- Email: [email protected]
Analysis of Fgf15 expression pattern in the mouse neural tube
Brain Research Bulletin, Vol. 57, Nos. 3/4, pp. 297–299, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/02/$–see front matter
PII S0361-9230(01)00717-1
Analysis of Fgf15 expression pattern in the mouse neural tube L. Gimeno,1 R. Hashemi,2 P. Bruˆlet2 and S. Martı´nez1* 1
Institut of Neurosciences, CSIC-UMH (Miguel Hernandez University), Alicante, Spain; and 2Unite´ d’Embryolodie Mole´culaire, Institut Pasteur, Paris, France
ABSTRACT: The dynamic process of neural tube regionalization in vertebrates is regulated by the expression of developmental genes which appear in characteristic patterns at neuroepithelial transversal domains, which are called secondary organizers. The molecular code present in these neuroepithelial organizers controls the generation of morphogenetic signals that induce and maintain regional characteristics in the surrounding neuroepithelium. The product of the Fgf8 gene is a secreted protein that has been demonstrated to be the key molecule for the isthmic organizer and is also expressed in two other organizer regions: the zona limitans and the anterior neural ridge. Here we analyze the expression of Fgf15 at different stages of mouse development in relation to Fgf8 and Otx2 expression patterns. © 2002 Elsevier Science Inc.
and colocalizes in organizer regions with Fgf8, we have decided to explore the neural expression of Fgf15 in relation to Fgf8 and Otx2 expression patterns. MATERIALS AND METHODS Mouse Embryos Mid-day of the day of the vaginal plug was considered as 0.5 day postcoitum in the timing of embryos. Staging of embryos used morphological criteria [6] . Timed pregnant Swiss mice were sacrificed and embryos dissected from decidual tissue in chilled phosphate-buffered saline 1⫻ solution and fixed in 4% paraformaldehyde (PFA) at 4°C overnight. In addition to wholemount analysis, 125-m-thick agarose gel vibratome sections were made for gene expression studies at several stages of development.
KEY WORDS: Fgf15, Fgf8, Otx2, Fibroblast growth factor, Secondary organizers, Developmental genes.
RNA Probes INTRODUCTION
Digoxigenin-UTP and fluorescein-UTP labelled single-strand antisense RNA probes were prepared according to instructions of the manufacturer (Boehringer-Mannheim). The Fgf15 probe was used as previously used in our laboratory [15]. The following plasmids were also used, which were kindly provided by A. Simeone (Fgf8 and Otx2) and A. MacMahon (Shh).
The dynamic process of neural tube regionalization in vertebrates is regulated by the combined expression of developmental genes [11]. Characteristic expression patterns of some genes have been found in transversal neuroepithelial domains that separate the main neural tube tagma (segments). These domains regulate local morphogenetic processes during brain development and are known as secondary organizers [8]. The molecular code present in each neuroepithelial organizer controls the generation of morphogenetic signals that induce and maintain regional characteristics in the surrounding neuroepithelium [4,8]. Three areas have been demonstrated as secondary organizers: the isthmic region (IsO) [8,14], the zona limitans (ZLI) ([8], Echevarria et al., in preparation) and the anterior neural ridge (ANR) [5,13]. The product of the Fgf8 gene is a secreted protein and was demonstrated to be the key molecule for the isthmic organizer activity [3,7]. This gene is also expressed in the other two organizer regions: the ZLI and the ANR [2,8]. Otx2 is a transcription factor related to the induction of the anterior part of neural plate and the specification of the IsO [1,4,10,14]. Fgf15 is a gene of the fibroblast growth factor family, which has been recently discovered in mouse embryos [9], and was proposed to be positively regulated by Otx2 expression [15]. In the present work we have carefully analyzed the expression of Fgf15 at different stages of the mouse embryo. Because the Fgf15 gene has been reported to be regulated by Otx2 expression
