Abnormalities in Neural Crest Cell Migration in Laminin A5 Mutant Mice

Developmental Biology 289 (2006) 218 – 228 www.elsevier.com/locate/ydbio

Abnormalities in neural crest cell migration in laminin a5 mutant mice

Edward G. Coles a, Laura S. Gammill a, Jeffrey H. Miner b, Marianne Bronner-Fraser a,*

a Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA b Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110, USA

Received for publication 14 June 2005, revised 11 October 2005, accepted 16 October 2005

Abstract

Although numerous in vitro experiments suggest that extracellular matrix molecules like laminin can influence neural crest migration, little is known about their function in the embryo. Here, we show that laminin a5, a gene up-regulated during neural crest induction, is localized in regions of newly formed cranial and trunk neural folds and adjacent neural crest migratory pathways in a manner largely conserved between chick and mouse. In laminin a5 mutant mice, neural crest migratory streams appear expanded in width compared to wild type. Conversely, neural folds exposed to laminin a5 in vitro show a reduction by half in the number of migratory neural crest cells. During gangliogenesis, laminin a5 mutants exhibit defects in condensing cranial sensory and trunk sympathetic ganglia. However, ganglia apparently recover at later stages. These data suggest that the laminin a5 subunit functions as a cue that restricts neural crest cells, focusing their migratory pathways and condensation into ganglia. Thus, it is required for proper migration and timely differentiation of some neural crest populations. D 2005 Elsevier Inc. All rights reserved.

Keywords: Extracellular matrix; Neural crest; Chick; Mouse; Laminin; Ganglia; Cell migration

Introduction (ECM) (Le Douarin and Kalcheim, 1999; Perris and Perissi- notto, 2000). A number of candidate permissive and inhibitory Presumptive neural crest cells form at the border between ECM molecules have been implicated in aspects of neural crest the neural plate and non-neural ectoderm by an inductive migration. Matrix glycoproteins thought to promote neural crest interaction between these two tissues (Moury and Jacobson, migration include fibronectin (Duband et al., 1986; Newgreen, 1990; Selleck and Bronner-Fraser, 1995; Knecht and Bronner- 1989), collagen types I and IV (Perris et al., 1993a,b), tenascin Fraser, 2002). During the transformation of the neural plate into (Tan et al., 1987; Bronner-Fraser, 1988) and laminin (Duband the brain and spinal cord, these precursors come to lie in the and Thiery, 1987). All of these components contribute to the dorsal portion of the neural tube. Subsequently, neural crest highly organized ECM scaffolding contacted by neural crest cells delaminate from the neuroepithelium by undergoing an cells as they migrate. In contrast, non-permissive cues are epithelial-to-mesenchymal transition. After emigration, these present in regions where crest cells fail to migrate and may act as migratory cells move in a highly patterned fashion through barriers to migratory crest cells. Extracellular matrix molecules neighboring tissues and arrive at specific sites, where they that prevent the attachment and migration of neural crest cells differentiate into a wide variety of derivatives including include aggrecan, versican (Landolt et al., 1995; Perris et al., neurons and glia of the peripheral nervous system (Bronner- 1991, 1996b) and collagen type IX (Ring et al., 1996). Fraser and Fraser, 1988; Stemple and Anderson, 1992; The laminin family of glycoproteins represents a major Christiansen et al., 2000; Basch et al., 2004). component of the interstitial ECM as well as basement Separation of neural crest cells from the dorsal neural membranes (basal laminae) ECM. Laminins have been epithelium and their locomotion though the embryo involves implicated in a broad array of biological processes including alterations in cell–cell adhesion, changes in cell–matrix cell adhesion, migration, angiogenesis, differentiation, tumor interactions and reorganization of the extracellular matrix growth and metastasis (Colognato and Yurchenco, 2000; Miner and Yurchenco, 2004). Laminins exist in the ECM as * Corresponding author. Fax: +1 626 395 7717. heterotrimers containing one a-, one h- and one g-chain that E-mail address: [email protected] (M. Bronner-Fraser). assemble with each other via their coiled-coil domains to form

