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Expression of the homeobox gene Hex during early stages of chick embryo development
Mechanisms of Development 80 (1999) 107–109
Gene expression pattern
Expression of the homeobox gene Hex during early stages of chick embryo development Tatiana A. Yatskievych a, Sharon Pascoe a, Parker B. Antin a , b ,* a
Department of Nutritional Sciences, University of Arizona, Tucson, AZ 85721, USA Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85721, USA
b
Received 23 July 1998; revised version received 8 October 1998; accepted 20 October 1998
Abstract Whole mount in situ hybridization studies were performed to investigate the expression pattern of the homeobox gene Hex (also known as Prh) during early stages of chick embryogenesis. At the time of laying, cHex transcripts are detected in Koller’s sickle and the forming hypoblast. During gastrulation (HH stage 4), cHex is expressed in anteriorly-displaced hypoblast cells. At stage 6, cHex transcripts are observed within endoderm in an anterior arc that overlaps the cardiogenic region. Later cHex expression is observed within pharyngeal endoderm immediately adjacent to the forming myocardium, in the endocardium and in the liver and thyroid gland primordia. cHex transcripts are also detected within blood islands beginning at stage 4, and in extraembryonic and intraembryonic vascular endothelial cells as vessels form. 1999 Elsevier Science Ireland Ltd. All rights reserved Keywords: Angiogenesis; Blood islands; Chicken; Endocardium; Hex; Homeobox; Hypoblast; In situ hybridization; Koller’s sickle; Liver; Mesoderm; Myocardium; Pharyngeal endoderm; Prh; Thyroid; Vascular endothelium
1. Results The homeobox gene Hex was first isolated from hematopoietic cell lines in birds and mammals (Crompton et al., 1992; Bedford et al., 1993; Hromas et al., 1993) and subsequently from two independent hybridization screens in Xenopus (Newman et al., 1997) and mouse (Harrison et al., 1995). Recent studies have implicated Hex in early anterior-posterior patterning and in development of the cardiovascular system (Newman et al., 1997; Thomas et al., 1998). Since a large literature has focused on cardiovascular development in avians, in this study we examined the expression pattern of Hex during early developmental stages of the chick. A chicken Hex cDNA was first cloned by Crompton et al. (1992), and was called Prh. For consistency with other studies in Xenopus and mouse, we have designated the avian gene cHex. cHex transcripts were detected at EGK St. XII in Koller’s * Corresponding author. Tel.: +1-520-621-5993; fax: +1-520-621-9435; e-mail: [email protected]
0925-4773/99/$ - see front matter PII S0925-4773 (98 )0 0204-4
sickle and in all cells of the forming hypoblast (Fig. 1A). cHex expression remained high within all hypoblast cells as the complete layer formed. During mid to late gastrulation (HH stages 4–5), cHex transcripts were detected in hypoblast cells that have been displaced anterior to the primitive streak, and weak expression was occasionally observed within the prechordal mesoderm (Fig. 1B,J). Expression was also detected at this stage within scattered foci of cells in the lateral and posterior area opaca that appear to mark future blood islands. At stage 6, anterior cHex expression was localized to endoderm within an anterior crescent extending bilaterally and posteriorly from the head process to the level of Hensen’s node (Fig. 1C,K). cHex-positive blood islands were observed throughout the lateral and posterior extrambryonic mesoderm, interspersed between chords of cHex-expressing endothelial cells (Fig. 1C). During the next several hours of development (stages 8–10), anterior expression became localized to the cardiogenic crescent along the ridge of the anterior intestinal portal (Fig. 1D,E). Sagittal sections showed that cHex transcripts were restricted to the pharyngeal endoderm immediately
1999 Elsevier Science Ireland Ltd. All rights reserved
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T.A. Yatskievych et al. / Mechanisms of Development 80 (1999) 107–109
Fig. 1. Whole mount in situ hybridization’s showing cHex expression in chick embryos. Ventral views of (A) EGK St. XII, (B) HH St. 5-, (C) St. 6, (D) St. 9, (E) St. 10, (F) St. 12, (G) St. 14 embryos. (H,I) Sagittal sections through the St. XII embryo in (A) at the levels indicated. (J) Transverse section through anterior region of embryo in (B) at level indicated. (K) Transverse section through lateral region of embryo in (C). (L) Sagittal section of embryo in (E) through the anterior intestinal portal. (M–P) Transverse sections through embryo in (F). Arrowhead in (M) indicates the thyroid anlage. (Q) High magnification view of boxed area in (O); expression just lateral to the somite is restricted to intermediate mesoderm in the region of the forming kidney and cardinal vein. (R,S) High magnification dorsal views of embryo in (F), showing cHex transcripts in the forming posterior cardinal and intersomitic vessels (R), and in the vascular network caudal to the youngest somite (S). DA, dorsal aorta; ECT, ectoderm; ENDOC, endocardium; END, endoderm; EPI, epiblast; HYP, hypoblast; KS, Koller’s sickle; MES, mesoderm; MYO, myocardium; SOM, somite.
