Programmed Cell Death in the Developing Somites Is Promoted By

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Developmental Biology 228, 326–336 (2000) doi:10.1006/dbio.2000.9948, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE Programmed Cell Death in the Developing Somitesprovided by Elsevier - Publisher Connector Is Promoted by Nerve Growth Factor via Its p75NTR Receptor

Marı´a L. Cotrina, Maritza Gonza´lez-Hoyuela, Julio A. Barbas, and Alfredo Rodrı´guez-Te´bar1 Instituto Cajal de Neurobiologı´a, CSIC, Avenida Doctor Arce, 37, E-28002 Madrid, Spain

Neurotrophins control neuron number during development by promoting the generation and survival of neurons and by regulating programmed neuronal death. In the latter case, the cell death induced by nerve growth factor (NGF) in the developing chick retina is mediated by p75NTR, the common neurotrophin receptor (J. M. Frade, A. Rodriguez-Tebar, and Y.-A. Barde, 1996, Nature 383, 166–168). Here we show that NGF also induces the programmed death of paraxial mesoderm cells in the developing somites. Both NGF and p75NTR are expressed in the somites of chick embryos at the time and the place of programmed cell death. Moreover, neutralizing the activity of endogenous NGF with a speciﬁc blocking antibody, or antagonizing NGF binding to p75NTR by the application of human NT-4/5, reduces the levels of apoptotic cell death in both the sclerotome and the dermamyotome by about 50 and 70%, respectively. Previous data have shown that Sonic hedgehog is necessary for the survival of differentiated somite cells. Consistent with this, Sonic hedgehog induces a decrease of NGF mRNA in somite explant cultures, thus showing the antagonistic effect of NGF and Sonic hedgehog with respect to somite cell survival. The regulation of programmed cell death by NGF/p75NTR in a mesoderm-derived tissue demonstrates the capacity of neurotrophins and their receptors to inﬂuence critical developmental processes both within and outside of the nervous system. © 2000 Academic Press Key Words: apoptosis; programmed cell death; p75NTR; NGF; somite development.

INTRODUCTION chord and the ventral spinal cord on the one hand and the surface ectoderm and dorsal spinal cord on the other. The Somites are condensations of the paraxial mesoderm that influence of the notochord on the differentiation of scle- initially form as cell clusters flanking the notochord and rotome tissue can be seen following the grafting of a the neural tube of vertebrates. Subsequently, these cells supernumerary notochord. Such manipulations induce the undergo a mesenchyme-to-epithelial transformation before development of sclerotome and inhibit the formation of differentiating into dermamyotome and sclerotome, situ- dermamyotome (Watterson et al., 1954; Brand-Saberi et al., ated dorsolaterally and ventromedially, respectively (re- 1993; Pourquie et al., 1993). Conversely, when the noto- viewed in Keynes and Stern, 1988; Gossler and Hrabe de chord is removed prior to the induction of the floor plate, Angelis, 1998). Later in development, deepithelialized scle- sclerotome tissue does not differentiate (Grobstein and rotome cells migrate ventrally and contribute largely to the Holtzer, 1955; Avery et al., 1956). The main notochord- formation of the vertebrae, intervertebral discs, and ribs derived factor responsible for sclerotome differentiation in (Christ and Ordahl, 1995). vitro is Sonic hedgehog (Shh; Fan and Tessier-Lavigne, Both the differentiation of somites and the establishment 1994; Fan et al., 1995; Johnson et al., 1994; Mu¨nsterberg et of their dorsoventral polarity are influenced by signals al., 1995), whereas Wnt-1, BMP-4, ␤-catenin, and emanating from neighboring tissues. These events occur N-cadherin have all been implicated in dermamyotome concomitantly, the sources of the signals being the noto- differentiation (Capdevila et al., 1998; Linask et al., 1998; Marcelle et al., 1997; Pourquie et al., 1996). However, the 1 To whom correspondence should be addressed. Fax: ϩ34 91 585 action of Shh is more complex and, in combination with the 4754. E-mail: [email protected]. Wnts, it is also capable of inducing myotomal markers

