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Keywords:

  • ADAM;
  • spinal cord;
  • motoneuron;
  • gene expression;
  • chicken development

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The expression patterns of seven members of the ADAM (a disintegrin and metalloprotease) family, including ADAM9, ADAM10, ADAM12, ADAM13, ADAM17, ADAM22, and ADAM23, were analyzed in the developing chicken lumbar spinal cord by in situ hybridization and immunohistochemistry. Results show that each individual ADAM is expressed and regulated spatiotemporally in the lumbar cord and its surrounding tissues. ADAM9, ADAM10, ADAM22, and ADAM23 are expressed predominantly by motoneurons in the motor column and by sensory neurons in the dorsal root ganglia, each with a different expression pattern. ADAM12 and ADAM13 are mainly expressed in the meninges around the lumbar cord and in the condensed sheets of chondroblasts around the vertebrae. ADAM17 expression is strong in the ventricular layer and limited to early stages. The differential expression of the ADAMs in the lumbar cord suggests that the ADAMs play a regulatory role in development of the spinal cord. Developmental Dynamics 239:1246–1254, 2010. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Members of the ADAM family are type I trans-membrane proteins (Wolfsberg et al.,1995; Black and White,1998; Schlöndorff and Blobel,1999), and they are involved in proteolysis, adhesion, cell fusion, and in cell signal transduction based on their multiple domains (Blobel,2002,2005; Seals and Courtneidge,2003; White,2003; Edwards et al.,2008). So far, approximately 38 members of ADAM protein family have been identified from different species (Edwards et al.,2008). An exhaustive member list of ADAMs has been shown in the homepage of the White Lab (http://people.virginia.edu/%7Ejw7g/Table_of_the_ADAMs.html).

During embryonic development, ADAMs play a role in morphogenesis and tissue formation (Edwards et al.,2008; Alfandari et al.,2009). ADAM10-mutant mice die of multiple morphological defects in the cardiovascular system and in developing brain at the embryonic stage (Hartmann et al.,2002). ADAM17- and ADAM19-deficient mice die at birth because of multiple cardiovascular defects (Horiuchi et al.,2005). ADAM22-deficient mice show ataxia and prominent hypomyelination of the peripheral nerves (Sagane et al.,2005), while ADAM23-deficient mice demonstrate tremor and ataxia and die during early postnatal times (Leighton et al.,2001). ADAMs are widely involved in neurogenesis, cell migration, axon outgrowth and guidance, and cell differentiation during development (Yang et al.,2006; Edwards et al.,2008). For example, knockdown of ADAM19 in Xenopus decreases the numbers of neurons and neural crest cells (Neuner et al.,2009). ADAM2 contributes the neuroblasts migrating to the olfactory bulb (Murase et al.,2008). Proteolytic processing of the neural cell adhesion molecule (NCAM) by ADAM17 is involved in NCAM-mediated neurite outgrowth (Kalus et al.,2006). ADAM10 sheds ephrinA2 inducing ephrinA2-mediated axon repulsion (Hattori,2000) and ADAM10 is required for the formation of the optic projection to the tectum (Chen at al.,2007). Finally, ADAM23 is involved in neuronal differentiation of P19 cells (Sun et al.,2007).

The vertebrate spinal cord develops from the posterior neural tube and differentiates along the dorsoventral and rostrocaudal axes (Edlund and Jessell,1999). Motor neurons in the spinal cord are derived from neural plate cells induced by Sonic hedgehog (Shh; Ericson et al.,1996). Motoneurons in the lateral motor column (LMC) are clustered into different motor pools (Hollyday,1980), where motoneurons project their axons into targeted limb muscle (Jackson and Frank,1987). Several members of the classic cadherin (Cad) play a role in the formation of distinct pools of motor neurons (Price et al.,2002). Of interest, ADAM10 is the major protease responsible for cadherin shedding and modulates cell–cell adhesion and signal transduction (Reiss et al.,2005,2006; Maretzky et al.,2005). Trunk neural crest cells migrate along a ventromedial route and form the dorsal root ganglia (DRG) and sympathetic ganglia (SG) (Le Douarin and Kalcheim,1999; Kasemeier-Kulesa et al.,2005). Wnt/β-catenin signaling promotes sensory neurogenesis in early neural crest cells (Lee et al.,2004), while bone morphogenic protein (BMP) signals induce neural crest cells to differentiate into sympathetic neurons (Reissmann et al.,1996). Semaphorin3A, axonin-1/SC2, and netrin-1 are required as repulsive guidance cues for the formation of sensory axonal projections to the spinal cord (Masuda et al.,2003,2008).