T.A. Yatskievych et al. / Mechanisms of Development 80 (1999) 107–109
adjacent to the epithelial layer of the forming heart tube (Fig. 1L). This cHex-expressing endoderm will ultimately form the liver. At stage 12, cHex expression was observed in the endocardium, thyroid anlage in the floor of the pharynx, and in the liver primordium just posterior to the heart (Fig. 1F,M). More posteriorly, expression was observed in the endoderm adjacent to the vitelline veins (Fig. 1N), within endothelial cells of the dorsal aortae, in cells just lateral to the somites at the sites of forming posterior cardinal veins and intersomitic vessels (Fig. 1O–Q), and in the posterior and lateral region vasculature (Fig. 1R,S.). At stage 14, cHex expression was observed in the thyroid and liver primordia, kidney, tail bud and scattered blood islands in the posterior area pellucida (Fig. 1G).
2. Methods 2.1. Whole mount in situ hybridization and histology Embryos were collected as described in Yatskievych et al. (1997) and staged according to Eyal-Giladi and Kochav (1976) for pregastrula stages and according to Hamburger and Hamilton (1951) for gastrula stages onward. Following fixation in 4% paraformaldehyde in PBS for 2–24 h at 4°C, embryos were processed as described according to Nieto et al. (1996), except that proteinase K treatment was omitted for pregastrula and early gastrula stage embryos. Digoxigenin-labeled antisense cHex probe was generated according to the manufacturer’s instructions (Boehringer Mannheim; Indianapolis, IN) from full-length chicken Hex (Prh cDNA gift of G.H. Goodwin, Institute of Cancer Research, London). This cDNA recognizes a single mRNA species on Northern blots (Crompton et al., 1992; Hromas et al., 1993). For histology, stained embryos were embedded in 7.5% gelatin/15% sucrose in PBS, frozen in an isopentane bath, and serially sectioned.
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Acknowledgements We thank Dr. G.H. Goodwin for providing the chicken Hex cDNA, and Andrea Ladd for comments regarding the manuscript. This work was supported by grants to P.B.A. from the NIH (HL54133 and HL20220). References Bedford, F.K., Ashworth, A., Enver, T., Wiedemann, L.M., 1993. HEX: a novel homeobox gene expressed during haematopoiesis and conserved between mouse and human. Nucl. Acids Res. 21, 1245–1249. Crompton, M.R., Bartlett, T.J., MacGregor, A.D., Manfioletti, G., Buratti, E., Giancotti, V., Goodwin, G.H., 1992. Identification of a novel vertebrate homeobox gene expressed in hematopoietic cells. Nucl. Acids. Res. 20, 5661–5667. Eyal-Giladi, H., Kochav, S., 1976. From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. Dev. Biol. 49, 321–337. Hamburger, V., Hamilton, H.L., 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49–92. Harrison, S.M., Dunwoodie, S.L., Arkell, R.M., Lehrach, H., Beddington, R.S.P., 1995. Isolation of novel tissue-specific genes from cDNA libraries representing the individual tissue components of the gastrulating mouse embryo. Development 121, 2479–2489. Hromas, R., Radich, J., Collins, S., 1993. PCR cloning of an orphan homeobox gene (PRH) preferentially expressed in myeloid and liver cells. Biochem. Biophys. Res. Commun. 195, 976–983. Newman, C.S., Chia, F., Krieg, P.A., 1997. The xHex homeobox gene is expressed during development of the vascular endothelium: overexpression leads to an increase in vascular endothelial cell number. Mech. Dev. 66, 83–93. Nieto, M.A., Patel, K., Wilkinson, D.G., 1996. In situ hybridization analysis of chick embryos in whole mount and tissue sections. Methods Cell Biol. 51, 219–235. Thomas, P.Q., Brown, A., Beddington, R.S.P., 1998. Hex: a homeobox gene revealing peri-implantation asymmetry in the mouse embryo and an early transient marker of endothelial cell precursors. Development 125, 85–94. Yatskievych, T.A., Ladd, A.N., Antin, P.B., 1997. Induction of cardiac myogenesis in avian pregastrula epiblast: the role of the hypoblast and activin. Development 124, 2561–2570.