(Mu¨nsterberg et al., 1995; Marcelle et al., 1997). More recent data indicate that one of the main effects of Shh on somite cells is that of a strong mitogen (Marcelle et al., 1999). In addition to its role in the differentiation of somite cells, the notochord is also needed for their survival: if the notochord is removed during or after somite differentiation, sclerotome cells die (Rong et al., 1992; Teillet and Le Douarin, 1983; Teillet et al., 1998). The loss of cells caused by extirpation of the notochord can be prevented by the application of exogenous Shh, thus demonstrating that this FIG. 1. NGF and its receptors are expressed in the somites from morphogen also acts as a survival factor for differentiated early developmental stages. Somites 6 to 10 were dissected from sclerotome cells later in their development (Teillet et al., chicken embryos at the developmental stages indicated on top, and 1998). However, under physiological conditions, naturally RT-PCR was performed on mRNA extracted from the tissues using occurring programmed cell death (PCD) affects both differ- specific primers from the NGF, TrkA, p75NTR, or GAPDH cDNA entiating and differentiated somite cells (Jeffs and Osmond, sequence. The amounts of total mRNA in the amplification mixtures 1992; Sanders, 1997), reflecting its key role in normal were made equivalent according to their GAPDH content. animal development (Glucksmann, 1951; Saunders, 1966; Sanders and Wride, 1995). In the nervous system, neurotrophins have been long and used to detect migratory neural crest cells. The anti-NT-3 mAb recognized as neuron survival factors (Lewin and Barde, was a gift from Y.-A. Barde (Martinsried, Germany) and its blocking activity has been previously assessed both in vitro and in vivo 1996). However, more recent results suggest that the neu- (Gaese et al., 1994; Barres et al., 1994; Bovolenta et al., 1996). The rotrophins are also capable of controlling neuron number by anti-␣6 chick integrin subunit was obtained from the Iowa Hybrid- inducing PCD. Specifically, nerve growth factor (NGF) kills oma Bank. This mAb recognizes only the denatured protein antigen neurons (Frade et al., 1996b; reviewed in Casaccia-Bonnefil and, therefore, does not demonstrate blocking activity (Frade et al., et al., 1998) by stimulating the common neurotrophin 1996a). This mAb was used in this study as a control antibody for receptor, p75NTR (Rodrı´guez-Te´bar et al., 1990) in cells embryo injections. devoid of TrkA, the specific receptor for NGF (Kaplan et Embryo manipulation. Manipulations of embryos were per- al.,1991; Klein et al., 1991). Here we present evidence that formed at st 11–12 via a lateral window in the shell of the eggs. NGF also activates PCD in both the sclerotome and the Fifty microliters of a phosphate-buffered saline (PBS) solution ␮ dermamyotome during somite morphogenesis. We show containing 100 g of protein A–Sepharose-purified anti-NGF 27/21 mAb, 100 ␮g of protein A–Sepharose-purified anti-NT-3, 100 ␮gof that both p75NTR and NGF are expressed in the somites at protein A–Sepharose-purified anti-␣6 integrin subunit mAb, or 2.5 the time and place of cell death and that NGF induces PCD ␮ NTR g of recombinant human neurotrophin 4/5 (a gift from Y.-A. of somite cells acting via the p75 receptor. Thus, in vivo Barde, Martinsried, Germany) was layered onto the embryos. The and in vitro, the mechanisms regulating cell death during eggs were sealed and returned to the incubator until the embryos neurogenesis also appear to be exploited in the morphogen- were sacrificed at st 16ϩ–17. esis of mesodermal structures. Histology. Chick embryos were fixed by immersion in 4% paraformaldehyde/PBS (w/v) overnight at 4°C and subsequently immersed in 100 mM sodium phosphate, pH 7.3, containing 30% MATERIALS AND METHODS sucrose (w/v) for 36 h. Eight-micrometer cryostat sections were mounted on gelatin-coated slides. Sections were incubated in PBS Chick embryos. Fertilized eggs from White Leghorn hens were with 10% (v/v) fetal calf serum for 30 min prior to immunostaining obtained from a local supplier and were incubated at 38.5°C at 70% with either the anti-p75NTR mouse polyclonal antibody (ascites humidity. Embryos were staged according to Hamburger and Ham- fluid, 1:4000 diluted) or the anti-HNK-1 mAb (ascites fluid diluted ilton (1951). 1:4000) in PBS containing 1% fetal calf serum for 16 h at 4°C. After Antibodies. The 27/21 anti-NGF monoclonal antibody (mAb) being rinsed, sections were incubated for1hatroom temperature specifically blocks the biological activity of NGF in ovo (Rohrer et with either Alexa 488 goat anti-mouse IgG (Molecular Probes) or al., 1988). Anti-p75NTR polyclonal antibody was obtained by immu- Cy3 goat anti-mouse IgM (Jackson Laboratories), both diluted nizing mice with a p75NTR receptor-Fc chimeric protein (5 ␮g per 1:1000 in the same buffer. For nuclear labeling, sections were injection) as described previously (Ker-hwa Ou et al., 1993). The counterstained with bisbenzimide (Hoechst 33258). localization of p75NTR immunoreactivity in somites detected with Cell death determination and quantification. Apoptotic cells this antibody coincided with the mRNA localization as seen by in were determined either by the TUNEL assay as previously de- situ hybridizations (compare Fig. 2K with 6C). In addition, the scribed (Frade et al., 1996b) or by observation of shrunken, de- nascent neurons of the basal diencephalon, the migratory cephalic formed, or broken, intensely fluorescent bisbenzimide-labeled nu- neural crest cells, and the primordium of the trigeminal ganglia clei. The TUNEL assay was applied to 18-␮m transverse sections were similarly stained with both techniques in st 18 embryos (not from st 16ϩ–17 embryos following the manufacturer’s instructions shown). The Fc chimeras of chick p75NTR, containing the extracel- (Boehringer Mannheim; Cat. No. 1684817). The same assay was lular domain of the receptor, were a gift from G. Dechant (Martins- also applied to proteinase K-treated embryos for whole-mount ried, Germany).The anti-HNK-1 mAb was obtained from ATCC staining.