In our previous studies, we have shown that ADAM13 is expressed in neural crest cell-derived structures and digestive organs (Lin et al.,2007), while several other ADAMs, including ADAM9, ADAM10, ADAM12, ADAM22, and ADAM23, demonstrate a spatiotemporally regulated expression in developing brain (Lin et al.,2008). The expression profiles of ADAMs in the developing spinal cord and its surrounding tissues remain unknown. Therefore, in the present study, we continue to analyze the expression patterns of these ADAMs at the developing lumbar cord during chicken embryonic development. Our results show that each of the ADAMs is expressed spatiotemporally in distinct parts of the developing lumbar cord, although the expression patterns overlap partially.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Structures of the developing spinal cord and its surrounding tissues in chicken embryos can be distinguished easily by nuclear staining (Nu) and hematoxylin and eosin (HE) staining. For example, the ventricular layer formed by neuroepithelial cells in the spinal cord and the dermomyotome, sclerotome, and the notochord around the spinal cord are clearly seen at embryonic incubation day (E) 3 by 4′-6-diamidino-2-phenylindole (DAPI) staining (Fig. 1A). At E6, the ventricular layer, motor column, and the DRG are clearly detectable by HE staining (Fig. 1B) or DAPI staining (Fig. 1C). At E10, the mantle layer, the ventral and dorsal horns of the spinal cord, the arches and bodies of vertebra and the DG are also clearly visible by HE staining (Fig. 1D).

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Figure 1. Expression of ADAM9 and ADAM10 in transverse sections of the developing chicken spinal cord at the lumbar level from incubation day 3 (E3) to E11 (marked). A–D: Nuclear staining (Nu; A,C) and hematoxylin and eosin (HE) staining (B,D) show structures of the spinal cord. E–Z: In situ hybridization for ADAM9 (A9; E,F,I,J,M,P,Q,T,U) and for ADAM10 (A10; G,H,K,L,N,O,R,S,V–Z). The asterisks in E, Q, and T indicate artificial folds of the tissue and bubbles, respectively. Arrows point to strong ADAM10 expression in cell clusters in G and H, and to blood vessels in S and X–Z; the arrow in W indicates axon fascicles inside the DRG. A′,B′: In situ hybridization using sense cRNA-probes for ADAM9 (A′) or ADAM10 (B′) as negative controls. dh, dorsal horn; dm, dermomyotome; drg, dorsal root ganglion; ep, epithelial cells of the ventricular layer; fp, floor plate; lm, lateral motor neuron column; mc, motor neuron column; me, meninges; ml, mantle layer; mt, myotome; mm, medial motor neuron column; nc, notochord; sp, spinal cord; sc, sclerotome; sg, sympathetic ganglion; ve, vertebrae; vh, ventral horn; vr, ventral root. Scale bars = 100 μm in C and in F (applies in F,H,J,L,X,Z), 200 μm in A (applies in A,E,G,I,K,O,Q,S,U,W,Y,A′), 400 μm in B (applies in B,D,M,N,P,R,T,V,B′).

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The aim of this study is to analyze the expression patterns of seven members of the ADAMs in adjacent transverse sections of the spinal cord at the lumbar level from E3 to E11. Expression patterns of each ADAM are shown according to developmental stages (early to late) and compared with each other, e.g., ADAM9 vs. ADAM10 (Fig. 1), ADAM12 vs. ADAM13 (Fig. 2), and ADAM22 vs. ADAM23 (Fig. 3). Sense RNA probes are used as negative controls (e.g., Fig. 1A′,B′). In general, each of the ADAMs investigated shows a spatially restricted and temporally regulated expression pattern with a partial overlay between each other.