Gene expression pattern
Expression of the homeobox gene Hex during early stages of chick embryo development Tatiana A. Yatskievych a, Sharon Pascoe a, Parker B. Antin a , b ,* a
Department of Nutritional Sciences, University of Arizona, Tucson, AZ 85721, USA Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85721, USA
b
Received 23 July 1998; revised version received 8 October 1998; accepted 20 October 1998
Abstract Whole mount in situ hybridization studies were performed to investigate the expression pattern of the homeobox gene Hex (also known as Prh) during early stages of chick embryogenesis. At the time of laying, cHex transcripts are detected in Koller’s sickle and the forming hypoblast. During gastrulation (HH stage 4), cHex is expressed in anteriorly-displaced hypoblast cells. At stage 6, cHex transcripts are observed within endoderm in an anterior arc that overlaps the cardiogenic region. Later cHex expression is observed within pharyngeal endoderm immediately adjacent to the forming myocardium, in the endocardium and in the liver and thyroid gland primordia. cHex transcripts are also detected within blood islands beginning at stage 4, and in extraembryonic and intraembryonic vascular endothelial cells as vessels form. 1999 Elsevier Science Ireland Ltd. All rights reserved Keywords: Angiogenesis; Blood islands; Chicken; Endocardium; Hex; Homeobox; Hypoblast; In situ hybridization; Koller’s sickle; Liver; Mesoderm; Myocardium; Pharyngeal endoderm; Prh; Thyroid; Vascular endothelium
1. Results The homeobox gene Hex was first isolated from hematopoietic cell lines in birds and mammals (Crompton et al., 1992; Bedford et al., 1993; Hromas et al., 1993) and subsequently from two independent hybridization screens in Xenopus (Newman et al., 1997) and mouse (Harrison et al., 1995). Recent studies have implicated Hex in early anterior-posterior patterning and in development of the cardiovascular system (Newman et al., 1997; Thomas et al., 1998). Since a large literature has focused on cardiovascular development in avians, in this study we examined the expression pattern of Hex during early developmental stages of the chick. A chicken Hex cDNA was first cloned by Crompton et al. (1992), and was called Prh. For consistency with other studies in Xenopus and mouse, we have designated the avian gene cHex. cHex transcripts were detected at EGK St. XII in Koller’s * Corresponding author. Tel.: +1-520-621-5993; fax: +1-520-621-9435; e-mail: [email protected]
0925-4773/99/$ - see front matter PII S0925-4773 (98 )0 0204-4
sickle and in all cells of the forming hypoblast (Fig. 1A). cHex expression remained high within all hypoblast cells as the complete layer formed. During mid to late gastrulation (HH stages 4–5), cHex transcripts were detected in hypoblast cells that have been displaced anterior to the primitive streak, and weak expression was occasionally observed within the prechordal mesoderm (Fig. 1B,J). Expression was also detected at this stage within scattered foci of cells in the lateral and posterior area opaca that appear to mark future blood islands. At stage 6, anterior cHex expression was localized to endoderm within an anterior crescent extending bilaterally and posteriorly from the head process to the level of Hensen’s node (Fig. 1C,K). cHex-positive blood islands were observed throughout the lateral and posterior extrambryonic mesoderm, interspersed between chords of cHex-expressing endothelial cells (Fig. 1C). During the next several hours of development (stages 8–10), anterior expression became localized to the cardiogenic crescent along the ridge of the anterior intestinal portal (Fig. 1D,E). Sagittal sections showed that cHex transcripts were restricted to the pharyngeal endoderm immediately
1999 Elsevier Science Ireland Ltd. All rights reserved
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T.A. Yatskievych et al. / Mechanisms of Development 80 (1999) 107–109
Fig. 1. Whole mount in situ hybridization’s showing cHex expression in chick embryos. Ventral views of (A) EGK St. XII, (B) HH St. 5-, (C) St. 6, (D) St. 9, (E) St. 10, (F) St. 12, (G) St. 14 embryos. (H,I) Sagittal sections through the St. XII embryo in (A) at the levels indicated. (J) Transverse section through anterior region of embryo in (B) at level indicated. (K) Transverse section through lateral region of embryo in (C). (L) Sagittal section of embryo in (E) through the anterior intestinal portal. (M–P) Transverse sections through embryo in (F). Arrowhead in (M) indicates the thyroid anlage. (Q) High magnification view of boxed area in (O); expression just lateral to the somite is restricted to intermediate mesoderm in the region of the forming kidney and cardinal vein. (R,S) High magnification dorsal views of embryo in (F), showing cHex transcripts in the forming posterior cardinal and intersomitic vessels (R), and in the vascular network caudal to the youngest somite (S). DA, dorsal aorta; ECT, ectoderm; ENDOC, endocardium; END, endoderm; EPI, epiblast; HYP, hypoblast; KS, Koller’s sickle; MES, mesoderm; MYO, myocardium; SOM, somite.