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.10.031 E.G. Coles et al. / Developmental Biology 289 (2006) 218–228 219 a cross-like structure (Colognato and Yurchenco, 2000). To litter can vary, embryos were staged in accordance with described criteria date, 5 distinct a-chains, 4 h-chains and 3 g-chains have been (Kaufman and Bard, 1999). Embryos were genotyped by PCR using genomic DNA extracted from the allantois as previously described (Miner et al., 1998). shown to form at least 15 different laminin isoforms (Miner et al., 1997; Patton et al., 1997; Miner and Yurchenco, 2004). Whole-mount in situ hybridization Laminin proteins have been suggested to play a role in neural crest cell emigration, specifically at cranial levels, as well as Briefly, to determine gene expression patterns for both chick and mouse during gangliogenesis (Bronner-Fraser, 1985, 1986; Bronner- embryos, linearized cDNAs were used to synthesize digoxigenin labeled Fraser and Lallier, 1988; Lallier et al., 1994; Perris and antisense RNA probes and whole-mount in situ hybridizations performed using F _ Perissinotto, 2000). Moreover, different laminin heterotrimers Protocol Four as previously described (Xu and Wilkinson, 1998). Antisense probes against chick laminin a5 (5kb), mouse laminin a5 (392bp), mouse have varying effects on neural crest cell migration in vitro; Sox10 (2.2kb), mouse Snail (¨500bp) and mouse NeuroD (1.6kb) were laminin-1 (a1 h1 g1) and -8 (a4 h1 g1) promote early neural transcribed. The 2.2kb mouse Sox10 probe was hydrolyzed prior to use. crest cell migration, whereas laminin-2 (a2 h1 g1), -4 (a2 h2 Stained embryos were imaged in 70% glycerol using a camera mounted on a g1) and -9 (a4 h2 g1) do not (Lallier et al., 1994; Desban and Zeiss Stemi SV11 microscope. Images were processed using Adobe Photoshop Duband, 1997). More recently, the distribution of individual 7.0 (Adobe Systems). laminin chains has been reported in the chick; a1 chain Immunohistochemistry transcripts are widely distributed in the early chick embryo, a4 chain is in the neural tube, and a5 chain is in the dorsal aspect Chick embryos processed by whole-mount in situ hybridization were of the neural tube (Gammill and Bronner-Fraser, 2002). subsequently stained with the migratory chick neural crest cell marker HNK-1. Despite the evidence demonstrating the importance of laminins Embryos were cryostat sectioned at 14 Am and then incubated with mouse-anti- in neural crest development, it has been difficult to identify the HNK-1 (American Type Culture, 1:200), which recognizes chick migratory neural crest cells (Sechrist et al., 1995). Mouse neural fold explants were precise function(s) of any one laminin subunit due to incubated with rabbit-anti-p75 (gift from Lou Reichardt, 1:2000), which compensation by other chains and/or laminin heterotrimer(s) recognizes migratory mouse neural crest cells and nuclei were stained with masking or buffering the phenotype that would otherwise result DAPI. Bright-field and fluorescent images of sections were captured using a from the loss of one subunit alone. Little information is Zeiss Axiocam mounted on a Zeiss Axioskop 2 microscope and processed available regarding laminin loss-of-function phenotypes during using Adobe Photoshop. Mouse embryos were processed with the rabbit-anti-laminin a5 antibody (J. stages of neural crest migration. Miner, 1:600) with a Fluorochrome-conjugated Alexa-Fluor 488 donkey-anti- It is likely that different laminin isoforms individually and/or rabbit secondary antibody (Molecular Probes, 1:500) as previously described in combination will influence different aspects of neural crest (Miner et al., 1997). Sections were stained with DAPI and mounted in development from maturation to emigration, migration or PermaFluor Mountant Medium (Thermo) prior to imaging. The mouse anti- h cessation. To address these issues, we have examined the neuron-specific class III -tubulin antibody, Tuj1 (IgG2a, BABCO, 1:500), was used as previously described (De Bellard et al., 2002), and a Alexa-Fluor 488 function of the a5 subunit of laminin during neural crest cell goat-anti-mouse IgG2a secondary antibody (Molecular Probes, 1:200). Embryos development. This subunit was previously identified in a screen were z scanned using a Zeiss LSM410 confocal microscope (Beckman Imaging for genes up-regulated in response to neural crest induction Center, Caltech) and projected (NIH-Image) into a single image. (Gammill and Bronner-Fraser, 2002). In chick and mouse, the temporal and spatial expression pattern of laminin a5 is Cell counts of Sox10 expressing cells consistent with a function in the emigration and/or migration of neural crest cells from the dorsal neural tube to their final E8.5 wild-type and laminin a5 null mouse embryos that had been hybridized with the Sox10 probe were imaged, then embedded in gelatin and location in the developing embryo. In laminin a5 null mice, there sectioned at 14 Am. These sections were stained with DAPI to enable the is an expansion in neural crest migratory streams, suggesting that identification of individual nuclei and mounted for analysis. In cranial regions the normal function of this subunit may be to focus or restrict where neural crest cell migration had initiated, a minimum of 10 images of neural crest migration pathways. Conversely, culturing murine serial sections at the same cranial axial level were captured for 3 (8, 9 and 10 neural folds in the presence of laminin a5 subunit in vitro results somites) wild-type and 3 (8, 9 and 10 somites) laminin a5 mutant embryos. Every DAPI-positive nuclei surrounded by cytoplasmic Sox10 staining was in a reduction in the numbers of migratory neural crest cells by marked, counted and the numbers recorded. The number of Sox10-positive half. Laminin a5-deficient mice exhibit a delay in gangliogen- neural crest cells was determined for each embryo and averaged with like stage esis of the peripheral ganglia compared to wild type. These data and genotyped embryo. To determine the variability of the data set, the standard demonstrate that proper regulation of the a5 subunit of laminin deviation based on the entire population was calculated (Microsoft Excel). is critical for normal neural crest emigration and timely differentiation. In vitro migration assay