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Figure 2. A–X: Expression of ADAM12 (A12; A,D,E,G,H,K,L,N,O), ADAM13 (A13; B,C,F,I,J,M,P,Q) and ADAM17 (A17; R–X) as detected by in situ hybridization in transverse sections of the chicken lumbar spinal cord from incubation day 3 (E3) to E11 (marked). Arrows in D, E, and in the insert of E indicate the neurites of the DRG, and in I point to the tissues above the roof plate. cc, condensed chondroblasts; da, dorsal aorta; de, epidermis; dm, dermomyotome; dr, dorsal root; drg, dorsal root ganglion; ep, epithelial cells of the ventricular layer; fp, floor plate; lm, lateral motor neuron column; mc, motor neuron column; me, meninges; mt, myotome; ncs, neural crest cell; rp, roof plate; sc, sclerotome; sn, spinal nerve; ve, vertebrae; vr, ventral root. Scale bars = 100 μm in E (applies in C,E,S,U), 200 μm in A (applies in A,B,D,F,H,J,L,N,Q,R,T,V,X), 400 μm in G (applies in G,I,K,M,O,P,W).

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Figure 3. Expression of ADAM22 and ADAM23 in transverse sections of the chicken lumbar spinal cord from incubation day 3 (E3) to E11 (marked). A–J: In situ hybridization for ADAM22 mRNA (A22; A,D,E,H,I) and ADAM23 mRNA (A23; B,C,F,G,J) at stages E3–E6. Thin dotted lines in I outline the DRG and thick dotted lines indicate the boundary between the dorsomedial part and the ventrolateral part of the DRG. K–M: Double-label immunohistochemistry for ADAM23 protein (A23; green in K,M) and Islet-1 (red in L,M) at stage E6. Arrows in K and L indicate the motor axons. N–U: In situ hybridization for ADAM22 mRNA (N,O,R,S) and ADAM23 mRNA (P,Q,T,U) at stages E8–E11. Thin dotted lines in O and S outline the DRG and thick dotted lines in O, S, Q, and U indicate the boundary between the dorsomedial part and the ventrolateral part of the DRG. V–X: Schematic diagrams outline the expression patterns of the seven investigated ADAMs at E4 (V), E6 (W), and E11 (X), respectively. The different ADAMs are represented by distinct color cycles. d-m, dorsomedial region of dorsal root ganglion; dm, dermomyotome; drg, dorsal root ganglion; lm, lateral motor neuron column; ml, mantle layer; mc, motor neuron column; mm, medial motor neuron column; mt, myotome; nc, notochord; sc, sclerotome; sg, sympathetic ganglion; v-l, ventrolateral region of dorsal root ganglion; vr, ventral root. Scale bars = 100 μm in A (applies in A,C,E,G,I,) and in K (applies in K–M), 200 μm in D (applies in B,D,F,O,Q,S,U), 400 μm in H (applies in H,J,N,P,R,T).

