I. Sites of serotonin uptake and -binding protein immunoreactivity in the
midgestation mouse embryo
J. M. LAUDER1, H. TAMIR2 and T. W. SADLER1
1 Department of Cell Biology and Anatomy, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA2Department of Neuroscience, New York State Psychiatric Institute, New York, NY 10032, USA
Summary
The possible involvement of the neurotransmitterserotonin (5-HT) in morphogenesis of the craniofacialregion in the mouse embryo has been investigatedusing the method of whole-embryo culture. Day-12embryos were incubated for 3-4 h in the presence of5-HT or its precursors L-tryptophan (L-TRP) or5-hydroxytryptophan (5-HTP), followed by fixation,sectioning and staining with a specific antiserum to5-HT. Sites of 5-HT immunoreactivity were found in avariety of locations in tissues of the head and neck,which are either epithelia derived from the non-neuralectoderm or are non-neuronal midline brain struc-tures. These sites include the surface epithelia of thehead, face, nasal prominences, branchial arches, oralcavity and associated parts of the nasal epithelium, theepithelium covering the eye, parts of the otic vesicle,the epiphysis and roof of the diencephalon. With theexception of the oral cavity, sites of immunoreactivity
for serotonin-binding protein were identified in themesenchyme adjacent to these sites. This mesenchymeconsists of ectodermally derived neural crest cells,which are known to receive inductive influences fromthe epithelia with which they interact during theirmigration through the craniofacial region. The pres-ence of 5-HT uptake sites in epithelia and adjacentsites of SBP in the underlying mesenchyme raises thepossibility that 5-HT might be involved in thoseepithelial-mesenchymal interactions known to be im-portant for the development of structures in thecraniofacial region.
Morphogenesis is the process whereby cell move-ments, changes in cell shape and cell-cell interactionsresult in the formation of tissues and organs in theembryo. The idea that classical neurotransmitters,such as 5-HT, the catecholamines or acetylcholinemight be involved in the control of morphogenesishas arisen from several lines of evidence, beginningwith the work of Buznikov and co-workers in the1960s, who demonstrated the presence of these sub-stances in the sea urchin embryo during cleavage andgastrulation (Buznikov et al. 1964, 1968; reviewed byBuznikov, 1980, 1984). Subsequent studies in whichvarious neurochemicals were applied to sea urchin
embryos led to the suggestion that dopamine, acetyl-choline and 5-HT (or related substances), acting atintracellular binding sites, are important triggers forcleavage divisions and particular phases of gastru-lation, such as the migration of primary mesenchymeand the onset of coelomic contractile activity (Buz-nikov etal. 1970; Buznikov & Shmukler, 1981; Deeb,1972; Gustafson & Toneby, 1970, 1971; Toneby,1911b; Manukhin et al. 1981; Markova et al. 1985;Renaud et al. 1983. See also reviews by Buznikov,1980 and Toneby, 1977a).
Similarly, in the vertebrate embryo, neurotrans-mitters appear to be present early in development(Baker & Quay, 1969; Burden & Lawrence, 1973;Ignarro & Shideman, 1968a,b). For example, specific
710 J. M. Lauder, H. Tamir and T. W. Sadler
regions of the neural tube and notochord of neurulat-ing chick and amphibian embryos have the capacity toaccumulate 5-HT or catecholamines (Allan & New-green, 1977; Godin, 1866; Godin & Gipouloux, 1986;Kirby & Gilmore, 1972; Lawrence & Burden, 1973;Newgreen et al. 1981; Rais et al. 1981; Sako et al.1986; Sims, 1977; Wallace, 1982) and the notochordand yolk have been identified as probable sites ofsynthesis of these substances (Emanuelsson, 1974;Godin, 1986; Ignarro & Shideman, 1968a; Strudel etal. 1911a,b; Wallace, 1982; Wallace et al. 1985).Accumulation of 5-HT in some regions of the chickembryo has characteristics similar to the high affinityreuptake system present in adult 5-HT neurones(Azmitia & Markovitz, 1980; Dreyfus et al. 1977;Wallace, 1982). Also, neurochemical disruption of5-HT synthesis and/or uptake in the chick embryoresults in neural tube defects and other teratologies ofthe neuraxis (Palen et al. 1979) suggesting that 5-HTmay be important in controlling morphogeneticmovements related to gastrulation and neurulation.
