Developmental Brain Research 136 (2002) 43–49www.elsevier.com/ locate /bres

Research report

Expression of nerve growth factor during the development of nervoussystem in early chick embryo

a , b*Shobha Bhargava , S.P. ModakaMolecular Embryology Laboratory, Department of Zoology, University of Pune, Pune 411 007, India

bDepartment of Zoology, Karnatak University, Dharwad, India

Accepted 8 March 2002

Abstract

In the chick gastrula, nerve growth factor (NGF) is localized to the endoblast mesoblast presumptive head ectoderm but not in thepresumptive neuroectoblast. During early morphogenesis the dorsal body ectoderm presumptive neural crest cells exhibit strong NGFpositive cell surface reaction. NGF appears to be a marker of cells participating in morphogenetic movements but not early neuraldifferentiation. NGF is localized where neural folds fuse and cells die allowing detachment of the neural tube from head ectoderm as wellas in dead cells in the neurocoele. NGF reactivity in cells lining the evaginated extremities of the optic vesicle the floor of the neural tubethe splanchnopleure heart primordia the inner outer surfaces of somites is suggestive of the role of NGF in primitive organ shaping. 2002 Published by Elsevier Science B.V.

Theme: Development and regeneration

Topic: Neurotrophic factors: expression and regulation

Keywords: Nerve growth factor; Neurogenesis; Cell death

1. Introduction population of neurons in the peripheral nervous system [8],control of neuroblast proliferation in the inner ear [37],

During early development of the nervous system, local- dorsal root ganglion [11] and continued survival ofized cell population growth, cell rearrangements and cell horizontal cells in developing avian retina [20]. Treatmentdeath are involved in shaping both structurally and func- with NGF promotes cell proliferation in embryonic pri-tionally discrete regions of the brain [40]. Morphogenetic mary sympathetic ganglion (PSG) suggesting that NGFcell death is a necessary process during embryonic de- acts as a mitogen [3] and reduces the level of apoptosisvelopment [12] and occurs during the formation of the [13]. NGF also appears to be a target-derived survival orneural tube [18] lens vesicle [27] and digit formation in differentiation factor for neural crest cells of the sympatho-developing limb buds [4,5]. Temporally and spatially adrenal lineage [13].defined cell death signals the end of histogenesis in NGF binds to two different receptors, namely, tryosineterminally differentiating lens fibres [1,23–26,28,29,31] kinase (trkA) and p75NTR or neurotropin [8]. trkA is aand keratocytes [30]. Immediately after birth, excess high affinity preferential receptor of NGF and associatedneurons in specific regions of mouse brain die to ensure with the survival promoting activity of NGF in neuralcorrect region-specific patterning [32]. cells. While p75NTR acts as a non-catalytic receptor [8],

Nerve growth factor (NGF), a glycoprotein with two the formation of a receptor–ligand complex creates highidentical 13-kD subunits has been ascribed a wide variety affinity binding sites for NGF and the activation of trkAof roles during development and differentiation [21]. These leads to an anti-apoptotic signal that dominates over anyinclude prevention of programmed cell death in specific proapoptotic signal generated by p75NTR [41]. In the

absence of trkA, NGF causes neuronal cell death throughthe activation of p75NTR [8]. Thus, the dual and opposite*Corresponding author.

E-mail address: shobhabhargava@hotmail.com (S. Bhargava). role of NGF is explained by its unique property of binding

0165-3806/02/$ – see front matter 2002 Published by Elsevier Science B.V.PI I : S0165-3806( 02 )00346-2

44 S. Bhargava, S.P. Modak / Developmental Brain Research 136 (2002) 43 –49

and activation of p75NTR [9]. Furthermore, NGF/p75ntr 0.02% H O dissolved in 0.05 M Tris (pH 7.2). Slides2 2

control the cell number [9]. Biologically active NGF is were rinsed in tap water (5 min), dehydrated in alcohol,present in chick embryos [2] and expressed in the central cleared in xylene and mounted in Permount or DPX. Bothnervous system during mid and late development in ovo fixatives were equally effective for immunohistochemical[6,7]. In this manuscript we report, for the first time, the detection and localization of NGF. Control sections werelocalization of NGF in cells involved in morphogenetic incubated with PBS without the primary antibody.cell death and shaping of the neural tube and bodyectoderm during early chick development.