FIG. 2. p75NTR mRNA is expressed in both neuroectodermal and mesodermal structures in chick embryos. In situ hybridization detects p75NTR mRNA in st 10 whole-mount embryos (A), in the rhombencephalic neural crest (F, transverse section at the rhombencephalic postotic level), and in the intermediate mesoderm (A, arrows, and G, transverse section at the level of somites 6–7). Expression increases at st 11 (B), being more apparent in the cervical neural tube. In st 12 embryos (C), p75NTR mRNA is also observed in nasal placodes (arrows) and in rhombencephalic neural crest, possibly of the enteric lineage (H, transverse section of the postotic region), and is also seen in somites, notably in the dorsal dermamyotome and sclerotome (I, section at the level of somite 5). At this stage, expression increases in the ventral neural tube (H, I). At st 14 (D) p75NTR mRNA is additionally seen in the basal diencephalon and more anterior cephalic neural crest (arrow and arrowhead, respectively). At this stage, expression increases in somites (J, transverse section at the level of somite 10 of st 14 embryos). At st 17 (E), expression is intense in the dermamyotome as shown in K (low cervical level) and L (dorsal level). FIG. 3. NGF expression in st 14 (A) and st 17 (B) chick embryos. Its mRNA appears in dermamyotome, surface ectoderm, roof plate, and the dorsalmost neural tube. NGF/p75NTR in Somite Cell Death 329

FIG. 4. Expression of TrkA mRNA in somites. The transcript is ﬁrst seen in the midline ﬁssure of somites at st 15 (A). Expression increases at st 18 (B). A transverse section at the low cervical level of st 18 embryos reveals high expression of TrkA mRNA in the dermamyotome (C). FIG. 5. PDC colocalizes with p75NTR expression but not with TrkA expression. Whole-mount st 17 chick embryos were labeled by TUNEL (A) to detect apoptotic cells. p75NTR (B) and TrkA (C) transcripts were detected by in situ hybridization. Note the segmental pattern of cell death which predominantly occurs in the rostralmost and caudalmost areas of the somites coinciding with p75NTR expression. In contrast, TrkA expression is restricted to the midline (rostral is left, caudal is right). 330 Cotrina et al.