In situ Hybridization in situ hybridization on mouse whole-mounts and agarose brain sections (125 m) were performed following the protocol described by Shimamura et al. [12]. For one-color staining NBT/ BCIP (Boehringer-Mannheim) was used as chromogenic alkaline phosphatase substrate and for two-color staining NBT/BCIP and INT/BCIP were used (Boehringer-Mannheim). RESULTS AND DISCUSSION We explored the expression of Fgf15 in the neural tube of mouse embryos from early neurulation stages (E8) to later stages of development (E15.5), when most of the neural regions are present and identifiable. At E9.5 Fgf15 transcripts were detected at both sides of the IsO, showing a rostrocaudally decreasing gradient of expression in the alar plate of rhombomere 1, where the cerebellar plate is localized, and an inverse caudorostrally decreasing gradient in the mesencephalic alar plate, where the inferior colliculus primordium
* Address for correspondence: Salvador Martı´nez Pe`rez, Institut of Neurosciences, CSIC-UMH, San Juan, P.O. Box 18, 03550 Alicante, Spain. Fax: ⫹965919555; E-mail: [email protected]
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FIG. 1. (A–C) Lateral view of E9.5 mouse embryos processed by in situ hybridization to detect Fgf15 (A), Fgf8 (B), and Otx2 (C) transcripts. (D) Vibratome sections of an E12.5 mouse brain processed by in situ hybridization showing Fgf15 expression pattern. (E) Schema of gene expressions in an E9.5 mouse brain. Different genes are represented by different grey scales. Abbreviations: ANR, anterior neural ridge; Cp, choroid plexus; DT, dorsal thalamus; F, forebrain; H, hindbrain; Is, isthmus; M, midbrain; os, optic stalk; otv, otic vesicle; PT, pretectum; R, rhombencephalon; VT, ventral thalamus; ZLI, zona limitans intrathalamic.
is localized (Fig. 1A). Between these two inverted gradiential domains of Fgf15 expression a negative transverse band appeared, at the central area of the isthmic constriction. Conversely, this Fgf15 negative tissue stripe was filled by the isthmic expression domain of Fgf8 (Fig. 1B). In the caudal mesencephalon, Fgf15 is coexpressed with Otx2, and their expression domains abruptly end at the same caudal limit (Fig. 1C). Therefore, the central gap of Fgf15 signal is filled by Fgf8 expression, coinciding with strong Gbx2 expression [4] and the IsO region. These differentially gradiential domains of Fgf15 expression may be regulated by the IsO, and coexpress Gbx2 in the cerebellar plate and Otx2 in the mesencephalon. While the mesencephalic expression domain of Fgf5 is known to be under the regulation of Otx2 expression [15], the relation of Fgf8, Gbx2 or other rhombencephalic gene expressions with Fgf15 needs further analysis. In the dorsal area of the anterior diencephalon, in relation with the dorsal end of the ZLI Fgf15 is expressed in a more extense domain than that of Fgf8 expression in dorsal P3 (Fig. 1A). In the ANR, Fgf15 was expressed in the presumptive area of the commissural plate and lamina terminalis, largely in coincidence with the domain of Fgf8 expression (Fig. 1A).
At E11.5–12.5 the expression pattern of Fgf15 around mesencephalico-rhombencephalic junction appeared similar to observations at E9.5. In addition, the alar plate of the pretectal region (P1) expressed Fgf15 (Fig. 1D). At the dorsal diencephalon the Fgf15 positive domain extended both caudal and rostral to the dorsal end of the ZLI (Fig. 1D). Here, similarly to the isthmic region, inverted gradients were detected in the dorsal thalamus (from rostral to caudal) and in the ventral thalamus (from caudal to rostral), showing a negative transverse strip at the level of the ZLI (Fig. 1D). This negative gap was filled by the expression of Shh (Fig. 1E). The most anterior expression of Fgf15 coincided with Fgf8 and Shh expressions in the telencephalic commissural plate (Figs. 1D,E). At later stages, E13.5–15.5, Fgf15 expression was localized along the whole mesencephalic alar plate, showing a specular expression gradients decreasing from maxima at its rostral and its caudal limits (data not shown). In the thalamus, the expression was observed in the neuroepithelium of dorsal and ventral thalamus, with a negative gap in the ZLI (data not shown). The ANR expression was localized to the commissural plate, the medial septum and the olfactory bulb (data not shown).
