- Email: [email protected]
Delta expression in the developing mouse lung
Mechanisms of Development 98 (2000) 95±98
www.elsevier.com/locate/modo
Gene expression pattern
Notch/Delta expression in the developing mouse lung Laura C. Post a,b, Melissa Ternet a,b, Brigid L.M. Hogan a,b,* a
Howard Hughes Medical Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA b Department of Cell Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA Received 6 July 2000; accepted 24 July 2000
Abstract Factors controlling the differentiation of the multipotent embryonic lung endoderm and mesoderm are poorly understood. Recent evidence that Delta-like 1 (Dll1) and other genes in the Notch/Delta signaling pathway are expressed in the embryonic mouse lung suggests that this pathway is important for cell fate decisions and/or the differentiation of lung cell types. Here, we report the localization of transcripts of several genes encoding members of the Notch/Delta pathway in the early mouse lung. Most genes are expressed in speci®c populations and so may contribute to cell diversi®cation. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Embryonic; Lung; Notch; Delta; Jagged; Radical fringe; Lunatic fringe; Endoderm
1. Results Previous studies of the lung expression of Notch/Delta genes relied primarily on Northern analyses and RNase protection experiments and did not address the temporal and spatial localization of transcripts in speci®c cell populations. Null mutations have been generated for the genes Notch1 (Swiatek et al., 1994; Conlon et al., 1995), Notch2 (Hamada et al., 1999), Notch4 (Krebs et al., 2000), Jagged1 (Xue et al., 1999), Jagged2 (Jiang et al., 1998), and Dll1 (Hrabe de Angelis et al., 1997). Notch4 null mutant mice are viable and fertile (Krebs et al., 2000) and Jagged2 (Jag2) null mice die shortly after birth from cleft palate (Jiang et al., 1998), but neither have been reported to have any lung abnormalities. Homozygous null mutations in the other genes are embryonic lethal, precluding analysis of their importance in lung development. To further explore the role of Notch/Delta genes, the expression of several members of this pathway was examined by whole-mount and section in situ hybridization and LacZ staining. Notch1 expression is seen in the distal endoderm at all times examined (E11.5±E13.5; Fig. 1A). By contrast, Notch2 and Notch3 are expressed throughout the mesenchyme, although Notch3 transcripts are also found at low levels in the endoderm (Fig. 1B,C,I±L). Notch4 expression
* Corresponding author. Tel.: 11-615-343-6418; fax: 11-614-343-2033. E-mail address: [email protected] (B.L.M. Hogan).
is con®ned to the endothelium of the blood vessels (data not shown; Uyttendaele et al., 1996). Dll1 was shown previously to be expressed within the respiratory epithelium after E13.5 using a LacZ reporter construct (Fig. 1D; Beckers et al., 1999). Dll3 expression was never observed in the developing mouse lung at any age examined (data not shown). Jagged1 (Jag1) transcripts are present in the mesenchyme and blood vessels (Fig. 1E), while Jag2 expression is observed in the peripheral mesenchyme underlying the surface mesothelium (Fig. 1F). Radical and Lunatic fringe proteins are believed to modulate the binding of ligand to the Notch receptors (Cohen et al., 1997; Johnston et al., 1997; Irvine, 1999). Expression of the genes encoding these proteins was also examined. Lfng RNA is localized to the developing endoderm of both the trachea and respiratory tree (Fig. 1G) while Rfng expression is ubiquitous throughout the endoderm and mesenchyme (Fig. 1H). The localized expression of Dll1 during early lung development was studied in more detail using lungs from Dll1 LacZ heterozygous animals (kindly provided by Dr Achim Gossler). Two distinct positive cell populations were found. First, LacZ-positive cells are observed in isolated clusters in the secondary bronchi after E13.5 (Fig. 1D). As gestation progresses, positive cells increase in number and can be found within more terminal branches of the bronchioles, often at branch points (Fig. 2B,C, and data not shown). Postnatally, fewer positive endodermal cells are found only within the deepest parts of the lung tissue; this pattern is maintained in the adult (Fig. 2D±G). The early expression
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00432-9
96
L.C. Post et al. / Mechanisms of Development 98 (2000) 95±98
Fig. 1. Gene expression in whole-mount mouse lungs. (A±H) Whole-mount in situ hybridization of lungs at E13 except for (D) which is LacZ staining of an E14.5 lung from a Dlll Lacz heterozygous mouse. Sense control for whole-mount probes were negative (data not shown). Section in situ hybridization of Notch2 (I,J), Notch3 (K,L), and a sense probe (M,N). Panels (J,L,N) are dark®eld images of (I,K,M) respectively. Scale bar, 200 mM (I±N).
pattern of Dll1 and the similar temporal-spatial pattern of expression seen for Mash1, encoding a mediator of Dll1 function (Borges et al., 1997), suggests that the Dll1-positive cells are neuroendocrine cells (NE cells). Mash1 null mice are viable but lack NE cells in the adult lung (Borges et al., 1997). A second pattern of Dll1 expression was seen after postnatal day 7 throughout the lung tissue (Fig. 2B). Upon sectioning, positive cells were identi®ed as endothelial cells lining the lung vasculature (Fig. 2F,G; Beckers et al., 1999).
2. Materials and methods 2.1. In situ hybridization Lungs were collected from E11.5 to E13.5 embryos obtained by timed matings of ICR mice (Harlan), ®xed for 4 h in 4% paraformaldehyde/PBS, and dehydrated. Lungs were then assayed for whole-mount or radioactive section in situ hybridization (Hogan et al., 1994). Probes used were Notch1 (4 Kb), Notch2 (0.9 Kb), Notch3 (2.6 Kb), Notch4
L.C. Post et al. / Mechanisms of Development 98 (2000) 95±98
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Fig. 2. b -Galactosidase staining of lungs from Dlll Lacz heterozygous mice at different stages of development. (A) Whole E14.5 lung, (B) left lobe of adult lung, (C) E14.5, (D) E18.5, (E) P4, (F) P7, (G) P8 and (H) adult. Arrowheads in (A) indicate Lacz positive cells in bronchi. Arrows in(D,E,G) indicate positive bronchiolar cells. Note absence of positive cells in blood vessels until P8 (G). Scale bar, 500 mM (B); 250 mM (C±G).
(1.8 Kb), Dll3 (2.1 Kb), Jag2 (1.4 Kb), Rfng (2.1 Kb), and Lfng (1.2 Kb). 2.2. b -Galactosidase expression Dll1 LacZ heterozygous mice (kindly provided by Drs Johannes Beckers and Achim Gossler) were mated with ICR mice. Embryos from timed matings and mice after birth were harvested at designated ages, the lungs were ®xed in 4% paraformaldehyde/PBS, permeabilized and stained for LacZ from overnight to 2 days (Gannon et al., 2000). Heterozygous lungs were paraf®n embedded, sectioned at 7 mm, and counterstained with eosin.
Acknowledgements Probes were generously donated by Drs Thomas Gridley (Notch2, Notch3, Notch4), Achim Gossler (Delta3), Janet Rossant (Notch1), and David Ish-Horowitz (Jag1). We thank Dr Mildred Stahlman and Dr Joyce Johnson for helpful discussions, and the members of the Hogan laboratory for technical assistance and critical reading of the manuscript. References Beckers, J., Clark, A., Wunsch, K., Angelis, M.H.D., Gossler, A., 1999. Expression of the mouse Delta1 gene during organogenesis and fetal development. Mech. Dev. 84, 165±168.
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L.C. Post et al. / Mechanisms of Development 98 (2000) 95±98
Borges, M., Linnoila, R.I., Velde, H.J.K.v.d., Chen, H., Nelkin, B.D., Mabry, M., Baylin, S.B., Ball, D.W., 1997. An achaete-scute homologue essential for neuroendocrine differentiation in the lung. Nature 386, 852±855. Cohen, B., Bashirullah, A., Dagnino, L., Campbell, C., Fisher, W.W., Leow, C.C., Whiting, E., Ryan, D., Zinyk, D., Boulianne, G., Hui, C.-c., Gallie, B., Phillips, R.A., Lipshitz, H.D., Egan, S.E., 1997. Fringe boundaries coincide with Notch-dependent patterning centres in mammals and alter Notch-dependent development in Drosophila. Nat. Genet. 16, 283±288. Conlon, R.A., Reaume, A.G., Rossant, J., 1995. Notch1 is required for the coordinate segmentation of somites. Development 121, 1533±1545. Gannon, M., Ray, M.K., Zee, K.V., Rausa, F., Costa, R.H., Wright, C.V.E., 2000. Persistent expression of HNF6 in islet endocrine cells causes disrupted islet architecture and loss of beta cell function. Development 127, 2883±2895. Hamada, Y., Kadokawa, Y., Okabe, M., Ikawa, M., Coleman, J.R., Tsujimoto, Y., 1999. Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality. Development 126, 3415±3424. Hogan, B., Beddington, R., Costantini, F., Lacy, E., 1994. Manipulating the mouse embryo: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Hrabe de Angelis II, M.J.M., Gossler, A., 1997. Maintenance of somite borders in mice requires the Delta homologue Dll1. Nature 386, 717± 721.