Mouse cranial neural folds from 8 dpc (5–9 somites) were isolated and Materials and methods cultured as previously described (De Bellard et al., 2003). Glass culture dishes were coated with 10 Ag/ml of either CultrexR mouse laminin-1 (R&D Systems) Analysis of mutant embryos or the purest commercially available form of laminin-10, derived from human placenta (SigmaR). Neural crest cells cultured on laminin-1 in vitro have Mutant mice were generated as previously described (Miner et al., 1998). properties very similar to fibronectin (Desban and Duband, 1997). Explanted Heterozygous laminin a5 mice were crossed to obtain homozygous progeny. tissue was cultured in complete neural crest media without retinoic acid Embryos were dissected between E8 and E11 days of development in Ringers (Stemple and Anderson, 1992) for 24 h, the next day, migratory neural crest solution and fixed in 4% paraformaldehyde prior to processing by in situ cells could be seen as a halo around the explants. Cells were fixed for 1 h with hybridization or immunohistochemistry. Since the developmental stage of a 4% PFA prior to immunological processing. 220 E.G. Coles et al. / Developmental Biology 289 (2006) 218–228

For analysis, cultures were viewed with phase optics to visualize migratory its distribution pattern by whole-mount in situ hybridization. neural crest cells, p75 immunohistochemistry to confirm migratory neural crest Laminin a5 transcripts are expressed in a number of tissues cell type and DAPI to stain individual nuclei. The total number of migratory neural crest cells was counted using ImageJ. during chick neural crest cell development (Figs. 1A–L). At Hamburger and Hamilton (1951) (HH) stage 8À, expression of Results laminin a5 is detected in the surface ectoderm and neural folds (Fig. 1A), specifically in the dorsal most regions (Fig. 1C), Distribution of laminin a5 transcripts during chick neural crest with strongest expression in the anterior neural folds. By HH development stage 8+, laminin a5 expression is up-regulated and becomes restricted to the dorsal aspect of the neural folds along the As a first step in understanding the function of laminin a5 in entire anterior–posterior axis (Fig. 1B). Also at this stage, neural crest development, we performed a detailed analysis of expression is detected in lateral plate regions but is absent from