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ADAM9 and ADAM10

At E3, ADAM9 mRNA is expressed strongly by neuroepithelial cells in the ventricular layer and by motor neurons in the motor column (mc) of the lumbar cord (Fig. 1E,F). Expression is also seen in tissues around the lumbar cord, for example, strongly in the notochord (nc) and dermomyotome (dm) and weakly in the sclerotome (sc). At this stage, ADAM10 transcription is strong in the ventricular layer, the floor plate (fp) and also in the dermomyotome (Fig. 1G,H), but weak in the sclerotome except of some cell clusters, which express ADAM10 strongly (arrows in Fig. 1G,H). At E4, ADAM9 (Fig. 1I,J) and ADAM10 (Fig. 1K,L) maintain strong expression in the ventricular layer and by the cells in the floor plate, but expression decreased slightly in the myotome (mt), when compared with that at E3, respectively (Fig. 1E–H). In the motor column, ADAM9 and ADAM10 become gradually restricted to the motoneurons in the LMC. The DRG starts to express ADAM9 and ADAM10 with a few strongly positive portions in the dorsal parts. At this stage, ADAM9 is expressed weakly in the notochord (Fig. 1I) and first ADAM10 signals are seen in the meningeal tissues (me in Fig. 1K,L). At E6, both ADAM9 (Fig. 1M) and ADAM10 (Fig. 1N,O) mRNAs are expressed prominently by the motoneurons in the LMC and by the sensory neurons in the DRG, but weakly by the neuroepithelial cells in the ventricular layer. At this stage, the meninges around the spinal cord express ADAM10 clearly (me in Fig. 1N,O). From E8 to E11, the LMC, DRG, and the SG show moderate to strong staining for ADAM9 (Fig. 1P,Q,T,U), and strong staining for ADAM10 (Fig. 1R,S,V,W). The mantle layer shows diffuse and weak to moderate staining for both ADAM9 and ADAM10 (e.g., Fig. 1P,R). Remarkably, in contrast to ADAM9, ADAM10 is expressed by sensory neurons predominantly in the dorsomedial part of the DRG as the embryo develops, although weak expression is also seen in the ventrolateral part (border indicated by dotted lines in Fig. 1S,W). ADAM10 expression in the ventral roots is found moderately at E8 (Fig. 1R,S), but weakly at E11 (Fig. 1V,W). Analogously to the blood vessels of the brain (Lin et al.,2008), ADAM10 is also observed to be strongly expressed in the microvessels of the spinal cord (arrows in Fig. 1S,X–Z). At E11, ADAM9 is expressed weakly by the motoneurons of the medial motor column (MMC; e.g., Fig. 1T).

ADAM12 and ADAM13

In contrast to the expression patterns of ADAM9 and ADAM10 in the spinal cord, the signals of ADAM12 and ADAM13 are found mainly in the structures around the spinal cord. At E3, ADAM12 expression is moderate in the epidermis (de) but undetectable in the spinal cord (Fig. 2A). ADAM13 is expressed moderately to strongly in the dermomyotome and sclerotome (Fig. 2B,C). At E4, ADAM12 signals are weak in the DRG (Fig. 2D and insert in Fig. 2E) but strong in the ventral roots (vr in Fig. 2D,E), in the distal dorsal roots (arrows in Fig. 2D,E) and in the spinal nerves (sn in Fig. 2D,E). At this stage, ADAM13 signals remain strong in the myotome and decreased in the sclerotome (Fig. 2F). Weak ADAM13 expression arises in the meninges around the spinal cord. At E6, ADAM12 expression appears strong in the roof plate (rp), the myotome and moderate in the meninges (Fig. 2G,H), while ADAM13 is expressed strongly in the tissues above the roof plate (arrow in Fig. 2I), in the myotome, the meninges, and in the chondroblasts around the vertebrae (Fig. 2I,J). From E8 to E10, ADAM12 expression persists in the roof plate and the meninges, and appears in the arches of the vertebrae (ve in Fig. 2K,L). Weak expression of ADAM12 in the ventral roots can still be seen at E8 (Fig. 2K). At E8, ADAM13 signals are clearly seen in the meninges and the chondroblasts around the vertebrae (Fig. 2M). At E11, ADAM12 is no longer detectable in the tissues around the spinal cord (Fig. 2O) but strong expression of ADAM 13 is still found in the meninges (Fig. 2P,Q).

ADAM17

At E3, ADAM17 is expressed strongly in the ventricular layer, the dermomyotome, and in the floor plate, but weakly in the sclerotome (Fig. 2R,S). From E4 to E6, it maintains strong expression in the ventricular layer and floor plate, but is weak in the myotome and the LMC (Fig. 2T–V). At E8, weak expression is observed in the ventricular layer, the DRG, the LMC, and in the ventral roots (Fig. 2W,X). ADAM17 signals are no longer detectable in the spinal cord at E11 (data not shown).