At present, no information is available concerningthe presence of sites for uptake or synthesis of 5-HTor catecholamines in the mammalian embryo duringorganogenesis. However, Burden & Lawrence (1973)reported that at the 4-cell stage, the blastomeres ofthe rat embryo have uptake mechanisms for thesesubstances, and Schlumpf & Lichtensteiger (1979)demonstrated that the visceral yolk sac of the ratembryo can synthesize catecholamines. Moreover, anumber of pharmacological studies have indicatedthat 5-HT, L-TRP or tricyclic antidepressants (par-ticularly those that act through serotonergic mechan-isms) can cause malformations of the skull, brain,spinal cord or vertebral column in rodents andhumans (Guram et al. 1980, 1982; Idanpaan-Heikkila& Saxen, 1973; Jurand, 1980; Van Cauteren et al.1986).
Recently, we have undertaken studies to determinewhether 5-HT may play a role in the control ofmorphogenetic cell movements in the mouse embryo(Lauder et al. 1987; Lauder & Zimmerman, 1988;Zimmerman & Lauder, 1987). For this purpose, themethod of whole-embryo culture was employedtogether with immunocytochemistry with a specific5-HT antiserum to analyse sites of uptake and/orsynthesis of 5-HT in mouse embryos grown in thepresence of 5-HT or its precursors. In addition, aspecific antiserum to the purified 45K form of seroto-nin-binding protein (SBP; Kirschgessner et al. 1987;Liu et al. 1985, 1987; Tamir, 1983) has been used tostudy possible sites of 5-HT binding in these embryos.This acidic protein binds 5-HT with high affinity(Tamir & Huang, 1974), acts as a storage protein,much like chromogranin, which binds 5-HT in synap-tic vesicles (Gershon et al. 1983; Tamir & Gershon,
1979) and is released together with 5-HT duringexocytosis (Jonakait et al. 1979). Once in the extra-cellular milieu, 5-HT dissociates from SBP, an eventwhich may be modulated by such molecules as ionsand gangliosides (Tamir & Liu, 1982; Tamir et al.1980). In the present study, we have identified sites ofSBP-like immunoreactivity in specific regions of mes-enchyme which are directly adjacent to sites of 5-HTuptake in epithelial structures of the craniofacialregion and in certain dorsally located midline struc-tures of the developing brain. These 5-HT uptake andbinding sites may have functional significance formorphogenesis of the craniofacial region and brainduring mouse embryogenesis.
Materials and methods
Whole-embryo cultureEmbryos from timed pregnant ICR mice were culturedusing the technique of New (1978), as modified by Sadler(Sadler, 1979; Sadler & New, 1981). In brief, embryos(45-48 somites) were removed from mothers on day 12 ofgestation (plug day = day 1), and dissected free fromuterine and placental tissues with their surrounding mem-branes and ectoplacental cones intact. The visceral yolk sacand amnion were opened to facilitate diffusion of nutrientsto the conceptus (Cockcroft, 1973). Each embryo was thenplaced in a 30ml sterile, disposable flask containing 2-25 mlof freshly centrifuged rat serum, which had been heatinactivated at 56°C for 30min, and 0-75 ml of Tyrode'ssolution (3:1 v/v). Flasks were gassed with a mixture of95% O2, 5% CO2, tightly stoppered and placed on arotator wheel (30revsmin~') at 38°C at the beginning ofthe treatments described below. Before beginning thetreatment period, viability was monitored by the presenceof circulating blood cells in the visceral yolk sac and a rapidheart beat, and nonviable embryos were discarded. A fewembryos were removed from mothers at day 14 andprocessed directly for immunocytochemistry with anti-SBPas described below.
Treatment of embryos with 5-HT and relatedsubstancesEmbryos were cultured in the presence of substances listedin Table 1 at the concentrations indicated. When themonoamine oxidase inhibitor nialamide, the antioxidant L-cysteine or the 5-HT uptake inhibitor fluoxetine were used,embryos were exposed to these agents for 1 h prior to a 3 hperiod of incubation in 5-HT or its precursors, 5-HTP orL-TRP. When nialamide, L-cysteine or fluoxetine were usedwithout 5-HT or precursors, embryos were exposed to themfor a total of 4h. Control embryos cultured in the absenceof any additives were also incubated for a period of 4h.Cultures were terminated by removing embryos from theirroller bottles, rinsing them in Tyrode's and placing theminto cold 4% paraformaldehyde in 0-67 M-phosphatebuffer.
Serotonin and morphogenesis 711
Table 1. Non-neuronal sites of 5-HT uptake in the day-12 mouse embryo following culture in the presence of5-HT or related substances
Treatment
5-HT+NC(10- 5 /10 - 7 M)
5-HTP+NC(10- 5 /10" 7 M)L-TRP+NC(10^ 5 /10- 6 M)
NC
cCult, only5-HT+C(10~5M)
5-HTP+C(10" 5 M)L-TRP+C(10" 5 M)
5-HT+NC+F(10" 5 M)
5-HTP+NC+F(10" 5 M)
L-TRP+NC+F(10" 5 M)
NC+F
Head
+ +/ ++ +/ +
++000000000
Location and relative
Face/arches
++/+++/ +
++000000000
Oral cav.