Fig. 1. Expression pattern of laminin a5 during neural crest cell development in the chick embryo. Whole-mount in situ hybridizations of chick embryos with the laminin a5 probe (A, B, F, G and I) and histological sections of laminin a5 hybridized chick embryos (C–E, H and J–L). Immunohistochemistry of chick embryos with HNK-1 (red staining; D, JV,KV,LV and M). (A) HH 8À (3 somite stage) laminin a5 transcripts are detected in the dorsal neural folds predominantly in cranial regions and at lower levels in the surface ectoderm (C). (B) HH 8+ (5 somite stage), expression is up-regulated in the dorsal neural folds along the anterior–posterior axis, which corresponds to the pre-migratory neural crest cell population. Expression is also detected in posterior lateral plate regions. (F) HH 10 (10 somite stage), laminin a5 expression is reduced in the dorsal neural tube, coinciding with the onset of neural crest cell migration, but is detected in cranial surface ectoderm overlying HNK-1 expressing migratory crest cells (D; red staining is HNK-1), and in the closing dorsal neural folds (E). (G) HH 11À (12 somite stage), laminin a5 transcripts are down-regulated in the dorsal neural tube, correlating with the onset of neural crest cell migration. (H) Transverse section of another HH stage 11-embryo demonstrates the down-regulation of laminin a5 in ectoderm overlying the neural tube (H; arrowheads). (I) HH 17 (32 somite stage), expression is lost in cranial regions, however, expression is detected in the caudal portion of the Hyoid arches. (J, JV,K,KV, L and LV) Longitudinal sections of embryo (I) at different dorsal–ventral axial levels. (J, K and L) identify laminin a5 expression in the dermomyotome lateral to the neural tube (J); in the anterior and ventral sclerotome adjacent to the notochord (K) but lacking from the sclerotome at the level of the dorsal aorta, where it is expressed in the ventral dermomyotome (L, LV). (K(V) and L(V)) arrowheads identify somite boundaries. HNK-1 staining overlays identify discrete laminin a5 staining from HNK-1 dorsally (JV) and ventrally (LV) but partially overlapping at intermediate levels (KV). (M) Transverse section of a similar stage chick embryo (I) stained for HNK-1 to illustrate the different widths of the neural crest migratory stream at different axial levels. Abbreviations; da, dorsal aorta; dnf, dorsal neural fold; ha, Hyoid arch; lp, lateral plate; r, rhombomere; nt, neural tube; no, notochord. E.G. Coles et al. / Developmental Biology 289 (2006) 218–228 221 the primitive streak, the site of secondary gastrulation. At HH dermomyotome is not contiguous with migratory neural crest stage 10, when neural crest cells have initiated migration from cells (Figs. 1J and JV). At intermediate levels adjacent to the the midbrain and anterior hindbrain (rhombomere 1/2), laminin notochord, laminin a5 expression appears to overlap with a few a5 transcripts are expressed in cranial surface ectoderm HNK-1 expressing cells, but the number is much lower than overlying migratory neural crest cells (Figs. 1F and D). By that at other levels of the sclerotome (Figs. 1K and KV). Thus, HH stage 11À, laminin a5 expression is down-regulated in the this apparent partial overlap coincides with the more restricted ectoderm immediately dorsal to the cranial neural tube (Figs. migratory stream of neural crest cells that occurs at the level of 1G and H, arrowheads) but persists in more lateral regions the notochord (see Fig. 1M). At more ventral levels, where the (Figs. 1G and H) until at least HH stage 16 (data not shown). migratory neural crest streams once again become more Also at HH stage 16, strong expression is detected in surface broadly distributed (Fig. 1M), laminin a5 is restricted to the ectoderm of the trunk and in the nephric rudiment, but only dermomyotome and does not overlap with migratory neural weak expression is seen in the dorsal neural tube (data not crest cells (Figs. 1L and LV). At later stage of development, HH shown). By HH stage 17, laminin a5 transcripts are lost from stage 23/24, when the neural crest cell derived melanoblasts cranial regions except in the posterior portion of the second start to invade the epidermis, laminin a5 expression is not branchial arch (Fig. 1I). Interestingly in the trunk at this stage, detected in the ectoderm (data not shown). the expression pattern of laminin a5 differs along the dorsal– ventral axis (Figs. 1J(V),K(V) and L(V)). At both the most dorsal Laminin a5 mRNA and protein are expressed during chick and (adjacent to the neural tube) and most ventral (adjacent to the mouse neural crest migration dorsal aorta) levels, laminin a5 expression is localized to the dermomyotome (Figs. 1J and L, respectively). However, at The expression pattern of laminin a5 transcripts during intermediate dorsoventral levels near the notochord, it is murine neural crest development is largely similar to that in the distributed in the anterior half of the sclerotome (Fig. 1K). chick (Figs. 2A–F). At E8.5 (11 somite stage), laminin a5 To determine the relationship between the laminin a5 transcripts are detected in the dorsal third of the neural distribution and migratory neural crest cells, sections (Figs. epithelium of the closing cranial neural folds (Fig. 2B) where 1J, K and L) were stained with the neural crest cell marker neural crest cells have initiated migration. Slightly later at stage HNK-1 (Figs. 1JV,KV and LV). At dorsal levels lateral to the E8.5, however, the relative expression levels of laminin a5 are neural tube, the domain of laminin a5 expression in the elevated in trunk neural epithelium compared with cranial

Fig. 2. Expression pattern of laminin a5 during neural crest cell development in the mouse embryo. In situ hybridizations with the antisense laminin a5 probe of wild-type mouse embryos (A–F) and laminin a5 mutant embryos (I). Immunohistochemistry on cranial sections of wild-type mouse embryo with an anti-laminin a5 antibody (green) and stained with DAPI (blue) (G and H). (A) E8.5 (11 somite stage), laminin a5 transcripts are localized to the dorsal third of the cranial neural epithelium (B). (C) At more caudal levels, where crest cells have yet to initiate migration, expression is detected in the dorsal half of the neural tube. (D) E9 (14 somite stage), expression of laminin a5 is detected in a sub-set of migratory neural crest cells in the branchial arches (E; arrow). (F) E9.5 (22 somite stage), expression is reduced in cranial regions, but expression is still detected in the posterior of the embryo, a region where neural crest cells have yet to commence migration. (G) E8.5, laminin a5 protein is detected in basal laminae. At E9.5 (H), laminin a5 protein expression persists in the surface ectoderm and ventral neural epithelium but is no longer detected in basal laminae surrounding the dorsal neural epithelium. (I) No laminin a5 transcripts were detected in laminin a5 mutant embryos. Abbreviations; r, rhombomere; se, surface ectoderm. 222 E.G. Coles et al. / Developmental Biology 289 (2006) 218–228 neural epithelium (Figs. 2B and C). Moreover, the domain of of the anterior neural folds to close in approximately 60% of expression in the neural tube extends further ventrally in the laminin a5À/À embryos) and craniofacial abnormalities, two trunk (Fig. 2C), an axial level where crest have yet to initiate phenotypes that can result from abnormal neural crest migration. At E9 (14 somite stage), transcripts are detected in development (Mansouri et al., 1996; Houzelstein et al., 1997; the neural epithelium and in some migratory neural crest cells Kohlbecker et al., 2002). This coupled with the dynamic spatial entering branchial arches 1 and 2 and/or the mesenchyme and temporal expression pattern in both chick and mouse through which they migrate (Fig. 2E; arrow). At E9.5 (22 during neural crest development is suggestive of a functional somite stage), as neural crest cell migration has initiated in the role for laminin a5 during this process. In order to characterize trunk, laminin a5 transcripts are detected in branchial arches a possible neural crest migratory phenotype, stage-matched but have been down-regulated in all other cranial regions; mutant and wild-type embryos were hybridized for expression however, expression persists in more caudal regions (Fig. 2F) of the migratory neural crest cell marker, Sox 10. where neural crest cells have yet to initiate migration. At E8.5, In wild-type embryos at E8 (7 somites), Sox10-positive and as previously reported (Miner et al., 1998), immunohisto- neural crest cells have initiated migration from the midbrain chemistry identifies laminin a5 localized to the basal side of prior to the fusion of the neural folds. The timing of neural epithelial cells (Fig. 2G). At E9.5, when neural crest cell crest emigration appeared similar in mutant laminin a5 migration is well underway, laminin a5 protein is detected in embryos (Fig. 3), suggesting there is no change in initiation basal laminae of the ventral neural epithelium and surface of migration. Furthermore, no change in Snail expression was ectoderm but is down-regulated surrounding the dorsal neural identified in pre- and early-migratory neural crest cell popula- tube (Fig. 2H). Thus, although we note laminin a5 mRNA tions in laminin a5 null embryos when compared to wild type expression throughout the cytoplasm, the protein expression is (data not shown). However, analysis of Sox10 staining of more discrete and more delimited. transverse sections suggested an increase in the breadth of the neural crest stream and/or intensity of staining in cranial Laminin a5 null mice have defects in neural crest migration migratory neural crest cells of laminin a5 null mice when compared to wild type (Fig. 3; compare B and C to H and I). Mice homozygous for a laminin a5 mutation exhibit This was most apparent at later stages (E9.5), when the multiple developmental defects including exencephaly (failure migratory stream appeared approximately twice the breadth in