For quantification of cell death in the sclerotome and the (Figs. 2A and 2F) and in the intermediate mesoderm or dermamyotome, apoptotic, TUNEL-positive-nuclei of somites pronephros (Figs. 2A and 2G) of st 10 chick embryos. Some 6–13 were counted. About 70 sections were analyzed from both expression was also detected in the neural tube at the level control and treated embryos. The number of apoptotic nuclei of the rostral somites (Fig. 2G). Unlike PCR amplification, counted in each control embryo was around 1000 in the sclerotome in situ hybridization failed to show p75NTR mRNA expres- and around 250 in the dermamyotome. sion in somites of st 10 embryos, presumably because of the cRNA probes and in situ hybridization. BlueScript plasmids NTR containing the full coding sequences of either chicken NGF (Eben- different sensitivities of the techniques. At st 11, p75 dal et al., 1986) or chicken p75NTR (Large et al., 1989) or bases mRNA expression was quantitatively more intense than at 1744–2329 of chicken TrkA (Backstrom et al., 1996) were used to st 10, but qualitatively similar (Fig. 2B). By st 12 (Fig. 2C), produce digoxigenin-labeled RNA probes for in situ hybridization p75NTR transcripts were clearly detected in branchial arches on either tissue sections or whole-mount embryos. (most likely due to the cephalic neural crest) and in rostral Hybridization of whole-mount embryos was carried out at 65°C somites as well as in the olfactory placodes. Expression for 48 h, following the procedure of Nieto et al. (1996). Labeled persisted in the intermediate mesoderm. Transverse sec- embryos were photographed, embedded in paraffin, and sectioned tions at the rhombencephalic postotic level showed intense ␮ at 20 m. Hybridization to tissue sections followed the method of labeling of the enteric neural crest migrating to the pe- Schaeren-Wiemers and Gerfin-Moser (1993). Briefly, 20-␮m-thick ripharynx (Fig. 2H). Transverse sections at the cervical level cryostat sections were hybridized with the NGF probe for 16 h at NTR 65°C and then washed three times at high stringency [50% form- show p75 expression in the somites and in the ventral amide (v/v), 65°C for 40 min]. The location of hybridized probes neural tube. In somites, expression was seen first in me- was revealed by incubation with an alkaline phosphatase-coupled diodorsal dermamyotome and more diffusely in the scle- anti-digoxigenin mAb (Boehringer). Development of the reaction rotome (Fig. 2I). At st 14, p75 mRNA is more intensely seen required 24–36 h. in somites (Figs. 2D and 2J). Later in development (st 17), Tissue explant cultures. Tissue pieces containing somites p75NTR mRNA was found in all somites, displaying a strong 6–10 were excised from st 11–12 embryos and cultured in DMEM/ rostrocaudal gradient (Figs. 2E, 2K, and 2L). At stages 14 and F12 medium supplemented with N2 (Bottenstein and Sato, 1979). 17, transcripts were also seen in putative migratory ce- Comparative PCR. The expression of different genes was deter- phalic neural crest and in the basal diencephalon. In trans- mined by RT-PCR of mRNA extracted from freshly dissected chicken verse sections of st 17 embryos at the posterior cervical somites or cultured explants. The primers used for PCR amplifica- NTR tions were the following: 5Ј-GTGTGTGACAGTGTCAGCATG-3Ј level, p75 mRNA was abundantly detected in the der- and 5Ј-ATTTACAGGCTGAGGTAGGGG-3Ј (377 bp) for NGF mamyotome and, at lower levels, in the sclerotome and in (Ebendal et al., 1986); 5Ј-ACGAGGTGATGGTGAAGGAG-3Ј and the ventral spinal cord neuroepithelium (Figs. 2K and 2L). 5Ј-TACAGGCTCTCAGCGATGTC-3Ј (727 bp) for p75NTR (Large et Previous data indicated that NGF mRNA can be detected al., 1989); 5Ј-GAGCAACTTCATCGAGAACC-3Ј and 5Ј-GCTTC- by PCR amplification in the somites of st 14 quail embryos ATGTACTCGAAGACC-3Ј (320 bp) for TrkA (Backstrom et al., (Yao et al., 1994). As shown before, we clearly detected 1996); and 5Ј-GGCTGCTAAGGCTGTGGGGA-3Ј and 5Ј-TATC- NGF mRNA by PCR at st 10 (see Fig. 1). However, NGF Ј AGCCTCTCCCACCTCC-3 (546 bp) for GAPDH (Panabieres et al., mRNA could be unambiguously revealed by in situ hybrid- 1984). To make gene expression comparable in different samples, an ization only at st 14 (Fig. 3A). In stages 14 and 17, NGF appropriate number of cycles was determined for each gene (normally NTR mRNA was mostly expressed in the surface ectoderm, in 25 for GAPDH, 30 for NGF, 35 for p75 , and 40 for TrkA) within the exponential phase of amplification. The amounts of total mRNA in the dermamyotome, and in the dorsalmost part of the the amplification mixtures were made equivalent according to their neural tube. Lower expression levels were also seen in the GAPDH content. notochord and in the sclerotome. The control experiments with a sense riboprobe did not reveal any signal (not shown). RESULTS Using in situ hybridization, the expression of TrkA mRNA could be first visualized at st 15 and more intensely Expression of NGF mRNA and That of Its at st 18 (Figs. 4A and 4B). At both ages, TrkA transcripts Receptors p75NTR and TrkA in Somite Development were found in the midline of the somites, at the so-called Ebner fissure (von Ebner, 1888, quoted by Gossler and Previous data showed that NGF, p75NTR, and TrkA Hrabe de Angelis, 1998). Because of its special location, mRNA were detected in somites during early development TrkA mRNA appears only in cross sections coincident with of the quail embryo (Yao et al., 1994). Here we dissected the Ebner fissure, like in Fig. 4C, which shows the expres- somites from chick embryos from st 10 on and found that, sion mostly in the dermamyotome, although expression although the levels of the three transcripts increased with also occurred in the mesenchyme at lower levels. development, they could be detected as early as st 10 by PCR amplification (Fig. 1). The distribution of the three transcripts was also exam- PDC in Early Chick Embryos ined during early embryogenesis by in situ hybridization. Our analysis revealed that the p75NTR mRNA was first A segmental pattern of cell death has been observed in expressed in the rhombencephalon-derived neural crest chick embryos from st 15 on using vital dyes (Jeffs and