3. Results

2. Materials and methods 3.1. Primitive streak stage

Fresh fertilized White Leghorn chicken eggs were At stage 4, the NGF specific immunoreaction is firstincubated at 37 8C. Freshly laid white Leghorn chick detected in the area pellucida stage (Fig. 1A–C) andembryos was staged according to Hamburger and Hamilton localized to the cell surface of invaginated mesoblast and[16]. Early chick embryo undergoes gastrulation during the cytoplasm in invaginated embryonic endoblast underly-stages 1–4. By stage 5, the head process (presumptive ing the primitive streak. In contrast, the ectoblast in andnotochord) is laid down and induces the overlying ec- around the primitive streak and the presumptive neuro-toderm to neuralize and form the neural plate. Towards the ectoblast are NGF negative (Fig. 1A–C). Furthermore, theanterior end of the embryonic axis, neural plate and the presumptive head ectoderm and neural crest cells locatedunderlying endoderm begin to fold ventro-posteriorly at the anterior margin of the area pellucida show stronggiving rise to the head fold which is the precursor of the reaction.. We did not find NGF positive reaction in earlyforegut formation at stage 6. Between stages 7 and 8 the primitive streak stages 1–3.neural plate begins to fold dorsally in the anterior regionwhere the neural folds meet and fuse to form the forebrain 3.2. Head fold stage 6(stage 9) leaving behind the neuropore at the anterior endand open neural folds near the hindbrain and spinal cord. At stage 6 the ectoderm in the head fold exhibits strongBetween stages 10 and 12, the brain is fully closed and NGF positive reaction. Barring a few faintly positive cellsfurther regionalizes into fore, mid and hindbrain with the in the medullary plate, most neuroectoblast cells wereformer giving rise to optic vesicles as latero–posterior NGF negative. The presumptive head ectoderm and neuraloutgrowths. Embryos at the required developmental stage crest cells located immediately surrounding the neuralwere harvested by the filter paper ring technique [33], plate, as well as the embryonic endoblast are strongly NGFwashed and fixed for 24 h in Bouin’s fixative, or 4% positive (Fig. 1E). We also find NGF positive reaction inparaformaldehyde (in 0.1 M phosphate buffer (PB), pH yolk platelets and vesicles in the yolk endoderm (Fig. 1D).7.4, on ice). Embryos were washed to remove the fixatives,dehydrated, embedded in Paraffin and serially sectioned (6 3.3. Neurulationmm). Sections were mounted on clean glass slides anddeparaffinized. Sections on slides were treated with 0.3% At stages 7 and 8, the overall staining pattern resemblesH O in methanol for 40 min to deactivate endogenous that for stage 6. Cells at the center of the anterior2 2

peroxidase activity, washed in 0.01 M phosphate buffered medullary plate and the presumptive floor of the brainsaline (pH 7.5, 20 min), incubated for 30 min in PBS exhibit moderate membrane-associated NGF positive re-containing BSA (0.5%), gelatin (0.5%) and 0.1% NaN action while the adjoining head ectoderm including pre-3