Fig. 3. Loss of laminin a5 results in expansion of the neural crest cell migratory pathway. In situ hybridizations of E8.5 (9 somite stage) (A–C and G–I) and E9.5 (D–F and J–L) wild-type (WT) (A–F) and null laminin a5 mice (LA5) (G–L) with Sox10 ribo-probe in whole mount and transverse section. At E8, cranial regions are shown; the Sox10 staining domain is greater, or Sox10 is expressed over a larger area, in laminin a5 mutants than in wild-type embryos (G and A, respectively). Transverse sections identify an expansion in domain size in laminin a5 mice in migratory midbrain neural crest cells and in early neural crest cells migrating from rhombomere 4 than in wild-type mice (compare B and C to H and I) (note; images C and I are at a higher magnification to clearly identify individual Sox10-positive cells). In E9.5 embryos, loss of laminin a5 results in an increase in the staining intensity of Sox10-positive migratory neural crest cells, compare (D) to (J). Histological sections confirm this observation identifying an increase in Sox10 expressing migratory branchial arch neural crest cells (K) and vagal neural crest cells (L) in laminin a5 null embryos compared to wild-type embryos (E and F, respectively). Note: anterior neural folds fails to close (exencephaly) in 60% of laminin a5 null mice (see panels K and L). E.G. Coles et al. / Developmental Biology 289 (2006) 218–228 223 mutant embryos versus wild type. These data suggest either a same number of cells were more broadly distributed in laminin possible expansion in neural crest migratory pathway width or a5 mutants (Figs. 4C and D). This suggests that the a5 subunit an increase in the overall number of migratory neural crest of laminin may be required to focus the migratory stream, and cells. To distinguish between these possibilities, we determined that its presence may restrict the sites of emigration. if there was an increase in the number of migratory neural crest At slightly later stages (E9.5), neural crest migration is well cells. To this end, we counted the number of Sox10-positive underway in cranial regions but just initiating at trunk levels. cells at the midbrain to rhombomere 4 level in 8, 9 and 10 Whole-mount in situ hybridization revealed an apparent somite stage laminin a5-deficient embryos and compared them expansion in size of the migratory pathways into the branchial to like stage wild type (n = 6). For each embryo, a minimum of arches in laminin a5 mutants (Fig. 3J), though the overall 10 sections were taken in regions where Sox10-positive neural pattern appeared normal. Histological sectioning confirmed an crest cells had just initiated migration. Sections were aligned expanded domain of the Sox10 expressing neural crest cell according to their axial identity and DAPI nuclei surrounded by population in laminin a5 null embryos at cranial and vagal Sox10 staining were counted (for examples, see Figs. 4A(V),B(V) axial levels (Fig. 3 arrow heads; E and F compared to K and L). and E). Interestingly, no significant alterations were noted in It should be noted that the migratory neural crest cell the overall number of migratory neural crest cells between phenotypes described above are similar in laminin a5 mutants wild-type and laminin a5 null (26 and 22 Sox10-positive cells with and without exencephaly. per section, respectively) embryos (Fig. 4F). Instead, the region Similar to cranial levels, the timing of trunk neural crest occupied by migrating neural crest cells appeared to be emigration is not affected by the loss of laminin a5. Embryos expanded due to increased intercellular spacing, such that the with the laminin a5 mutation exhibit a more diffuse and