Osmond, 1992). Detection of apoptotic cell death by the The Roles of NGF and p75NTR in Somitic Cell TUNEL assay on st 17 whole-mount embryos (Fig. 5A) also Death revealed this segmental pattern of apoptosis. In somites NTR Both p75 and NGF, but not TrkA, are expressed at the 5–10, cell death was concentrated in both the rostral and time and place of cell death in the somites. At the same the caudal regions of the somite, whereas more caudally, NTR time, NGF induces cell death in nervous tissue via p75 the cell death mostly affected the rostral region, presum- in the absence of TrkA later in development (reviewed in ably the sclerotome (Sanders, 1997). NTR Casaccia-Bonnefil et al., 1998). Therefore, a series of func- A closer inspection of the distribution of p75 mRNA tional experiments was performed to determine whether also revealed its expression in the dorsal part of the p75NTR and/or NGF could elicit a similar phenomenon in somite (principally dermamyotome), whereas in the more mesoderm derivatives. ventral part (the sclerotome), the location of these tran- First, to assess the role of NGF in somite cell death in scripts was higher in both the rostral and the ventral vivo, endogenous NGF was neutralized by systemic appli- aspects of somites 5–10 and in the rostral part of the cation of the 27/21 anti-NGF mAb. This antibody has been more caudal somites. Therefore, both cell death and NTR previously shown to block endogenous NGF activity and to p75 mRNA expression appeared to colocalize in the interfere with the normal development of both sensory and sclerotome (compare Figs. 5A and 5B). In contrast, the sympathetic neurons in vivo (Rohrer et al., 1988). A single, expression of TrkA mRNA was absent from the regions of 100-␮g dose of antibody was applied to st 11–12 embryos the somite expressing p75NTR mRNA or undergoing PCD NTR that were subsequently examined at st 17. About 1000 and (Figs. 4 and 5C). As p75 can mediate the death of 250 apoptotic nuclei were counted in the sclerotome and specific cell populations in the absence of TrkA recep- the dermamyotome, respectively, in each control embryo. tors (Casaccia-Bonnefil et al., 1998), these data suggest NTR The neutralization of endogenous NGF resulted in a de- the involvement of both NGF and p75 in somite crease of about 50 and 70% of the number of TUNEL- cell death. positive cells in the sclerotome and dermamyotome, respectively (Fig. 7). To prove that the antiapoptotic activity of the anti-NGF mAb was specific, either a mAb against the anti-␣6 integrin subunit, devoid of blocking activity, or a Localization of PCD in Somites mAb against NT-3 with blocking activity was also injected and the level of cell death with respect to untreated em- When TUNEL assays were performed on transverse sec- bryos did not change significantly (Fig. 7). Hence, our tions of st 17 embryos (Figs. 6A and 6B), most apoptotic results indicate that endogenous NGF regulates the PCD nuclei were detected in the central region of the sclerotome, occurring in somite cells. although lower levels of cell death could also be seen in the In a second set of in vivo experiments, the participation of dermamyotome. Previous reports have reached contradic- the p75NTR receptor in somite cell death was investigated by tory conclusions about the identity of dying cells at the antagonizing the binding of endogenous NGF by the exog- level of the sclerotome. While Jeffs and Osmond (1992) enous application of NT-4/5. The application of 2.5 ␮g concluded that they were migratory neural crest cells, NT-4/5 to st 11–12 embryos also resulted in a decrease in Sanders (1997) identified them as sclerotome cells of the apoptotic cell death in the sclerotome and the dermamyo- chondrogenic lineage. We performed double-labeling ex- tome of about 55 and 65%, respectively, at st 17. Such a periments on transverse sections to show that cells from reduction in cell death was similar to that observed after both the dermamyotome and the sclerotome were p75NTR blocking endogenous NGF (Fig. 7). immunoreactive, confirming the in situ hybridization data Together, these data demonstrate that physiological cell (compare Figs. 6C and 2K). Nuclear labeling of the same death in the somites is, to a large extent, the result of the section with bisbenzimide (Figs. 6D and 6F) showed that interaction of NGF with its receptor p75NTR. some of these p75NTR-immunoreactive cells contained ab- normally shaped, condensed, irregular, or fragmented and intensely fluorescent nuclei, characteristic of apoptotic Shh Influences the Expression of NGF mRNA on cells (Figs. 6D and 6F). However, as migratory neural crest Somites cells are also p75NTR immunoreactive, we determined Shh influences the survival of differentiated sclerotome whether these apoptotic cells might correspond to neural cells (Teillet et al., 1998) and maintains the expression crest cells within the sclerotome. Thus, in sections of the levels of Pax1 (Fan and Tessier-Lavigne, 1994; Fan et al., caudal part of the somites, HNK-1 was used to identify 1995; Johnson et al., 1994), a gene needed for the matura- neural crest cells together with sympathetic precursors at tion of sclerotome cells (Koseki et al., 1993). To investigate the dorsolateral level of the dorsal aorta (Fig. 6G). Nuclear a mechanistic link between Shh and NGF with respect to staining of the same section (Figs. 6H and 6I) revealed that, somite cell death, we examined the effects ofShh on the as in Fig. 3A, apoptotic nuclei were found only in the expression of NGF and p75NTR mRNA in cultured somite central sclerotome in cells that were not recognized by explants. As shown in Fig. 8, Shh decreased the levels of HNK-1 and thus could not be neural crest cells. NGF mRNA, whereas no effect was observed on the p75NTR