T.A. Yatskievych et al. / Mechanisms of Development 80 (1999) 107–109
adjacent to the epithelial layer of the forming heart tube (Fig. 1L). This cHex-expressing endoderm will ultimately form the liver. At stage 12, cHex expression was observed in the endocardium, thyroid anlage in the floor of the pharynx, and in the liver primordium just posterior to the heart (Fig. 1F,M). More posteriorly, expression was observed in the endoderm adjacent to the vitelline veins (Fig. 1N), within endothelial cells of the dorsal aortae, in cells just lateral to the somites at the sites of forming posterior cardinal veins and intersomitic vessels (Fig. 1O–Q), and in the posterior and lateral region vasculature (Fig. 1R,S.). At stage 14, cHex expression was observed in the thyroid and liver primordia, kidney, tail bud and scattered blood islands in the posterior area pellucida (Fig. 1G).
2. Methods 2.1. Whole mount in situ hybridization and histology Embryos were collected as described in Yatskievych et al. (1997) and staged according to Eyal-Giladi and Kochav (1976) for pregastrula stages and according to Hamburger and Hamilton (1951) for gastrula stages onward. Following fixation in 4% paraformaldehyde in PBS for 2–24 h at 4°C, embryos were processed as described according to Nieto et al. (1996), except that proteinase K treatment was omitted for pregastrula and early gastrula stage embryos. Digoxigenin-labeled antisense cHex probe was generated according to the manufacturer’s instructions (Boehringer Mannheim; Indianapolis, IN) from full-length chicken Hex (Prh cDNA gift of G.H. Goodwin, Institute of Cancer Research, London). This cDNA recognizes a single mRNA species on Northern blots (Crompton et al., 1992; Hromas et al., 1993). For histology, stained embryos were embedded in 7.5% gelatin/15% sucrose in PBS, frozen in an isopentane bath, and serially sectioned.
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Acknowledgements We thank Dr. G.H. Goodwin for providing the chicken Hex cDNA, and Andrea Ladd for comments regarding the manuscript. This work was supported by grants to P.B.A. from the NIH (HL54133 and HL20220). References Bedford, F.K., Ashworth, A., Enver, T., Wiedemann, L.M., 1993. HEX: a novel homeobox gene expressed during haematopoiesis and conserved between mouse and human. Nucl. Acids Res. 21, 1245–1249. Crompton, M.R., Bartlett, T.J., MacGregor, A.D., Manfioletti, G., Buratti, E., Giancotti, V., Goodwin, G.H., 1992. Identification of a novel vertebrate homeobox gene expressed in hematopoietic cells. Nucl. Acids. Res. 20, 5661–5667. Eyal-Giladi, H., Kochav, S., 1976. From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. Dev. Biol. 49, 321–337. Hamburger, V., Hamilton, H.L., 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49–92. Harrison, S.M., Dunwoodie, S.L., Arkell, R.M., Lehrach, H., Beddington, R.S.P., 1995. Isolation of novel tissue-specific genes from cDNA libraries representing the individual tissue components of the gastrulating mouse embryo. Development 121, 2479–2489. Hromas, R., Radich, J., Collins, S., 1993. PCR cloning of an orphan homeobox gene (PRH) preferentially expressed in myeloid and liver cells. Biochem. Biophys. Res. Commun. 195, 976–983. Newman, C.S., Chia, F., Krieg, P.A., 1997. The xHex homeobox gene is expressed during development of the vascular endothelium: overexpression leads to an increase in vascular endothelial cell number. Mech. Dev. 66, 83–93. Nieto, M.A., Patel, K., Wilkinson, D.G., 1996. In situ hybridization analysis of chick embryos in whole mount and tissue sections. Methods Cell Biol. 51, 219–235. Thomas, P.Q., Brown, A., Beddington, R.S.P., 1998. Hex: a homeobox gene revealing peri-implantation asymmetry in the mouse embryo and an early transient marker of endothelial cell precursors. Development 125, 85–94. Yatskievych, T.A., Ladd, A.N., Antin, P.B., 1997. Induction of cardiac myogenesis in avian pregastrula epiblast: the role of the hypoblast and activin. Development 124, 2561–2570.