Fig. 4. Analysis of Sox10 expressing migratory neural crest cells in wild-type and laminin a5 mutant embryos. Examples of cranial sections taken through 8 somite stage embryo hybridized with Sox10 (A and B) and stained to identify DAPI nuclei (AV and BV) with cell count highlighted by individual red dots (A(V) and B(V)). The domain of migratory neural crest cell pathway is expanded in the mediolateral direction in laminin a5-deficient embryos (D, lines) compared to wild type (C, lines). (E) Bar-chart to illustrate total cell count of wild-type and laminin a5 mutant 10 somite stage embryos over 10 sequential sections. (F) Mean number of Sox10 expressing neural crest cells for wild type (n = 3) and laminin a5 mutant (n = 3) mice, error bars identify standard deviation based on the entire population. 224 E.G. Coles et al. / Developmental Biology 289 (2006) 218–228 scattered Sox10 expression pattern in early migratory neural Table 1 crest cells as they enter the somite (Fig. 3J). Histological Number of p75-positive migratory cells cultured in the presence or absence of the laminin a5 subunit analysis, however, shows that the rostrally restricted segmental migration of trunk neural crest cells is retained in laminin a5 Embryo Àalpha5 +alpha5 Fold difference null mice (data not shown). 1 632 263 2.4 2 689 351 1.96 The a5 subunit abrogates cranial neural crest cell migration in 3 780 275 2.84 vitro 4 620 189 3.28 5 574 222 2.59 To determine if the laminin a5 subunit is acting as a non- 6 530 252 2.1 permissive or restrictive cue, mouse cranial neural folds were 7 344 160 2.15 8 168 119 1.4 dissected and cultured in vitro on laminin substrates differing 9 606 177 3.42 in their laminin a subunits. To facilitate direct comparison, one 10 753 154 4.89 cranial neural fold was cultured on laminin-1, and the 11 644 204 3.16 contralateral neural fold from the same embryo was cultured 12 344 160 2.15 on laminin-10. Whereas laminin-1 contains the a1 subunit, 13 172 31 5.55 14 180 16 11.25 laminin-10 utilizes the a5 subunit. 15 191 91 2.1 After 24 h in culture, neural crest cell migration had occurred 16 291 133 2.19 on both substrates, but there was a notable difference in the Contralateral cranial neural fold from E8 murine embryos were dissected overall number of neural crest cells that had migrated away from, and cultured on laminin substrates differing in their laminin a subunits. and were surrounding, the cranial neural fold on laminin-1 The total number of p75-positive migratory neural crest cells, cultured in versus laminin-10 from the same embryo (representative image; vitro in the presence or absence of the laminin a5 subunit, was counted Figs. 5A and B). Analysis of the total number of p75-positive and the fold difference calculated. Statistical analysis identified a significant reduction in the number of migratory neural crest cells cultured on the migratory neural crest cells identified 14 out of 16 explants laminin a5 subunit compared to explants cultured in its absence ( P < cultured in the presence of the a5 subunit had a two-fold 0.0001; paired Student’s t test). reduction in the total number of migratory neural crest cells compared with similar explants cultured on substrate free of matched wild-type and laminin a5 mutant embryos, there is a laminin a5. The cell counts for each neural fold are presented in marked reduction in Sox10 staining in laminin a5 mutant Table 1. Paired Student’s t test demonstrated a significant embryos, though all cranial and trunk ganglia form in their difference in the number of migratory neural crest cells cultured correct locations (Fig. 6). This suggests that there is a reduction in the presence of the laminin a5 subunit (P < 0.0001; t test). and/or delay in ganglion formation. No apparent alterations in These data are consistent with the a5 subunit of laminin acting as cell death, as assayed by DAPI staining, were noted. Smaller a non-permissive signal for migratory neural crest cells. and less pronounced nodose and cervical ganglia are one of the most noticeable cranial phenotypes in laminin a5 mutants (Fig. Laminin a5 mutant mice have reduced/delayed formation of 6; compare B and F). At the level of the trunk, neural crest cells cranial ganglia and peripheral glia migrate through the somites and condense to form segmentally arranged sensory and sympathetic ganglia. However, laminin At E10, neural crest cells have already started to undergo a5 null mice have reduced Sox10 staining in regions that differentiation into neurons; at this stage, Sox10 marks correspond to sites of differentiating dorsal root ganglia, differentiating ganglionic and glial precursors (Kuhlbrodt et indicating greatly reduced numbers of condensing neural crest al., 1998; Pusch et al., 1998). When comparing E10 stage- cells (Fig. 6; compare black arrow in A to black arrow in E). To confirm these observations, we examined the expression of NeuroD, a marker of early neurogenesis. NeuroD mRNA is expressed in all placodes and both neural crest and placode- derived ganglia. In laminin a5 mutants, these structures appear misshapen and more diffuse compared with wild-type embryos (Fig. 6; compare C and D with G and H). To further investigate the apparent reduction or delay in ganglion formation in laminin a5 mutant embryos, we used an early pan-neuronal marker anti-h-tubulin III (Tuj1) antibody at E10, and later at E11 at a stage when neurogenesis is well underway (Figs. 7A–D). At E10, Tuj1 labeling reveals a reduction in cranial and trunk peripheral ganglia in mice with Fig. 5. Neural crest cell migration is reduced in the presence of the a5 subunit. the laminin a5 mutation (compare labels and arrow in Figs. 7A Wild-type murine cranial neural folds explanted and cultured on laminin substrate in the presence of the a5 subunit of laminin (B) have 2-fold less (14/ and C). This reduction in staining is suggestive of a decrease in 16) migratory p75-positive migratory neural crest cells than the contralateral the number of neurons being born and/or an alteration in the cranial neural fold cultured in the absence of the laminin a5 subunit (A). timing of gangliogenesis as a whole. However, Tuj1 expression E.G. Coles et al. / Developmental Biology 289 (2006) 218–228 225