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. 332 Cotrina et al. transcript. These results strongly suggest that the mecha- nism used by Shh to lend trophic support to somite cells relies in part on the downregulation of NGF in the somites.

DISCUSSION

Early Expression and Possible Functions of p75NTR in Nonneuronal Tissues By analyzing the sites of expression of the neurotrophin receptors, we hoped to gain a better idea of the potential roles of p75NTR outside of the nervous system. Both TrkA and p75NTR have been shown to be expressed in nonneuronal tissues during development, mainly in places where mesenchymal/epithelial interactions occur (Casaccia- Bonnefil et al., 1998). Hence, p75NTR expression is present in the developing maxillary pad, muscle, lung, testes, kidney, and teeth (Byers et al., 1990; Heuer et al., 1990; Huber and Chao, 1995; Mitsiadis et al., 1993; Sainio et al., 1997; Wheeler and Bothwell, 1992; Yan and Johnson, 1988). In a recent report, Wheeler et al. (1998) found that the different Trk receptors and p75NTR are not coexpressed in the same nonneuronal cells and inferred that these receptors may have distinct developmental functions. However, despite the large body of morphological data, an unambiguous function for mesodermal p75NTR during development has not been demonstrated. We show here that the expression of p75NTR in st 10 embryos is seen in both neural derivatives, such as neural crest, and in nonneural structures, such as the intermediate mesoderm. Shortly afterward, p75NTR expression extends to both the paraxial mesoderm and the neural tube. In st 14–17 embryos, p75NTR is highly expressed in the rostral and caudal parts of the somites 5–10, with little or no expression in the central region. p75NTR expression decreases caudally along the embryonic axis, being present mainly in the rostral parts of the more FIG. 6. PDC affects sclerotome cells but not HNK-1-positive caudal somites. In contrast, in situ hybridization of TrkA cells. Sections at the lower cervical level from st 17 chick embryos revealed that the expression of this specific receptor for were labeled by TUNEL to detect apoptotic nuclei (A, inset detailed NGF is concentrated in the midline of somites (von Ebner’s in B) in both the dermamyotome (arrows) and the sclerotome fissure), thus implying that a large number of somitic cells (arrowheads). A section at the same level was double labeled with an anti- p75NTR antibody (C) and with bisbenzimide (D) and their express only p75NTR as a receptor for NGF, a situation that respective insets are expanded in E and F. Note that apoptotic is also encountered in several other types of mesodermal nuclei locate to p75NTR-positive cells (arrowheads). HNK-1 labels cells later in development (Wheeler et al., 1998). neural crest derivatives (G) at the beginning of their migration Prior to this study, few details were available regarding (arrowhead), as well as sympathetic precursors at the dorsal aorta the topographical expression of NGF mRNA in early em- (arrow). The nuclear labeling of the same section (H, the inset of bryos although Yao et al. (1994) detected NGF mRNA by which is I) shows that apoptotic nuclei (arrowheads) are located in the RT-PCR in microdissected somites of st 12–14 quail em- central sclerotome in areas where no HNK-1-labeled cells are found. bryos. These authors also observed neurotrophin and TrkA receptor mRNA expression in most axial and paraxial structures of quail embryos from st 6 to st 14. We studied the expression of NGF, p75NTR, and TrkA in excised somites suggest a topographical and temporal association between by PCR and found that the three mRNAs were detected as the presence of NGF/p75NTR and PCD affecting the somites. early as st 10. In addition, our in situ hybridization study has further defined the location of NGF mRNA expression The Nature of Dying Cells in the Somites by showing that the highest levels of expression occur in the roof plate of the neural tube, in the surface ectoderm, The first comprehensive morphological study of cell and in the dermamyotome. These expression data strongly death in somites revealed a similarity between the pattern