ADAM22 and ADAM23

ADAM22 and ADAM23 are both uncatalytic members of the ADAM family and are closely related phylogenetically (Yang et al.,2006; Lin et al.,2008). At E3, ADAM22 mRNA is moderately expressed only by the motoneurons of the motor column (mc in Fig. 3A), while ADAM23 mRNA is expressed strongly by the motoneurons in the motor column and by the cells in the dermomyotome (Fig. 3B,C). At E4, ADAM22 expression is strong in the LMC, moderate in the DRG, and weak in the myotome and sclerotome (Fig. 3D,E). At this stage, ADAM23 maintains strong expression in the LMC and the myotome and starts to be expressed strongly in the DRG (Fig. 3F,G). At E6, ADAM22 and ADAM23 mRNAs are expressed mainly in the LMC and the DRG (Fig. 3H–J), and weak signals are found in the myotome for ADAM22 (Fig. 3H) and in the SG for ADAM23 (Fig. 3J). By double-label immunostaining, ADAM23 protein is shown mainly in the motoneurons of the LMC and in the sensory neurons of the DRG (green in Fig. 3K,M), where Islet-1 (red in Fig. 3L) is coexpressed (yellow in Fig. 3M). Islet-1 is an early neuronal differentiation marker in the motor neuron column (Ericson et al.,1992) and a neuronal marker in the DRG and SG (Cui and Goldstein,2000; Avivi and Goldstein,2003). The sympathetic neurons in the SG also coexpress ADAM23 and Islet-1 proteins (data not shown). Furthermore, ADAM23 protein is expressed by the ventral roots (e.g., arrows in Fig. 3K,M) and spinal nerves projecting to the limb muscles. From E8 to E11, the LMC and DRG express strongly ADAM22 mRNA (Fig. 3N,O,R,S) and ADAM23 mRNA (Fig. 3P,Q,T,U), while the mantle layer shows moderate and diffuse ADAM22 and ADAM23 signals (Fig. 3N–T). During these stages, ADAM23 mRNA transcript, but not ADAM22, is expressed moderately in the SG (Fig. 3P,T). It should be noted that from E6 onward, ADAM22 is expressed predominantly and strongly by the sensory neurons in the ventrolateral part of the DRG and that there is no ADAM22 expression in its dorsomedial part (indicated by thick dotted lines in Fig. 3I,O,S). This ADAM22 expression by the sensory neurons in the DRG is different from ADAM9 (Fig. 1P,Q,T,U), ADAM10 (Fig. 1R,S,V,W), and also from ADAM23, which is expressed strongly and predominantly by sensory neurons in the ventrolateral part, but moderately in the dorsomedial part (border indicated by dotted lines in Fig. 3Q,U). At E11, ADAM22 and ADAM23 mRNAs are expressed weakly by the motoneurons in the MMC (Fig. 3R,T).

In summary, each of the seven ADAMs is expressed in the lumbar spinal cord and/or its surrounding tissues (Fig. 3V–X). The expression patterns differ from each other but share partial overlap.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Expression of the ADAMs by Motor Neurons

In this study, ADAM9, ADAM10, ADAM22, and ADAM23 are found to be expressed strongly and predominantly by the motoneurons of the motor column at E3 and of the LMC from E4 onward in the lumber cord (Figs. 1, 3). Furthermore, the ventral roots of motor axons projecting to limb muscles also express ADAM23 protein (e.g., Fig. 3K,M). During motor neuron development, motor pool sorting in the chicken lumbar spinal cord begins at approximately E4.5 (stage 24; Lin et al.,1998; Price et al.,2002). Several classic cadherins are expressed by different subsets of motor neurons (Marthiens et al.,2002,2005; Price et al.,2002; Ju et al.,2004; Luo et al.,2006) and the combinatorial interaction of these classic cadherins triggers the formation of distinct pools of motor neurons (Price et al.,2002). Of interest, ADAMs have multiple functions involved in cell–cell interaction, cell–matrix interaction, and ectodomain shedding (Blobel,2002,2005; Seals and Courtneidge,2003; White,2003; Edwards et al.,2008). For example, ADAM19 has been identified to be involved in the differentiation of Schwann cells (Wakatsuki et al.,2009); and ADAM10 is responsible for the constitutive and regulated shedding of cadherins and controls the multiple functions of cadherins under physiological as well as pathological conditions (Maretzky et al.,2005; Reiss et al.,2005,2006; Kohutek et al.,2009). In the present study, ADAM9 and ADAM10, which have a proteolytic function, are expressed by motoneurons in the motor column from E3 onward. Therefore, it will be interesting to further study whether the expression of ADAMs in the motor column contributes to motor neuron specification and motor pool sorting mediated by regulation of the function of the cadherins.