and washed in PBS (10 min). Sections were then treated sumptive neural crest exhibit strong cytoplasmic reactionwith goat serum (1:40 dilution, 30 min, room temperature, (Fig. 2A and B). Again at the edge of the area pellucida,Vectastain, Burlingame, CA, USA). After blotting off the presumptive body ectoderm contiguous with the headexcess serum, sections were incubated (16 h, room tem- ectoderm is strongly NGF positive (Fig. 2C). The headperature) in a humid chamber with rabbit antisera to 2.5s mesenchyme (Fig. 2A and B) and splanchnopleure alsoNGF (Sigma, USA). The optimal dilution of anti-NGF exhibit NGF positive cell surface (Fig. 2C).antibody was found to be 1:2000 and used in all experi- At stages 9 –10, converging neural folds fuse and neuralments. Slides were washed in PBS (10 min) and treated in cells near fusion sites exhibit strong positive cytoplasmicbiotinylated goat anti-rabbit antibody (30 min), followed or nuclear reaction (Fig. 3C, E and F). In the neural tubefirst by a 10-min wash in PBS and then incubation (16 8C, lumen we detect highly NGF positive dead cells squeezed40 min) with ABC reagent (1:100 dilution, Vectastain, out of the crowded sites at the roof of the neural tube (Fig.Burlingame, CA, USA). After three washes in PBS (10 3E and F). Similarly, the head mesenchyme and areamin each), sections were reacted in the dark for 5–6 min pellucida endoderm are moderately positive. In the anteriorwith 0.05% 3,39-diaminobenzidine tetrahydrochloride and region of the developing neural tube, we notice strongly

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Fig. 2. Transverse sections (A, B, C) of head fold stage 7–8 chickembryo showing a strong NGF positive reaction in the presumptive headectoderm (phe) and presumptive neural crest (pnc) cell and the headmesenchyme (mc). Few faintly NGF positive cells are seen on the floor of

Fig. 1. Transverse sections of chick embryo of primitive streak stage medullary plate (mp) and extraembryonic ectoblast. Foregut (fg) is NGF(A–D) and stage 6 (E) showing NGF positive reaction (arrow) on the cell negative. Discernible reaction is seen along the midline of splanchnop-surface of the invaginated embryonic endoderm (ee) and mesoblast (m), leure (sp) nf, neural fold.(A, B,C). NGF positive granules (big arrow head) are seen in the yolkendoderm (D). Presumptive body ectoderm (pbec), and presumptiveneural crest (pnc), cells lateral to the neural plate margin and embryonic

At stages 11 and 12, NGF positive cells are abundant inendoblast (ee), immediately below the medullary plate (mp), show strongthe telencephalon, surround the neuropore (Fig. 4A,C andNGF reaction (E). ec, ectoblast; pg, primitive groove; ps, primitive streak.

D) and become restricted posteriorly to the roof of thebrain where it detaches from the head ectoderm. NGF

NGF positive cells surrounding the anterior neuropore positive cells are also located at the evaginated extremities(Fig. 3D) with cell surface or nuclear reaction while those of optic vesicles (Fig. 4B and E). Furthermore a diffusein the medullary plate are moderately positive (Fig. 3A and cytoplasmic reaction appears in the cells lining the floor ofB). The entire head ectoderm (Fig. 3A–E) and head the forebrain (Fig. 4E). It is notable that the neurocoelemesenchyme (Fig. 3C and E) show a strong cytoplasmic contains strongly NGF positive dead cells (Fig. 4B). AsNGF positive reaction while a strong NGF positive one examines the posterior neural tube, elongated NGFreaction is associated with the endo-mesodermal surface of positive cells, initially located at the roof of the brain in thethe splanchnopleure (Fig. 3C). mid and hindbrain, (Fig. 4F and G), become gradually

46 S. Bhargava, S.P. Modak / Developmental Brain Research 136 (2002) 43 –49

restricted to the middorsal line with NGF positive surface crest cells are again NGF positive. The head mesenchymeof the neural tube lumen. In contrast, the spinal cord cells is moderately NGF positive with diffuse cytoplasmicappear negative to anti-NGF serum (Fig. 4 H and I). The staining (Fig. 4F), while at the surface splanchnic endo-head ectoderm, body ectoderm and presumptive neural mesoderm strong NGF positive reaction continues to

manifest at the cell surface (Fig. 4H) just as at the outerand inner linings of the somites (Fig. 4I). In contrast, thepresumptive trunk ectoderm continues to exhibit strongNGF positive reaction.