differentiated sclerotome cells depends on external signals. For instance, Sanders (1997) has shown that the geometric pattern of cell death does not change after grafting somites in a reversed orientation. In addition, Teillet et al. (1998) have demonstrated that the ablation of the notochord from E2 embryos is followed by massive death of differentiated sclerotome cells and that this cell death is prevented by the application of Shh, thus revealing that this morphogen is needed not only for differentiation of somitic cells into sclerotome but also for a further survival of these cells. Moreover, Shh is also required for the maintenance of Pax1 expression in the somites (Fan and Tessier-Lavigne, 1994; Johnson et al., 1994) and for the maintenance and proliferation of sclerotome cells in vivo (Furumoto et al., 1999; Koseki et al., 1993; Marcelle et al., 1999). Our inhibition studies demonstrate that endogenous NGF, most likely derived from the dermamyotome, inter- acts with p75NTR to counteract the trophic effects of FIG. 7. Quantification of cell death in the somites in the absence notochord- and floor plate-derived Shh. Our experiments in or presence of anti-NGF antibodies or NT-4/5. Untreated st 17 vivo show that the neutralization of the NGF activity by chick embryos contain an average of 16 and 6 TUNEL-positive the application of the anti-NGF blocking antibody is fol- nuclei per section in the sclerotomal and dermamyotomal region, lowed by a 50 and 70% decrease of sclerotomal and der- respectively, between somite 6 and somite 13 (see Fig. 3A). Either mamyotomal cell death, respectively, an effect similar to ␣ in mock-injected (anti- 6) or in anti-NT-3-injected embryos, the what is observed after the blockade of the binding activity levels of cell death were not significantly altered. In contrast, the of p75NTR by human NT-4/5. This neurotrophin has not number of apoptotic nuclei per section was significantly reduced if NTR st 11/12 embryos were treated with either an antibody against NGF been found in the chick but it does bind to chick p75 , or NT-4/5, a neurotrophin not found in the chick but which binds thereby antagonizing the binding of NGF in vitro (Dechant with equal affinity to, and competes with, NGF for binding to the et al., 1997). Human NT-4/5 is not active on avian p75NTR receptor. *P Ͻ 0.05; **P Ͻ 0.01; ***P Ͻ 0.001. Analysis neurons, indicating that it does not induce any signaling of variance for group comparisons. We used the Student t test to after binding to p75NTR (Davies et al., 1993). Furthermore, establish the significance of differences. recent studies have shown that NT-4/5 competes with NGF to abolish the neurite-promoting activity of NGF in chick ciliary neurons, an activity mediated solely by the p75NTR receptor (Yamashita et al., 1999). Consistent with of migration of neural crest and cell death (Jeffs and Os- these data, the administration of NT-4/5 to the embryos mond, 1992). In contrast, in a more recent and detailed decreased the levels of cell death by 55 and 65% in the study, Sanders (1997) demonstrated that most dying cells sclerotome and dermamyotome, respectively. To further were not of neural crest origin. Our results are in agreement with the conclusion of Sanders and also exclude the possi- bility that the cells dying in the sclerotome are derived from the neural crest. The dying sclerotome cells are not HNK-1 immunoreactive and cell death does not appear in the posterior part of the somite, i.e., far from the migration pathway of the neural crest cells. These data demonstrate that cell death within the somites affects the sclerotome cells of chondrogenic lineage.