In developing embryos, motor axons extend from the spinal cord, grow into the limbs, and innervate targeted muscles (Jacob et al.,2001). The process of motor axon outgrowth and pathfinding is controlled by a combination of different factors (Schneider and Granato,2003), for example, by members of the Eph/ephrins family of molecules (Eberhart et al.,2000) and by NCAM (Landmesser,1996; Stoker,1996). It is of interest that ADAM10 can cleave ephrins in the Eph/ephrin binding-complex and regulate their functions (Janes et al.,2005). ADAM17 is involved in neurite outgrowth mediated by regulation of the proteolytic processing of NCAM (Kalus et al.,2006). ADAM10 is required for Xenopus retinal ganglion axons to recognize the targeted tectum (Chen et al.,2007). Furthermore, UNC-71, a member of the ADAM family in C. elegans, cooperates with integrins and UNC-6/netrin and provides distinct cues for motor axon guidance (Huang et al.,2003). Whether the expression of the ADAMs by motor neurons regulates motor axon outgrowth and pathfinding remains to be elucidated.

Expression of the ADAMs in Dorsal Root Ganglia and Sympathetic Ganglia

In the peripheral nervous system (PNS), the multipotent trunk neural crest can generate different types of neurons, for example, sensory neurons in the DRG and sympathetic neurons in the SG (Le Douarin and Kalcheim,1999). Two subtypes of sensory neurons in the developing chicken DRG are distinguished by their anatomical location from E6 onward: (1) a group of early differentiated large-diameter neurons located in the ventrolateral part expressing neurotrophic tyrosine receptor kinase C (TrkC), and (2) a group of late differentiated small-diameter neurons located in the dorsomedial part expressing TrkA (Hamburger et al.,1981; Avivi and Goldstein,2003). The distinct sensory neurons project their axons into the spinal cord and generate subtype-specific sensory–motoneuron connectivity.

In the present study, ADAM9, ADAM10, ADAM22, and ADAM23 were found to be expressed by sensory neurons in the DRG at E4. From E6 onward, each individual ADAM shows different expression patterns in the DRG, for example, ADAM9 is expressed homogenously by sensory neurons; ADAM10 is expressed predominantly by the sensory neurons in the dorsomedial part, and ADAM23 in the ventrolateral part, while ADAM22 is expressed by the sensory neurons only in the ventrolateral part without any expression in the dorsomedial part (Figs. 1, 3). We have not found any expression of these ADAMs by the migrating neural crest cells at early stages, but ADAM23 has been identified to regulate the differentiation of neural crest cells during embryonic development (Neuner et al.,2009). Therefore, the changes of the ADAM expression patterns coincide with the settlement of sensory neurons in the DRG, suggesting that the ADAMs play a role in the differentiation and specification of sensory neurons. Furthermore, it is possible that there is a high correlation of the ADAM expressions in the sensory and motor neurons that project to the same targeted muscle. In this regard, it will be interesting to investigate the effects of ADAMs on the selective connectivity of these neural elements in specific sensory–motor circuits.

Moreover, in the present study, we show for the first time that the ADAMs are also expressed by sympathetic neurons in the SG (Figs. 1, 3). The role of the ADAMs on the developing sympathetic neurons during embryonic development is still unclear.