Fig. 6. Laminin a5 is required for early development of cranial ganglia, peripheral glia and proper timing of ganglionic condensation. Whole-mount in situ hybridization of E10 embryos with Sox10 (A, B, E and F) and NeuroD (C, D, G and H) probes in control (WT; A–D) and laminin a5 (LA5; E–H) mutant mice. Cranial ganglia in WT (A and B) and laminin a5 mutant embryos (E and F) form at their correct positions, but laminin a5 null embryos show a marked reduction in Sox10 staining of cranial nerves (white arrows) suggesting a reduction or delay in peripheral glia formation. Mice mutant for laminin a5 have abnormally shaped trigeminal (t), vestibulo-cochlear (v), geniculate (g), petrosal (p) and nodose (n) ganglia as labeled by Sox10 and NeuroD (F and G) compared to wild type (B and C). In the trunk, Sox10 and/or NeuroD expressing condensing dorsal root ganglions are reduced in laminin a5 mutant embryos (black arrows in E and H versus A and D). appeared to occur on schedule in the mutants, making the latter combination of permissive and inhibitory cues whose expres- possibility unlikely. Slightly later in development at E11, sion is regulated in space and time. These may be detected by neurogenesis has continued and indeed ganglia appear largely migrating neural crest cells and facilitate their condensation recovered in laminin a5 null mice. The mutant embryos appear into discrete streams (Thiery et al., 1982; Newgreen, 1989; similar to the wild type, apart from its slightly smaller size and Oakley and Tosney, 1991). a slight expansion in the width of some neuronal projections Laminins, a family of heterotrimeric ECM glycoproteins, (Fig. 7; arrow head in B and D). Taken together, these data are required for the proper emigration or migration of some suggest a reduction and/or delay in ganglia condensation in neural crest populations (Bronner-Fraser, 1986; Bronner-Fraser laminin a5-deficient mice, followed by regulation and later and Lallier, 1988; Perris et al., 1990; Perris and Perissinotto, recovery. 2000). In vitro assays have demonstrated differences in neural In addition to elements of the peripheral nervous system, crest migratory properties when contacting different isoforms neural crest cells contribute to cartilage and bone of the face. of laminin (Perris et al., 1996a; Spessotto et al., 2001) and Therefore, we investigated possible skeletal malformations in more recently on individual laminin subunits (Desban and the laminin a5 mutant embryos. Consistent with previous Duband, 1997; Powell et al., 2000). Embryos with null studies (Miner et al., 1998), we saw no obvious change in bone mutations of the laminin a5 gene exhibit multiple develop- or cartilage deposits in laminin a5 null mice. These data mental defects including exencephaly and craniofacial abnor- suggest that there is no alteration of ossification and malities (Miner et al., 1998). This study demonstrates that some chondrogenesis of neural crest-derived bone and cartilage (data of these problems may result from defects in neural crest cell not shown), making unlikely a shift in neural crest potential migration. We find that the spatial and temporal expression of from neurogenic to skeletogenic in the laminin a5 null mice. laminin a5 during neural crest development is largely conserved between chick and mouse. Furthermore, loss of Discussion laminin a5 results in multiple migratory defects. Most notably laminin a5 mutant mice have more diffuse migratory streams Migration of neural crest through the periphery of the and exhibit a delay in ganglion formation. These results embryo involves interactions between motile neural crest cells suggest that laminin a5 may be required for proper routing of and the substrata that they traverse. The ECM contains a neural crest cells. In vitro experiments together with in vivo 226 E.G. Coles et al. / Developmental Biology 289 (2006) 218–228