Control reaction in sections treated with either primaryor secondary antibody alone followed by avidin–biotin andH O were NGF negative (not shown).2 2

4. Discussion

NGF positive cells were found in the invaginatedendomesoblast under the primitive streak in the areapellucida of full gastrula and early neurula of chickembryo, but not earlier. Similarly, presumptive head andbody ectoderm and presumptive neural crest exhibit strongNGF positive reaction while the presumptive or definitiveneuroectoblasts do not. Thus, NGF appears to play a rolein the ectoderm involved in body shaping but not in earlyneural differentiation. The NGF positive reaction in in-tracytoplasmic storage vesicles and yolk platelets in thevitelline endoderm suggests that NGF may also be a storedmaternal product.

By stages 6 and 8, NGF is localized primarily in thehead fold ectoderm, and appears first in few positive cellsalong the midline of the medullary plate. It is striking thatNGF-staining becomes extensive in neural cells whereneural folds converge, meet and fuse with a concomitantappearance of dead cells in the lumen of neural tube. Thisis in agreement with earlier report [17] on NGFR mRNAduring avian neural tube closure. Indeed, till stage 13,NGF-positive cells are located distinctly at the point ofjunction of the neural folds as well as the roof of the brainalong the midline. Thus, our results extend the earlier data[35,36] showing dorso-ventral distinction for b-NGF re-ceptor expression in developing dorsal root ganglia andbrain. Furthermore, appearance of NGF at the constrictionat the junction between the optic vesicle and the fore brain(Fig. 4E), as well as the tip of the optic vesicle, prior to itsinvagination to form the optic cup, suggests that cells

Fig. 3. Transverse sections of early neurula (stage 9, A–C) and mid-neurula (stage 10, D–F). The invaginating neural plate contains NGFpositive cells while presumptive head ectoderm (hec) and body ectoderm(bec) are strongly NGF positive (A, B). The head mesenchyme (hmc) isNGF positive (C,E). In stage 10 embryo, NGF positive reaction islocalized in the brain neuroblasts (nb), preferably along the roof of thebrain (D–F) where lateral head folds meet. Most of the neural tube (nt)floor is NGF negative excepting the ventral lining of the neurocoele (nc)(arrow). Dead cells (arrow head) at the roof of the neural tube are highlyNGF positive (E,F). Notochord (n), cells are uniformly NGF negative. t,telencephalon; m, mesenchyme; nf, neural fold; sp, splanchnopleure; fg,foregut.

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Fig. 4. Transverse sections of stage11 late neurula (A,B) and stage 12 embryo (C–I). The roof (r) of the telencephalon (t) and diencephalons containsneuroblasts (nb) with strong NGF positive intracellular reaction, while the surface of the neurocoele is mildly NGF positive. A few NGF positive cell debrisis also seen (B) (arrow head) in the neurocoele (nc). At stage 12 the entire telencephalon (t) contains NGF positive neuroblasts (nb) while posteriorwards,the NGF is localized in the roof (r) of the diencephalon along with the outer extremities of the optic vesicles (ov) as well as a smattering of cells along themidline of the brain floor (f) (C–E). In the hind brain (F) positive reaction is restricted to the roof (r) and floor (f) along the midline. The headmesenchyme (hmc), head ectoderm (hec) and somites (s) are mildly NGF positive (F,G). Notice strong NGF positive reaction along the endo-mesoblasticjunction in the splanchnopleure (sp), (H) as well as the heart (h) lining (G). In the posterior region of the stage 12 embryo (I), NGF positive reaction isseen in the body ectoderm (bec), lateral plate mesoblast (lpm), and the outer and inner lining of somites (s). Throughout the development, notochord (n)does not show a NGF positive reaction.

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