Expression of the ADAMs in Dermomyotome and Sclerotome

During embryonic development, the myotome forms most of the trunk and limb muscles, while the sclerotome builds the bones including the vertebrae. A previous study shows that the chicken dermomyotome expresses ADAM10, which may contribute to the epithelial-to-mesenchymal transformation during dermatome and myotome formation (Hall and Erickson,2003). ADAM12 and ADAM19 are also detected in the embryonic limb and tail bud (Lewis et al.,2004). In the present study, we demonstrate that the ADAMs are expressed in the dermomyotome from E3 onward (Figs. 1–3) and ADAM13 is also transcribed strongly in the sclerotome (Fig. 2). The general role of the ADAMs in the formation of the muscular system and of vertebrae remains to be defined.

Expression of ADAM10 in Blood Vessels

In the present study, ADAM10 is found to be expressed in blood vessels of the spinal cord from E8 onward (Fig. 1). A similar expression of ADAM10 in blood vessels of the chicken brain has been reported previously (Lin et al.,2008).

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Embryos

Fertilized eggs of White Leghorn chickens (Gallus gallus) were purchased from a local farm and incubated in a forced-draft incubator (Ehret, Emmendingen, Germany) at 37°C with 65% humidity. Embryos were studied at E3, E4, E6, E8, E10, and E11 (at least 3 embryos at each stage). For in situ hybridization, embryos were removed from the eggs and fixed in formaldehyde solution (4% in Hepes-buffered salt solution; HBSS) on ice for 6 to 24 hr, depending on the size of the embryos. After fixation, the brains were immersed in graded sucrose solutions (12%, 15%, and 18% in HBSS). Specimens were embedded in Tissue-Tec O.C.T. compound (Science Services, Munich, Germany), frozen in liquid nitrogen, and stored at −80°C, as described previously (Lin et al.,2008).

Probe Synthesis and In Situ Hybridization

Digoxigenin-labeled sense and antisense cRNA probes were synthesized in vitro according to the manufacturer's instructions (Roche, Mannheim, Germany). As templates, plasmids containing previously cloned ADAM sequences were used (Lin et al.,2007,2008). Sense cRNA probes were used as a negative control. In situ hybridization on cryosections was performed according to the protocol described previously (Lin et al.,2007). The sections were viewed and photographed under a transmission microscope (BX40; Olympus, Hamburg, Germany) equipped with a digital camera (DP70; Olympus). Photographs were adjusted in contrast and brightness by the Photoshop software (Adobe, Mountain View, CA).

Antibodies and Immunohistochemistry

Primary mouse monoclonal antibody against Islet-1 (39.4D5) and primary rabbit polyclonal antibody against chicken ADAM23 were used. 39.4D5 was obtained from the Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa, USA. Antibody against ADAM23 was produced by a custom service from a commercial company (Eurogentec, Seraing, Belgium). This antibody was generated to two portions (aa 198-213 and aa 715-729) of chicken ADAM23 (NP_001138702) and had a high titer by ELISA test. Preincubation of the antibody with its corresponding immunizing peptide abolished the specific bands measured by Western blot (data not shown).

Fluorescent double-labeling immunohistochemistry was performed on cryosections according to the method described previously (Luo et al.,2007). Briefly, after postfixation with 4% formaldehyde, cryosections were preincubated with blocking solution (5% skimmed milk, 0.3% Triton X-100 in TBS) at room temperature for 60 min. The sections were then incubated overnight at 4°C with antibody against ADAM23, followed by Alexa-488-labeled secondary antibody against rabbit IgG (Molecular Probes, Eugene, OR). After another incubation with blocking solution, the sections were incubated with antibodies against Islet-1 overnight at 4°C. Subsequently, Cy3-labeled secondary antibody against mouse IgG (Dianova, Hamburg, Germany) was applied. Finally, cell nuclei were counterstained with DAPI (Sigma). Fluorescence was imaged under a fluorescent microscopy system (BZ-8000; Keyence Deutschland GmbH, Neu-Isenburg, Germany).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Dr. E. Mix for critical reading of the manuscript, and Ms. S. Schreiber for technical assistance. J.L. was funded by a grant from the German Research Foundation (DFG).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES