Mesodermal defects and cranial neural crest ... - Semantic Scholar

4309

Development 124, 4309-4319 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 DEV3667

Mesodermal defects and cranial neural crest apoptosis in α5 integrin-null embryos Keow Lin Goh, Joy T. Yang and Richard O. Hynes* Howard Hughes Medical Institute, Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA *Author for correspondence

SUMMARY α5β1 integrin is a cell surface receptor that mediates cellextracellular matrix adhesions by interacting with fibronectin. α5 subunit-deficient mice die early in gestation and display mesodermal defects; most notably, embryos have a truncated posterior and fail to produce posterior somites. In this study, we report on the in vivo effects of the α5-null mutation on cell proliferation and survival, and on mesodermal development. We found no significant differences in the numbers of apoptotic cells or in cell proliferation in the mesoderm of α5-null embryos compared to wildtype controls. These results suggest that changes in overall cell death or cell proliferation rates are unlikely to be responsible for the mesodermal deficits seen in the α5-null embryos. No increases in cell death were seen in α5-null embryonic yolk sac, amnion and allantois compared with wild-type, indicating that the mutant phenotype is not due to changes in apoptosis rates in these extraembryonic tissues. Increased numbers of dying cells were, however, seen in migrating cranial neural crest cells of the hyoid arch and in endodermal cells surrounding the omphalomesenteric artery in α5-null embryos, indicating that these sub-

populations of cells are dependent on α5 integrin function for their survival. Mesodermal markers mox-1, Notch-1, Brachyury (T) and Sonic hedgehog (Shh) were expressed in the mutant embryos in a regionally appropriate fashion. Both T and Shh, however, showed discontinuous expression in the notochords of α5-null embryos due to (1) degeneration of the notochordal tissue structure, and (2) non-maintenance of gene expression. Consistent with the disorganization of notochordal signals in the α5-null embryos, reduced Pax-1 expression and misexpression of Pax-3 were observed. Anteriorly expressed HoxB genes were expressed normally in the α5-null embryos. However, expression of the posteriormost HoxB gene, Hoxb-9, was reduced in α5null embryos. These results suggest that α5β1-fibronectin interactions are not essential for the initial commitment of mesodermal cells, but are crucial for maintenance of mesodermal derivatives during postgastrulation stages and also for the survival of some neural crest cells.

INTRODUCTION

at high levels in early developing Xenopus and chicken embryos, but is later down-regulated in adult tissues (Muschler and Horwitz, 1991; DeSimone, 1994). It is found in mesenchymal and connective tissues, and in all muscle types in the chicken embryo. Studies have shown that α5β1-FN binding regulates a variety of cellular responses including gene induction (Huhtala et al., 1995), oncogenic transformation (Giancotti and Ruoslahti, 1990; Dedhar, 1995), FN assembly (Fogerty et al., 1990), differentiation (Adams and Watt, 1990; Boukamp and Fusenig, 1993), adhesion and migration (Akiyama et al., 1989; Giancotti and Ruoslahti, 1990), and proliferation and cell survival (Varner et al., 1995; Zhang et al., 1995). The targeted disruption of the α5 integrin subunit in mice has shown that α5β1 plays a crucial role in postgastrulation development (Yang et al., 1993). Mice lacking α5 integrin subunit (α5-nulls) fail to complete the turning process, and die at embryonic gestation day (E) 10-11 apparently due to vascular defects. Most strikingly, the α5-null embryos have

Cell-cell and cell-extracellular matrix (ECM) interactions have long been thought to play critical roles in developmental processes (Damsky and Werb, 1992; Adams and Watt, 1993). One class of cell surface receptors that mediate such interactions are the integrins (Hynes, 1992). Integrins are heterodimeric transmembrane glycoproteins consisting of noncovalently bound α and β subunits. ECM molecules recognized by integrins include collagens, laminins and fibronectin (FN). Many integrins localize to focal adhesions, sites of cell-ECM adhesion where integrin cytoplasmic domains bind to components of the actin cytoskeleton. These integrin-cytoskeletal networks are crucial in regulating changes in cell adhesion and shape that occur during cell migration and spreading, and also form the foundation for the construction of signalling complexes (Damsky and Werb, 1992; Clark and Brugge, 1995; Schwartz et al., 1995). The α5β1 integrin is a major fibronectin receptor expressed

Key words: α5-integrin, apoptosis, cell proliferation, mesoderm, neural crest, mouse

4310 K. L. Goh, J. T. Yang and R. O. Hynes normal anterior structures, forming heart, brain, otic and optic vesicles, but beyond the axial level of somites 7-10, possess distorted trunk regions, have a kinked neural tube and fail to form posterior somites. At E8-8.5, mutant embryos show a slightly shortened anterior-posterior axis but have normal numbers of somites. As the mutant embryos develop, their posterior defects become more pronounced and their tail regions fail to increase in size relative to the embryos’ anterior regions. At E9.5, mutant embryos are significantly delayed, equivalent in size to E9 wild-type embryos. In vitro studies demonstrated that embryonic cells derived from the α5-null mice remain capable of FN matrix assembly and migration on a FN substratum (Yang et al., 1993). Our aim in this study was to investigate the basis of the mesodermal deficit seen in α5-null embryos. The deficit might be due to defects in the migration, determination or differentiation of cells that make up the posterior trunk region of the embryo. Alternatively, a decrease in cell proliferation and/or increase in cell death might be responsible for the mutant phenotype. Previous studies, mentioned above, that have implicated α5β1FN interactions in all these processes, were based on experiments performed in cell culture systems, on studies of expression patterns and, in some cases, on the injection of inhibitory agents into embryos. In contrast, in this study, we have used in situ assays to analyze the in vivo functions of α5 integrin in early embryogenesis. We have examined the effects of the loss of α5 integrin on embryonic cell proliferation and cell death. Using markers, we have investigated the ability of mesodermal precursor cells to undergo determination and differentiation into axial and paraxial structures, and examined the extent of axis determination in α5-null embryos. To examine further the effects of α5 on the cell cycle, we investigated cell cycle status of embryonic fibroblasts derived from α5-null and wild-type embryos. Our results suggest that proliferation and survival of mesodermal cells are independent of the presence of α5β1 integrin, and that absence of α5 does not increase cell deaths in the visceral yolk sac, amnion and allantois. Survival of subpopulations of cranial neural crest and of posterior endodermal cells, however, was dependent on α5β1 integrin. Absence of α5 integrin resulted in alterations in the expression patterns of several mesodermal markers and posteriorly expressed HoxB genes. Most strikingly T and Shh expression were not maintained in the notochordal tissues. Concurrently, notochordal tissues underwent degeneration in the α5-null embryos. MATERIALS AND METHODS Genotyping of embryos E8.5-9.5 mouse embryos were dissected out as described by Cockroft (1990). Embryos were treated as described in the protocols below. Yolk sacs were removed and lysed in buffer containing 50 mM TrisHCl pH 8.0, 10 mM EDTA, 100 mM NaCl, 0.1% SDS, 1.2 mg/ml Proteinase K. DNA was subjected to PCR with Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT), using α5-specific primers as well as primers for the neoR gene (Yang et al., 1995), under the following conditions: 94°C, 1 minute; 60°C, 1 minute 30 seconds and 72°C, 1 minute 30 seconds for 33 cycles. Immunohistochemistry 8 µm frozen sections from wild-type embryos were made as described by Yang et al. (1995). Sections were fixed in acetone for 5 minutes at

−20° C, air dried for 1 hour and stored at −80°C. Sections were washed with PBS/0.1% bovine serum albumin (BSA), blocked in blocking buffer (5% normal goat serum in PBS/0.1% BSA) for 30 minutes, and incubated for 30 minutes with rat anti-mouse α5 integrin antibody (antiCD49e: Pharmingen, San Diego, CA) diluted 1:500 in blocking buffer. Control slides were treated with antibody isotype control, rat IgG2a (Zymed, San Francisco, CA). Sections were then washed with PBS and incubated with biotinylated goat anti-rat IgG secondary antibody (Vector Labs, Burlingame, CA). After 3 washes in PBS, sections were incubated with 0.3% H2O2 in methanol for 30 minutes, followed by Endoblock (Biomeda, Foster City, CA) for 5 minutes. Sections were then washed in PBS and incubated with ABC reagent mix (Vector Labs) for 30 minutes, followed by 3,3′-diaminobenzidine (DAB)/H2O2 mix (DAB substrate kit, Vector Labs) for 2 minutes. Sections were counterstained with hematoxylin for 10 seconds, dehydrated in ethanol and xylene, mounted with Permount (Fisher, Chicago, IL), and then photographed on an Axiophot microscope (Carl Zeiss, Thornwood, NY). In situ hybridization and histology Whole-mount RNA in situ hybridization was performed essentially as described by Wilkinson (1992). After hybridization procedures, embryos were fixed in 0.1% glutaraldehyde/4% paraformaldehyde, cleared in 50% glycerol/PBS, then 80% glycerol/PBS overnight. Whole-mount embryos were mounted in 80% glycerol/PBS and photographed using bright-field or Nomarski optics. For histology, fixed whole-mount embryos were dehydrated in ethanol, cleared in xylene and embedded in paraffin. 4 µm sections were counterstained with eosin, examined and photographed. In situ probes used were gifts from the following researchers: Brachyury, Bernhard Herrmannn (Herrmannn, 1991); Notch-1, Tom Gridley (Del Amo et al., 1992); HNF3β, Siew-Lan Ang; BMP-4, Brigid Hogan; Wnt3a and Shh, Andrew McMahon; Pax-1 (Deutsch et al., 1988) and Pax-3, Peter Gruss; Hoxb-1 (#866), Hoxb-4 (#486), Hoxb-5 (#267) and Hoxb-9 (#803) Robert Krumlauf;CRABP-1 (Stoner and Gudas, 1989) and Mox-1 (Candia et al., 1992), Pierre Chambon. Immunohistochemical analysis of cell proliferation DNA synthesis was determined using immunohistochemical detection of 5′ bromodeoxyuridine (BrdU) incorporation adapted from Morganbesser et al. (1995). Briefly, pregnant mice bearing E8.5-E9 embryos were injected intraperitoneally, 2 hours prior to killing, with a solution of 25 mg/ml BrdU (Sigma, St Louis, MO) in PBS at a final concentration of 250 mg BrdU per gram of mouse body weight. Embryos and sections of the mothers’ large intestine, as positive controls for mothers’ uptake of BrdU, were fixed in 10% formalin for 2 hours, paraffin-embedded and sectioned. Sections were then treated as described by Morganbesser et al. (1995). After exposure to BrdUspecific primary antibody (Becton-Dickinson, Bedford, MA) and biotinylated secondary antibody (Vector Labs), sections were stained with avidin/biotin complex and DAB (ABC and DAB substrate kits, Vector Labs). Photomicrographs of anterior, midsection and posterior regions of nine mutant and eight wild-type embryos were taken at ×40 magnification and labelling was scored without knowledge of the genotype. All mesenchymal cells in a 6.5×7 cm field of the photomicrographs were counted and scored as labelled or unlabelled. Paired groups of data were analyzed using the Mann-Whitney one-tailed test. Results were designated to be significant at P