FGF15 EXPRESSION IN THE NEURAL TUBE The expression of Fgf15 in the mouse neural tube is closely related to the expression of important developmental genes that have been related to active morphogenetic neuroepithelial territories: the IsO, the ZLI and the ANR. In addition, the Fgf15 transcripts were localized in specular gradients of decreasing concentration from the most active domains, suggesting that Fgf15 can be regulated by the active factors localized in the organizers. The active role of Fgf15 in the regulation controlled by these regions is now being studied by experimental embryology methods.
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8. 9.
ACKNOWLEDGEMENTS
We are grateful to A. Simeone for the Fgf8 and Otx2 plasmids and A. McMahon for the Shh plasmid. L. Gimeno was supported by the Seneca Foundation Fellowship BP 00396/CV/00. This work has been supported by the Seneca Foundation 00708/CV/99.
10.
11.
REFERENCES 1. Broccoli, V.; Boncinelli, E.; Wurst, W. The caudal limit of Otx2 expression positions the isthmic organizer. Nature 40:164 –168; 1999. 2. Crossley, P. H.; Martin, G. M. The mouse Fgf8 gene encode a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121:439 – 451; 1995. 3. Crossley, P. H.; Martı´nez, S.; Martin, G. M. Midbrain development induced by FGF8 in the chick embryo. Nature 380:66 – 68; 1996. 4. Garda, A.-L.; Echevarrı´a, D.; Martı´nez, S. Neuroepithelial coexpression of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer. Mech. Dev. 101:111–118; 2001. 5. Houart, C.; Westerfield, M.; Wilson, S. W. A small population of
12.
13.
14. 15.
anterior cells patterns the forebrain during zebrafish gastrulation. Nature 391:788 –792; 1998. Kaufman, M. H. The atlas of mouse development. New York: Academic Press; 1999. Martı´nez, S.; Crossley, P. H.; Cobos, I.; Rubenstein, J. L. R.; Martin, G. R. FGF-8 induces an isthmic organizer and isthmocerebellar development in the caudal forebrain via a repressive effect on Otx2 expression. Development 126:1189 –1200; 1999. Martı´nez, S. The isthmic organizer and brain regionalization. Int. J. Dev. Biol. 45:367–371; 2000. McWhirter, J. R.; Goulding, M.; Weiner, J. A.; Chun, J.; Murre, C. A novel fibroblast growth factor gene expressed in the developing nervous system is a downstream target of the chimeric homeodomain oncoprotein E2A-Pbx1. Development 124:3221–3232; 1997. Millet, S.; Campbell, K.; Epstein, D. J.; Losos, K.; Harris, E.; Joyner, A. L. A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer. Nature 40:161–164; 1999. Rubenstein, J. L. R.; Shimamura, K.; Martı´nez, S.; Puelles, L. Regionalization of the prosencephalic neural plate. Ann. Rev. Neurosci. 21:445– 478; 1998. Shimamura, K.; Hirano, S.; McMahon, A. P.; Takeichi, M. Wnt-1— Dependent regulation of local E-cadherin and ␣N-catenin expression in the embryonic mouse brain. Development 120:2225–2234; 1994. Shimamura, K.; Rubenstein, J. L. R. Inductive interactions direct early regionalization of the mouse forebrain. Development 124:2709 –2718; 1997. Wurst, W.; Bally-Cuif, L. Neural plate patterning: Upstream and downstream of the isthmic organizer. Nat. Rev. 2:99 –108; 2001. Zakin, L.; Reversade, B.; Virlon, B.; Rusniok, C.; Glaser, P.; Elalouf, J.-M.; Bruˆ let, P. Gene expression profiles in normal and Otx2 ⫺/⫺ early gastrulating mouse embryos. Proc. Natl. Acad. Sci. USA 97: 14388 –14393; 2000.
PII S0361-9230(01)00717-1
Analysis of Fgf15 expression pattern in the mouse neural tube L. Gimeno,1 R. Hashemi,2 P. Bruˆlet2 and S. Martı´nez1* 1
Institut of Neurosciences, CSIC-UMH (Miguel Hernandez University), Alicante, Spain; and 2Unite´ d’Embryolodie Mole´culaire, Institut Pasteur, Paris, France
ABSTRACT: The dynamic process of neural tube regionalization in vertebrates is regulated by the expression of developmental genes which appear in characteristic patterns at neuroepithelial transversal domains, which are called secondary organizers. The molecular code present in these neuroepithelial organizers controls the generation of morphogenetic signals that induce and maintain regional characteristics in the surrounding neuroepithelium. The product of the Fgf8 gene is a secreted protein that has been demonstrated to be the key molecule for the isthmic organizer and is also expressed in two other organizer regions: the zona limitans and the anterior neural ridge. Here we analyze the expression of Fgf15 at different stages of mouse development in relation to Fgf8 and Otx2 expression patterns. © 2002 Elsevier Science Inc.
and colocalizes in organizer regions with Fgf8, we have decided to explore the neural expression of Fgf15 in relation to Fgf8 and Otx2 expression patterns. MATERIALS AND METHODS Mouse Embryos Mid-day of the day of the vaginal plug was considered as 0.5 day postcoitum in the timing of embryos. Staging of embryos used morphological criteria [6] . Timed pregnant Swiss mice were sacrificed and embryos dissected from decidual tissue in chilled phosphate-buffered saline 1⫻ solution and fixed in 4% paraformaldehyde (PFA) at 4°C overnight. In addition to wholemount analysis, 125-m-thick agarose gel vibratome sections were made for gene expression studies at several stages of development.
KEY WORDS: Fgf15, Fgf8, Otx2, Fibroblast growth factor, Secondary organizers, Developmental genes.
RNA Probes INTRODUCTION
Digoxigenin-UTP and fluorescein-UTP labelled single-strand antisense RNA probes were prepared according to instructions of the manufacturer (Boehringer-Mannheim). The Fgf15 probe was used as previously used in our laboratory [15]. The following plasmids were also used, which were kindly provided by A. Simeone (Fgf8 and Otx2) and A. MacMahon (Shh).
The dynamic process of neural tube regionalization in vertebrates is regulated by the combined expression of developmental genes [11]. Characteristic expression patterns of some genes have been found in transversal neuroepithelial domains that separate the main neural tube tagma (segments). These domains regulate local morphogenetic processes during brain development and are known as secondary organizers [8]. The molecular code present in each neuroepithelial organizer controls the generation of morphogenetic signals that induce and maintain regional characteristics in the surrounding neuroepithelium [4,8]. Three areas have been demonstrated as secondary organizers: the isthmic region (IsO) [8,14], the zona limitans (ZLI) ([8], Echevarria et al., in preparation) and the anterior neural ridge (ANR) [5,13]. The product of the Fgf8 gene is a secreted protein and was demonstrated to be the key molecule for the isthmic organizer activity [3,7]. This gene is also expressed in the other two organizer regions: the ZLI and the ANR [2,8]. Otx2 is a transcription factor related to the induction of the anterior part of neural plate and the specification of the IsO [1,4,10,14]. Fgf15 is a gene of the fibroblast growth factor family, which has been recently discovered in mouse embryos [9], and was proposed to be positively regulated by Otx2 expression [15]. In the present work we have carefully analyzed the expression of Fgf15 at different stages of the mouse embryo. Because the Fgf15 gene has been reported to be regulated by Otx2 expression