+ +/ ++ +/ +
++000000000
intensity of immunoreactivity
Eye
+
(+)(+)(+)
000000000
Otocyst
+ +/ ++ +/ +
++000000000
BrainRoof dien/epiphysis
+ +/0000000000000
Total (n)
141268335443443
NC, nialamide+L-cysteine (10 5M) 1 h prior to treatment (3h); C, L-cysteine (10 5M) 1 h prior to treatment; L-TRP, L-tryptophan; 5-HTP, 5-hydroxytryptophan; F, fluoxetine (10"3M) 1 h prior to treatment; Oral cav., oral cavity and parts of nasal epithelium;face/arches, face and branchial arches 1 & 2; dien, diencephalon. Intensity of immunoreactivity was rated on a relative scale of 0(none) to ++ (very intense); (+) = less intense than + (10~5M).
Tissue processingEmbryos were fixed for 1-2 h in cold fixative, then rinsedseveral times in cold phosphate-buffered saline (PBS).Embryos were dehydrated in an ascending series of etha-nols, followed by toluene, and infiltrated with Paraplast-Plus using a Tissuematon. Using a Tissuetek embeddingstation, embryos were oriented in moulds filled with hotParaplast-Plus and placed on a cold plate to solidify. Serialsections were cut at 10 j*m using a rotary microtome andmounted on uncoated glass slides. Sections were baked at45 °C for 2-3 days to adhere them to the slides prior toperforming immunocytochemistry.
ImmunocytochemistrySections were deparaffinized in xylene, then rehydratedthrough a descending series of ethanols to PBS. Sectionswere immunostained using either anti-5-HT (diluted1:5000), an antiserum raised against formaldehyde coupled5-HT-haemocyanin conjugates, which is specific for thelocalization of 5-HT in paraformaldehyde-fixed tissues(Wallace et al. 1982), or anti-SBP 45K (diluted 1:100), anantiserum raised against the purified 45K form of SBP (Luiet al. 1985), which has recently been characterized (Kirch-gessner et al. 1987; Liu et al. 1987). The staining protocol(Towle et al. 1984) was a modification of the avidin-biotinperoxidase method of Hsu et al. (1981). Prior to applicationof primary antiserum, sections were incubated in trypsin(0-6mgml~' in PBS) for 2min to expose antigenic sites,then washed in PBS and coated with 2 % normal sheepserum. Sections were incubated in primary antiserum for48 h at 4°C in a moist chamber, followed by exposure tobiotinylated secondary antiserum for 2h at room tempera-ture. Slides were washed in PBS, then incubated in avidin--biotinylated peroxidase complex (ABC, Vector Labs) for
2h. Following additional PBS rinses, the diaminobenzidine(DAB) reaction was performed for lOmin, slides wererinsed in Tris buffer followed by PBS, then exposed toosmium vapours for 5min. Sections were washed in PBS,counterstained with toluidine blue, dehydrated in ethanolsand xylene, and coverslipped in Permount.
Analysis of immunoreactive sitesImmunostained transverse sections through the head andneck of embryos were viewed with a Leitz Orthoplanmicroscope at magnifications ranging from x63-160. Sitesof immunoreactivity for 5-HT were identified in treatedembryos and rated on a semiquantitative scale according tothe intensity of staining: 0, no staining; + + , very intense(see Table 1). Regions of SBP-like immunoreactivity werealso identified in sections adjacent to those where 5-HTimmunoreactivity was found (Table 2), as well as in nonad-jacent sections from untreated animals (Fig. 3). Immuno-reactive sites of both types were photographed and com-pared in terms of their locations (see Figs 1,2).
Results
Sites of serotonin uptake in epithelia and brainFollowing incubation of embryos in medium contain-ing 5-HT together with the monoamine oxidaseinhibitor nialamide and the antioxidant L-cysteine(Table 1), a number of sites of 5-HT immunoreac-tivity were observed in the head and neck, as shownin Figs 1, 2A,C,E,G). All of these sites were locatedin epithelia, including the surface epithelia of thehead, face, nasal prominences, branchial arches, oralcavity and parts of the nasal epithelium, the epithelial
712 J. M. Lauder, H. Tamir and T. W. Sadler
Fig. 1. Sites of 5-HT uptake (arrows) in the surface epithelium of the head, nose, jaw and mouth (left panels)visualized using anti-5-HT immunocytochemistry compared to the distribution of immunoreactivity for SBP in nearbymesenchyme on adjacent sections (right panels). (A,B) Head at the level of the trigeminal ganglion (5th cranial nerve;Kg). Bars, 180urn. (C,D) Nose (mn, In, medial and lateral nasal prominences). Bars, 180^m. (E,F) Jaw (md,mandibular arch; mx, maxillary process; Vn, trigeminal nerve, 5th cranial nerve). Bars, 280//m. (G,H) Mouth (oc, oralcavity; ns, nasal septum; n, nasal cavity; open arrow, continuity between nasal epithelium and oral epithelium). Bars,500^m. All sections are in the transverse plane.
