sites of expression were observed for clones XCG 2 and XCG 7 in older embryos (past stage 25). At least some of these expression sites were common to both of the genes. The most prominent secondary sites of expression were the pharynx (with the highest expression in the branchial arches) (Fig. 5A), a part of the olfactory placode that appears to be the olfactory pit (Fig. 2D), an endodermal region between the pharynx and heart mesoderm which is probably involved in the formation of the trachea or esophagus (Fig. 5C), and the ear vesicle (not shown). One of the requirements for understanding the molecular detail of the processes involved in inductive phenomena is the availability of defined model systems for induction. Induction of the cement gland was achieved by treating microdissected animal caps of Xenopus gastrulae in a solution of 10 mM-NI-LtCl for few hours. Fig. 6 shows a strong activation of the gene XCG 7 in the treated caps using Northern blot analysis of isolated RNA. The other two genes (XCG2 and XCG13) are activated by this treatment as well (not shown). In contrast, transcription of the epidermal cytokeratin gene XK 81 (Jonas et al. 1985; Miyatani et al. 1986; Jamrich et al. 1987) declined after this induction period (Fig. 6). Discussion We have studied the expression of three genes preferentially transcribed in the cement gland during the embry- 784 M. Jamrich and S. Sato B Fig. 5. (A) 7n sita hybridization of XCG 7 probe to a section of a stage-38 embryo. Hybridization is to the pharyngeal arches. Arrow shows the accumulation of pigment in the eye. There is no hybridization to this region. (B) Phase-contrast view of the same section; E, eye; NT, neural tube; P, pharynx. (C) In situ hybridization of XCG 7 to a section of a stage-28 embryo. Arrow indicates the pigment in the neural crest cells. Most of the hybridization is to the pharynx with a high concentration of grains dorsal to the heart mesoderm. (D) Phase-contrast view of the same picture; H, heart; N, notochord; NT, neural tube. onic development of Xenopus laevis. We found that these genes are initially expressed at stage 12 (gastrula) in the anterior neural plate. This result suggests that the induction of this gland coincides with, or is part of, neural induction. Furthermore, our results demonstrate that gene expression specific for the anterior neural plate is already in progress during gastrulation. We do not see expression of these genes in the ectoderm prior to the contact of ectoderm with chordamesoderm, suggesting that the chordamesoderm is inducing the transcription of cement-gland-specific genes in the ectoderm. This agrees well with transplantation exper- iments by Spemann & Mangold (1934) and Mangold (1933), suggesting that the contact of mesoderm and ectoderm is necessary to induce the ectoderm to form neural structures. More recently, it was shown that the entire ectoderm is programmed to express epidermisspecific products unless it is prevented from doing so by contact with chordamesoderm (Jones & Woodland, 1986; Jamrich et al. 1987). It was also demonstrated that N-CAM, a neural-specific marker, is not synthesized in presumptive neural ectoderm before induction by chordamesoderm (Kintner & Melton, 1987). However, the presumptive neural ectoderm might not be totally Gene expression in the neural plate during gastrulation S o o Q o i- o° ?9 °o XCG7 XK81 Fig. 6. Induction of XCG 7 gene by treatment of animal caps with NH4Cl. Northern blot analysis of RNA from NH4Cl-treated caps shows a strong induction of transcription of this gene whereas cytokeratin XK 81 RNA accumulation is suppressed. naive. It was shown that the dorsal ectoderm is more easily induced to transcribe XlHbox6, a homeoboxcontaining neural-specific gene, and the N-CAM gene than the ventral ectoderm (Sharpe etal. 1987). Similarly London et al. (1988) showed that the dorsal ectoderm is biased in its ability to express Epi 1 gene, an epidermisspecific marker. It is not clear at present what developmental significance these differences have in the embryo since it appears that this bias is neither sufficient nor necessary for the formation of neural tissue. The expression of the cement-gland-specific genes at stage 12 can be visualized in the region immediately posterior to the leading edge of the chordamesoderm, suggesting that this induction requires only a brief contact between the mesoderm and ectoderm. The expression of these genes is limited to a very defined region, implying that the induction process taking place during gastrulation is already imprinting a region identity on the overlying ectoderm. This specificity is immediately translated into regional differences in gene expression, although how this is accomplished is not easily understood. In the more posterior regions of the neural plate, which have also had contact with the leading edge of chordamesoderm, the expression of the cement-glandspecific genes was not observed. The following expla- 785 nations are possible: (1) the mesoderm was not transmitting the signal to the overlying ectoderm; (2) the ectoderm was not ready to receive the signal; (3) the signal was transmitted but immediately negated by additional signals coming from more posterior mesoderm; (4) only a certain area of ectoderm is predetermined to become cement gland and the contact with archenteron roof simply provides an initiating signal for this expression. It is unlikely that the fourth alternative is correct, since it was previously shown that any ectoderm can form a cement gland if properly induced (Spemann & Mangold, 1924; Spemann & Schotte, 1932). Furthermore, induction experiments by Picard (1975a,b) showed no difference between dorsal and ventral ectoderm in the ability to form cement gland if properly induced; uninduced animal caps will not form cement glands to any significant degree. Our experiments (Fig. 6) confirm these observations. While at stage 12 the expression of the cement-glandspecific genes is in the anterior dorsal region, at stage 17 the expression is more ventral. This suggests that the cells expressing these genes move ventrally in the process of elongation of the embryo during neurulation. Alternatively, the cells expressing these genes at stage 12 and 17 are not identical. It is possible that the hybridization signal at stage 12 visualizes a transient site of expression (see discussion above). After stage 25, clones XCG 2 and XCG 7 are also expressed in additional tissues, such as pharynx, esophagus, ear vesicle and olfactory pit. These tissues are not derived from the same germ layer and there is no reason to assume that they are of common origin. Most likely these tissues share a common physiological function or have similar physiological requirements. The clones XCG 2 and XCG 7 may be useful as markers for the induction of these tissues. One of the most exciting aspects of the study of cement gland induction is the ability to induce this structure by incubating the animal caps of gastrulae in lOmM-NELtCl. This treatment results in the expression of all three cement-gland-specific genes. At the same time, we observe a reduction in the expression of epidermal cytokeratin gene XK81. This agrees well with our previous finding that cytokeratin genes are turned off in the neural plate during the process of induction (Jamrich et al. 1987). In the future, we hope to use this induction system to isolate genes which are activated prior to the genes described here. 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