Trophic Interactions Affecting Somite Cell Death A large body of evidence has established that the differ- FIG. 8. Shh decreases the expression of NGF mRNA but it does NTR entiation of somites is greatly influenced by inductive not affect the expression of p75 mRNA in cultured somites. Tissue explants comprising the somites were cultured either in the signals between neighboring structures (Christ et al., 1998). absence or in the presence of 2 ␮g/ml Shh. After the times indicated In particular, the differentiation of the sclerotome is largely in culture, RT-PCR was performed on mRNA extracted from the driven by notochord- and floor plate-derived Shh. However, explants using specific primers from the NGF, p75NTR, or GAPDH much less is known about the later signaling that controls cDNA sequences. The amounts of total mRNA in the amplifica- maturation, proliferation, migration, and survival of scle- tion mixtures were made equivalent according to their GAPDH rotome cells. There is some evidence that the death of content.

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. 334 Cotrina et al. prove the specificity of the action of NT-4/5, we admin- Backstrom, A., Soderstrom, S., Kylberg, A., and Ebendal, T. (1996). istered an anti-NT-3 mAb to the embryos. Neural tube- Molecular cloning of the chicken trkA and its expression in early derived NT-3 is needed for the dermatome–dermis con- peripheral ganglia. J. Neurosci. Res. 46, 67–81. version later in development (Brill et al., 1995). However, Barres, B. A., Raff, M. C., Gaese, F., Bartke, I., Dechant, G., and the blockade of this neurotrophin failed to alter the levels Barde, Y.-A. (1994). A crucial role for neurotrophin-3 in oligoden- of cell death in the somite. These data demonstrate the drocyte development. Nature 367, 371–375. validity of the use of NT-4/5 as a blocker of p75NTR Bovolenta, P., Frade, J. M., Marti, E., Rodriguez-Penã, M. A., Barde, Y. A., and Rodriguez-Tebar, A. (1996). Neurotrophin-3 antibodies activity. disrupt the normal development of the chick retina. J. Neurosci. Prior to this study, no data existed to indicate that NGF 16, 4402–4410. could induce PCD in nonneuronal cells. Therefore, it re- Bottenstein, J. E., and Sato, G. H. (1979). Growth of a rat neuro- mains uncertain which signaling pathway might be trig- blastoma cell line in serum-free supplemented medium. Proc. gered by this neurotrophin to induce apoptosis. Further Natl. Acad. Sci. USA 76, 514–517. work will be needed to establish at what level NGF coun- Brand-Saberi, B., Ebensperger, C., Wilting, J., Balling, R., and Christ, teracts the Shh-induced signaling that provides trophic B. (1993). The ventralizing effect of the notochord on somite support to somite cells. However, our data do reveal that differentiation in chick embryos. Anat. Embryol. 188, 239–245. 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