Irvine, K.D., 1999. Fringe, Notch, and making developmental boundaries. Curr. Op. Gen. Dev. 9, 434±441. Jiang, R., Lan, Y., Chapman, H.D., Shawber, C., Norton, C.R., Serreze, D.V., Weinmaster, G., Gridley, T., 1998. Defects in limb, craniofacial, and thymic development in Jagged2 mutant mice. Genes Dev. 12, 1046±1057. Johnston, S.H., Rauskolb, C., Wilson, R., Prabhakaran, B., Irvine, K.D., Vogt, T.F., 1997. A family of mammalian Fringe genes implicated in boundary determination and the Notch pathway. Development 124, 2245±2254. Krebs, L.T., Xue, T., Norton, C.R., Shutter, J.R., Maguire, M., Sundberg, J.P., Gallahan, D., Closson, V., Kitajewski, J., Callahan, R., Smith, G.H., Stark, K.L., Gridley, T., 2000. Notch signalling is essential for vascular morphogenesis in mice. Genes Dev. 14, 1343±1352. Swiatek, P.J., Lindsell, C.E., Amo, F.F.d, Weinmaster, G., Gridley, T., 1994. Notch1 is essential for postimplantation development in mice. Genes Dev. 8, 707±719. Uyttendaele, H., Marazzi, G., Wu, G., Yan, Q., Sassoon, D., Kitajewski, J., 1996. Notch4/int3, a mammary proto-oncogene, is an endothelial cellspeci®c mammalian Notch gene. Development 122, 2251±2259. Xue, Y., Gao, X., Lindsell, C.E., Norton, C.R., Chang, B., Hicks, C., Gendron-Maguire, M., Rand, E.B., Weinmaster, G., Gridley, T., 1999. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum. Mol. Gen. 8, 723±730.
www.elsevier.com/locate/modo
Gene expression pattern
Notch/Delta expression in the developing mouse lung Laura C. Post a,b, Melissa Ternet a,b, Brigid L.M. Hogan a,b,* a
Howard Hughes Medical Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA b Department of Cell Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA Received 6 July 2000; accepted 24 July 2000
Abstract Factors controlling the differentiation of the multipotent embryonic lung endoderm and mesoderm are poorly understood. Recent evidence that Delta-like 1 (Dll1) and other genes in the Notch/Delta signaling pathway are expressed in the embryonic mouse lung suggests that this pathway is important for cell fate decisions and/or the differentiation of lung cell types. Here, we report the localization of transcripts of several genes encoding members of the Notch/Delta pathway in the early mouse lung. Most genes are expressed in speci®c populations and so may contribute to cell diversi®cation. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Embryonic; Lung; Notch; Delta; Jagged; Radical fringe; Lunatic fringe; Endoderm
1. Results Previous studies of the lung expression of Notch/Delta genes relied primarily on Northern analyses and RNase protection experiments and did not address the temporal and spatial localization of transcripts in speci®c cell populations. Null mutations have been generated for the genes Notch1 (Swiatek et al., 1994; Conlon et al., 1995), Notch2 (Hamada et al., 1999), Notch4 (Krebs et al., 2000), Jagged1 (Xue et al., 1999), Jagged2 (Jiang et al., 1998), and Dll1 (Hrabe de Angelis et al., 1997). Notch4 null mutant mice are viable and fertile (Krebs et al., 2000) and Jagged2 (Jag2) null mice die shortly after birth from cleft palate (Jiang et al., 1998), but neither have been reported to have any lung abnormalities. Homozygous null mutations in the other genes are embryonic lethal, precluding analysis of their importance in lung development. To further explore the role of Notch/Delta genes, the expression of several members of this pathway was examined by whole-mount and section in situ hybridization and LacZ staining. Notch1 expression is seen in the distal endoderm at all times examined (E11.5±E13.5; Fig. 1A). By contrast, Notch2 and Notch3 are expressed throughout the mesenchyme, although Notch3 transcripts are also found at low levels in the endoderm (Fig. 1B,C,I±L). Notch4 expression
* Corresponding author. Tel.: 11-615-343-6418; fax: 11-614-343-2033. E-mail address: [email protected] (B.L.M. Hogan).
is con®ned to the endothelium of the blood vessels (data not shown; Uyttendaele et al., 1996). Dll1 was shown previously to be expressed within the respiratory epithelium after E13.5 using a LacZ reporter construct (Fig. 1D; Beckers et al., 1999). Dll3 expression was never observed in the developing mouse lung at any age examined (data not shown). Jagged1 (Jag1) transcripts are present in the mesenchyme and blood vessels (Fig. 1E), while Jag2 expression is observed in the peripheral mesenchyme underlying the surface mesothelium (Fig. 1F). Radical and Lunatic fringe proteins are believed to modulate the binding of ligand to the Notch receptors (Cohen et al., 1997; Johnston et al., 1997; Irvine, 1999). Expression of the genes encoding these proteins was also examined. Lfng RNA is localized to the developing endoderm of both the trachea and respiratory tree (Fig. 1G) while Rfng expression is ubiquitous throughout the endoderm and mesenchyme (Fig. 1H). The localized expression of Dll1 during early lung development was studied in more detail using lungs from Dll1 LacZ heterozygous animals (kindly provided by Dr Achim Gossler). Two distinct positive cell populations were found. First, LacZ-positive cells are observed in isolated clusters in the secondary bronchi after E13.5 (Fig. 1D). As gestation progresses, positive cells increase in number and can be found within more terminal branches of the bronchioles, often at branch points (Fig. 2B,C, and data not shown). Postnatally, fewer positive endodermal cells are found only within the deepest parts of the lung tissue; this pattern is maintained in the adult (Fig. 2D±G). The early expression
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00432-9
96
L.C. Post et al. / Mechanisms of Development 98 (2000) 95±98
Fig. 1. Gene expression in whole-mount mouse lungs. (A±H) Whole-mount in situ hybridization of lungs at E13 except for (D) which is LacZ staining of an E14.5 lung from a Dlll Lacz heterozygous mouse. Sense control for whole-mount probes were negative (data not shown). Section in situ hybridization of Notch2 (I,J), Notch3 (K,L), and a sense probe (M,N). Panels (J,L,N) are dark®eld images of (I,K,M) respectively. Scale bar, 200 mM (I±N).
pattern of Dll1 and the similar temporal-spatial pattern of expression seen for Mash1, encoding a mediator of Dll1 function (Borges et al., 1997), suggests that the Dll1-positive cells are neuroendocrine cells (NE cells). Mash1 null mice are viable but lack NE cells in the adult lung (Borges et al., 1997). A second pattern of Dll1 expression was seen after postnatal day 7 throughout the lung tissue (Fig. 2B). Upon sectioning, positive cells were identi®ed as endothelial cells lining the lung vasculature (Fig. 2F,G; Beckers et al., 1999).
2. Materials and methods 2.1. In situ hybridization Lungs were collected from E11.5 to E13.5 embryos obtained by timed matings of ICR mice (Harlan), ®xed for 4 h in 4% paraformaldehyde/PBS, and dehydrated. Lungs were then assayed for whole-mount or radioactive section in situ hybridization (Hogan et al., 1994). Probes used were Notch1 (4 Kb), Notch2 (0.9 Kb), Notch3 (2.6 Kb), Notch4
L.C. Post et al. / Mechanisms of Development 98 (2000) 95±98
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Fig. 2. b -Galactosidase staining of lungs from Dlll Lacz heterozygous mice at different stages of development. (A) Whole E14.5 lung, (B) left lobe of adult lung, (C) E14.5, (D) E18.5, (E) P4, (F) P7, (G) P8 and (H) adult. Arrowheads in (A) indicate Lacz positive cells in bronchi. Arrows in(D,E,G) indicate positive bronchiolar cells. Note absence of positive cells in blood vessels until P8 (G). Scale bar, 500 mM (B); 250 mM (C±G).
(1.8 Kb), Dll3 (2.1 Kb), Jag2 (1.4 Kb), Rfng (2.1 Kb), and Lfng (1.2 Kb). 2.2. b -Galactosidase expression Dll1 LacZ heterozygous mice (kindly provided by Drs Johannes Beckers and Achim Gossler) were mated with ICR mice. Embryos from timed matings and mice after birth were harvested at designated ages, the lungs were ®xed in 4% paraformaldehyde/PBS, permeabilized and stained for LacZ from overnight to 2 days (Gannon et al., 2000). Heterozygous lungs were paraf®n embedded, sectioned at 7 mm, and counterstained with eosin.
Acknowledgements Probes were generously donated by Drs Thomas Gridley (Notch2, Notch3, Notch4), Achim Gossler (Delta3), Janet Rossant (Notch1), and David Ish-Horowitz (Jag1). We thank Dr Mildred Stahlman and Dr Joyce Johnson for helpful discussions, and the members of the Hogan laboratory for technical assistance and critical reading of the manuscript. References Beckers, J., Clark, A., Wunsch, K., Angelis, M.H.D., Gossler, A., 1999. Expression of the mouse Delta1 gene during organogenesis and fetal development. Mech. Dev. 84, 165±168.
98
L.C. Post et al. / Mechanisms of Development 98 (2000) 95±98
Borges, M., Linnoila, R.I., Velde, H.J.K.v.d., Chen, H., Nelkin, B.D., Mabry, M., Baylin, S.B., Ball, D.W., 1997. An achaete-scute homologue essential for neuroendocrine differentiation in the lung. Nature 386, 852±855. Cohen, B., Bashirullah, A., Dagnino, L., Campbell, C., Fisher, W.W., Leow, C.C., Whiting, E., Ryan, D., Zinyk, D., Boulianne, G., Hui, C.-c., Gallie, B., Phillips, R.A., Lipshitz, H.D., Egan, S.E., 1997. Fringe boundaries coincide with Notch-dependent patterning centres in mammals and alter Notch-dependent development in Drosophila. Nat. Genet. 16, 283±288. Conlon, R.A., Reaume, A.G., Rossant, J., 1995. Notch1 is required for the coordinate segmentation of somites. Development 121, 1533±1545. Gannon, M., Ray, M.K., Zee, K.V., Rausa, F., Costa, R.H., Wright, C.V.E., 2000. Persistent expression of HNF6 in islet endocrine cells causes disrupted islet architecture and loss of beta cell function. Development 127, 2883±2895. Hamada, Y., Kadokawa, Y., Okabe, M., Ikawa, M., Coleman, J.R., Tsujimoto, Y., 1999. Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality. Development 126, 3415±3424. Hogan, B., Beddington, R., Costantini, F., Lacy, E., 1994. Manipulating the mouse embryo: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Hrabe de Angelis II, M.J.M., Gossler, A., 1997. Maintenance of somite borders in mice requires the Delta homologue Dll1. Nature 386, 717± 721.
Irvine, K.D., 1999. Fringe, Notch, and making developmental boundaries. Curr. Op. Gen. Dev. 9, 434±441. Jiang, R., Lan, Y., Chapman, H.D., Shawber, C., Norton, C.R., Serreze, D.V., Weinmaster, G., Gridley, T., 1998. Defects in limb, craniofacial, and thymic development in Jagged2 mutant mice. Genes Dev. 12, 1046±1057. Johnston, S.H., Rauskolb, C., Wilson, R., Prabhakaran, B., Irvine, K.D., Vogt, T.F., 1997. A family of mammalian Fringe genes implicated in boundary determination and the Notch pathway. Development 124, 2245±2254. Krebs, L.T., Xue, T., Norton, C.R., Shutter, J.R., Maguire, M., Sundberg, J.P., Gallahan, D., Closson, V., Kitajewski, J., Callahan, R., Smith, G.H., Stark, K.L., Gridley, T., 2000. Notch signalling is essential for vascular morphogenesis in mice. Genes Dev. 14, 1343±1352. Swiatek, P.J., Lindsell, C.E., Amo, F.F.d, Weinmaster, G., Gridley, T., 1994. Notch1 is essential for postimplantation development in mice. Genes Dev. 8, 707±719. Uyttendaele, H., Marazzi, G., Wu, G., Yan, Q., Sassoon, D., Kitajewski, J., 1996. Notch4/int3, a mammary proto-oncogene, is an endothelial cellspeci®c mammalian Notch gene. Development 122, 2251±2259. Xue, Y., Gao, X., Lindsell, C.E., Norton, C.R., Chang, B., Hicks, C., Gendron-Maguire, M., Rand, E.B., Weinmaster, G., Gridley, T., 1999. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum. Mol. Gen. 8, 723±730.