In situ Hybridization in situ hybridization on mouse whole-mounts and agarose brain sections (125 m) were performed following the protocol described by Shimamura et al. [12]. For one-color staining NBT/ BCIP (Boehringer-Mannheim) was used as chromogenic alkaline phosphatase substrate and for two-color staining NBT/BCIP and INT/BCIP were used (Boehringer-Mannheim). RESULTS AND DISCUSSION We explored the expression of Fgf15 in the neural tube of mouse embryos from early neurulation stages (E8) to later stages of development (E15.5), when most of the neural regions are present and identifiable. At E9.5 Fgf15 transcripts were detected at both sides of the IsO, showing a rostrocaudally decreasing gradient of expression in the alar plate of rhombomere 1, where the cerebellar plate is localized, and an inverse caudorostrally decreasing gradient in the mesencephalic alar plate, where the inferior colliculus primordium
* Address for correspondence: Salvador Martı´nez Pe`rez, Institut of Neurosciences, CSIC-UMH, San Juan, P.O. Box 18, 03550 Alicante, Spain. Fax: ⫹965919555; E-mail: [email protected]
297
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FIG. 1. (A–C) Lateral view of E9.5 mouse embryos processed by in situ hybridization to detect Fgf15 (A), Fgf8 (B), and Otx2 (C) transcripts. (D) Vibratome sections of an E12.5 mouse brain processed by in situ hybridization showing Fgf15 expression pattern. (E) Schema of gene expressions in an E9.5 mouse brain. Different genes are represented by different grey scales. Abbreviations: ANR, anterior neural ridge; Cp, choroid plexus; DT, dorsal thalamus; F, forebrain; H, hindbrain; Is, isthmus; M, midbrain; os, optic stalk; otv, otic vesicle; PT, pretectum; R, rhombencephalon; VT, ventral thalamus; ZLI, zona limitans intrathalamic.
is localized (Fig. 1A). Between these two inverted gradiential domains of Fgf15 expression a negative transverse band appeared, at the central area of the isthmic constriction. Conversely, this Fgf15 negative tissue stripe was filled by the isthmic expression domain of Fgf8 (Fig. 1B). In the caudal mesencephalon, Fgf15 is coexpressed with Otx2, and their expression domains abruptly end at the same caudal limit (Fig. 1C). Therefore, the central gap of Fgf15 signal is filled by Fgf8 expression, coinciding with strong Gbx2 expression [4] and the IsO region. These differentially gradiential domains of Fgf15 expression may be regulated by the IsO, and coexpress Gbx2 in the cerebellar plate and Otx2 in the mesencephalon. While the mesencephalic expression domain of Fgf5 is known to be under the regulation of Otx2 expression [15], the relation of Fgf8, Gbx2 or other rhombencephalic gene expressions with Fgf15 needs further analysis. In the dorsal area of the anterior diencephalon, in relation with the dorsal end of the ZLI Fgf15 is expressed in a more extense domain than that of Fgf8 expression in dorsal P3 (Fig. 1A). In the ANR, Fgf15 was expressed in the presumptive area of the commissural plate and lamina terminalis, largely in coincidence with the domain of Fgf8 expression (Fig. 1A).
At E11.5–12.5 the expression pattern of Fgf15 around mesencephalico-rhombencephalic junction appeared similar to observations at E9.5. In addition, the alar plate of the pretectal region (P1) expressed Fgf15 (Fig. 1D). At the dorsal diencephalon the Fgf15 positive domain extended both caudal and rostral to the dorsal end of the ZLI (Fig. 1D). Here, similarly to the isthmic region, inverted gradients were detected in the dorsal thalamus (from rostral to caudal) and in the ventral thalamus (from caudal to rostral), showing a negative transverse strip at the level of the ZLI (Fig. 1D). This negative gap was filled by the expression of Shh (Fig. 1E). The most anterior expression of Fgf15 coincided with Fgf8 and Shh expressions in the telencephalic commissural plate (Figs. 1D,E). At later stages, E13.5–15.5, Fgf15 expression was localized along the whole mesencephalic alar plate, showing a specular expression gradients decreasing from maxima at its rostral and its caudal limits (data not shown). In the thalamus, the expression was observed in the neuroepithelium of dorsal and ventral thalamus, with a negative gap in the ZLI (data not shown). The ANR expression was localized to the commissural plate, the medial septum and the olfactory bulb (data not shown).