cue that may restrict and/or guide early motile neural crest cells to prescribed pathways. Interestingly, the migratory pathways appear to be expanded in laminin a5 mutant embryos when compared to wild-type embryos. Our cell counts in vivo suggest that the numbers of migratory neural crest cells are relatively constant between mutant and wild-type mice. These data favor the explanation that the a5 subunit functions as a non-permissive or adhesive cue for neural crest cells to confine and focus their migration to discrete pathways without necessarily affecting their total cell numbers in vivo. In contrast, although migration is not arrested, the numbers of neural crest cells emigrating from explanted neural folds are significantly less in the presence of the laminin a5 subunit. These data suggest that more cells may actually emigrate on substrates lacking laminin a5, but additional mechanisms are required to control the total cell number in the intact embryo as compared to the culture dish. Another possible explanation for the more limited migration in vitro of neural crest cells on substrates that contain the laminin a5 subunit is that it prevents neural crest cells from exiting the explanted neural fold prematurely. Alternatively, the presence of laminin a5 in the substrate could slow the rate of emigration from the neural fold. We were, however, unable to Fig. 7. Laminin a5 is required for early formation of the peripheral nervous detect an obvious change in the timing of emigration between system and proper timing of ganglionic condensation. Immunostaining of E10 mutant and wild-type mice (as identified by Snail and Sox10 and E11 wild-type (WT) (A and B) and laminin a5 (LA5) mutant (C and D) expression), making these scenarios unlikely. Interestingly, the mouse embryos with antibody against anti-h-tubulin III (Tuj1), an early pan- reduction in migratory ability of neural crest cells on a5 neuronal marker. The trigeminal (t), vestibulo-cochlear (v), geniculate (g), substrate is consistent with previous studies on axon growth petrosal (p) and nodose (n) ganglia form in the correct places in laminin a5 null mice, but at E10, patterning is affected (compare A to C). In the trunk, there is a where the a5 subunit has been shown to inhibit motor neurite reduction in fused ganglia in laminin a5 null mice (white arrow A and C). As outgrowth (Patton et al., 1997; Cho et al., 1998), Schwann cell development progresses (E11), gangliogenesis in laminin a5 mutant mice has progression (Patton, 2000) and tumor cell migration (Hibino et continued and, compared to control embryos, appeared grossly normal al., 2004). Our data together with these previous studies (compare B to D). There is, however, a slight expansion in the size of the suggest a conserved function for laminin a5 as a non- petrosal ganglion in laminin a5 mutant embryos (arrow head in B and D). permissive, rather than repulsive or adhesive, substrate for motile cells and axonal processes. data support the possibility that the laminin a5 may restrict Once neural crest cells have reached their destinations, they neural crest cells onto discrete pathways. differentiate into a number of derivatives along the rostrocaudal In both chick and mouse, laminin a5 transcripts are axis including neurons and glia of the peripheral nervous transiently expressed adjacent to the basal laminae overlying system. In laminin a5 null mice, the number of migratory the neural tube. The breakdown of this basal lamina facilitates neural crest cells was similar to stage-matched wild-type initial emigration of neural crest cells (Erickson, 1987). In embryos, but the dorsal root ganglia were much smaller chick, the down-regulation of laminin a5 transcripts in regions suggestive of a possible delay in some peripheral ganglion immediately ventral to the dorsal neural tube at the onset of formation in laminin a5-deficient mice. A delay in this vital neural crest emigration is consistent with a possible inhibitory process could be attributed to a decrease in the number of role. Indeed, the a5 subunit of laminin-11 (a5h2g1) has been migratory neural crest cells, or that the full complement of reported to act as a barrier preventing Schwann cells from neural crest cells do not reach, or stop, at their proper site of entering into the synaptic cleft (Patton, 2000). Although differentiation. In this scenario, neural crest cells in mutant laminin a5 is expressed in the dorsal neural folds at the onset embryos may be diffuse or malpositioned such that ganglionic of murine neural crest cell migration, loss of laminin a5 does condensation takes longer. Alternatively, the reduction in not affect the timing of emigration from the neural tube. One Sox10 staining and apparent delay in gangliogenesis could be possible explanation is that the a5 subunit does not function attributed to more or earlier neuronal differentiation. This in during this process. Alternatively, other laminin subunits and/ turn could result in premature loss of the early glial marker or molecules may be compensating in its absence. Sox10 in laminin a5 null embryos. This seems unlikely, At later stages of neural crest cell migration in the chick and however, because no differences in the timing of neurogenesis mouse, laminin a5 transcripts have minimal overlap with was detected at E10 in laminin a5 mutants when compared to migratory neural crest cells, further corroborating the hypoth- wild-type embryos, as identified by the pan-neuronal marker esis that laminin a5 may act as a non-permissive or inhibitory Tuj1. Therefore, these data suggest that laminin a5 is not E.G. Coles et al. / Developmental Biology 289 (2006) 218–228 227 required for neurogenesis per se but rather may be important References for proper positioning and timely formation of some neural crest derivatives. Basch, M.L., Garcia-Castro, M.I., Bronner-Fraser, M., 2004. Molecular No neural crest cells were detected at the level of the mechanisms of neural crest induction. Birth Defects. Res. Part C Embryo Today 72, 109–123. sympathetic ganglia in laminin a5 mutant embryos. 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