FGF15 EXPRESSION IN THE NEURAL TUBE The expression of Fgf15 in the mouse neural tube is closely related to the expression of important developmental genes that have been related to active morphogenetic neuroepithelial territories: the IsO, the ZLI and the ANR. In addition, the Fgf15 transcripts were localized in specular gradients of decreasing concentration from the most active domains, suggesting that Fgf15 can be regulated by the active factors localized in the organizers. The active role of Fgf15 in the regulation controlled by these regions is now being studied by experimental embryology methods.
299
6. 7.
8. 9.
ACKNOWLEDGEMENTS
We are grateful to A. Simeone for the Fgf8 and Otx2 plasmids and A. McMahon for the Shh plasmid. L. Gimeno was supported by the Seneca Foundation Fellowship BP 00396/CV/00. This work has been supported by the Seneca Foundation 00708/CV/99.
10.
11.
REFERENCES 1. Broccoli, V.; Boncinelli, E.; Wurst, W. The caudal limit of Otx2 expression positions the isthmic organizer. Nature 40:164 –168; 1999. 2. Crossley, P. H.; Martin, G. M. The mouse Fgf8 gene encode a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121:439 – 451; 1995. 3. Crossley, P. H.; Martı´nez, S.; Martin, G. M. Midbrain development induced by FGF8 in the chick embryo. Nature 380:66 – 68; 1996. 4. Garda, A.-L.; Echevarrı´a, D.; Martı´nez, S. Neuroepithelial coexpression of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer. Mech. Dev. 101:111–118; 2001. 5. Houart, C.; Westerfield, M.; Wilson, S. W. A small population of
12.
13.
14. 15.
anterior cells patterns the forebrain during zebrafish gastrulation. Nature 391:788 –792; 1998. Kaufman, M. H. The atlas of mouse development. New York: Academic Press; 1999. Martı´nez, S.; Crossley, P. H.; Cobos, I.; Rubenstein, J. L. R.; Martin, G. R. FGF-8 induces an isthmic organizer and isthmocerebellar development in the caudal forebrain via a repressive effect on Otx2 expression. Development 126:1189 –1200; 1999. Martı´nez, S. The isthmic organizer and brain regionalization. Int. J. Dev. Biol. 45:367–371; 2000. McWhirter, J. R.; Goulding, M.; Weiner, J. A.; Chun, J.; Murre, C. A novel fibroblast growth factor gene expressed in the developing nervous system is a downstream target of the chimeric homeodomain oncoprotein E2A-Pbx1. Development 124:3221–3232; 1997. Millet, S.; Campbell, K.; Epstein, D. J.; Losos, K.; Harris, E.; Joyner, A. L. A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer. Nature 40:161–164; 1999. Rubenstein, J. L. R.; Shimamura, K.; Martı´nez, S.; Puelles, L. Regionalization of the prosencephalic neural plate. Ann. Rev. Neurosci. 21:445– 478; 1998. Shimamura, K.; Hirano, S.; McMahon, A. P.; Takeichi, M. Wnt-1— Dependent regulation of local E-cadherin and ␣N-catenin expression in the embryonic mouse brain. Development 120:2225–2234; 1994. Shimamura, K.; Rubenstein, J. L. R. Inductive interactions direct early regionalization of the mouse forebrain. Development 124:2709 –2718; 1997. Wurst, W.; Bally-Cuif, L. Neural plate patterning: Upstream and downstream of the isthmic organizer. Nat. Rev. 2:99 –108; 2001. Zakin, L.; Reversade, B.; Virlon, B.; Rusniok, C.; Glaser, P.; Elalouf, J.-M.; Bruˆ let, P. Gene expression profiles in normal and Otx2 ⫺/⫺ early gastrulating mouse embryos. Proc. Natl. Acad. Sci. USA 97: 14388 –14393; 2000.