Serotonin and morphogenesis 713
covering of the developing eye and parts of the oticvesicle. In addition, there were also two dorso-medially located regions in the developing brain: theroof of the diencephalon and the epiphysis (pineal),which exhibited 5-HT immunoreactivity (Fig. 2E,G).With the exception of the brain sites, all of these areashad characteristics similar to sites of 5-HT reuptakefound in mature 5-HT neurones (Azmitia & Marko-vitz, 1980; Dreyfus et al. 1977), since they could beseen after incubation of embryos with 5-HT at con-centrations in the high affinity range (10~7 M) andtheir appearance was prevented by fluoxetine(Table 1), a specific inhibitor of neuronal 5-HT up-take (Wong et al. 1974). Moreover, the amount ofaccumulated 5-HT appeared to increase in a dose-dependent manner at these sites as the concentrationof 5-HT in the medium was increased. In the brain,however, neither of the dorsomedial sites could beseen with low concentrations of 5-HT, even thoughtheir appearance after higher concentrations of 5-HTwas blocked by fluoxetine.
As shown in Table 1, these sites have been charac-terized in terms of their ability to synthesize 5-HT, byexposing embryos to 5-HT precursors with or withoutfluoxetine, followed by immunocytochemical analysisto assess the appearance of 5-HT immunoreactivity inthe regions described above. With the exception ofthe brain sites, all of these regions could be visualizedfollowing incubation of embryos in medium contain-ing one of the precursors of 5-HT: 5-HTP or L-TRP,
together with nialamide and L-cysteine. Moreover,these sites could be seen when nialamide and L-cysteine were added to the medium without thesesubstances, but not when embryos were cultured inthe absence of nialamide or 5-HT precursors.
Of the two 5-HT precursors, the intensity ofimmunoreactivity was greatest with 5-HTP, andincreased in a dose-dependent manner as the amountof 5-HTP was increased, whereas the amount ofstaining seen with L-TRP was not greater than thatobserved following incubation with nialamide and L-cysteine alone, even at the highest concentration of L-TRP. However, when either these precursors ornialamide and L-cysteine alone were administeredtogether with fluoxetine, no 5-HT accumulation wasobserved (Table 1).
Another characteristic of this system is the abilityto rapidly metabolize 5-HT, either in the embryo,associated structures (e.g. the yolk sac or ectoplacen-tal cone) or the medium, since without nialamide nodetectable accumulation of 5-HT was seen, even inthe presence of 5-HT (Table 1).
Localization of serotonin-binding protein-likeimmunoreactivityImmunocytochemistry with an antiserum to the 45Kform of purified SBP (Kirchgessner et al. 1987; Liu etal. 1985, 1987) performed on sections from treatedand untreated embryos revealed sites of SBP-likeimmunoreactivity in specific regions of the cranio-facial mesenchyme. As shown in Table 2 and Figs 1,2B,D,F, many of these sites were located in closeproximity to sites of 5-HT uptake in adjacent epi-thelia or brain structures. In fact, with the exceptionof the oral cavity and associated parts of the nasalepithelium, where no mesenchymal SBP immuno-reactivity was seen (Fig. 1H), all of the observed sitesof 5-HT uptake were located next to regions of SBPimmunoreactivity in the underlying mesenchyme,with which they generally seemed to be in closecontact. However, there were other regions of SBPimmunoreactivity which were not located in proxim-ity to sites of 5-HT uptake. These sites included theperinotochordal sheath and surrounding mesen-chyme (Fig. 3A,B), portions of the caudal somites(Fig. 3A), the mesenchyme surrounding the brain(part of which overlies the roof of the diencephalonand epiphysis; Fig. 2F,H) and the meningeal coveringof the fourth ventricle (Fig. 3C). In the developingeye, regions of SBP-like immunoreactivity were ob-served in the mesenchyme surrounding the lens(Fig. 2B), both adjacent and nonadjacent to theoverlying epithelial site of 5-HT uptake.
Investigations in older embryos (day 14) showedthat the sites of 5-HT uptake observed at day 12 hadlargely disappeared, except for remnants in the oralcavity (e.g. the palatal shelves; Lauder & Zimmer-man, 1987; Zimmerman & Lauder, 1987) and that theregions of SBP immunoreactivity in the mesenchyme
Table 2. Comparison of non-neuronal sites of 5-HTuptake in epithelia and brain to sites of SBP-likeimmunoreactivity in adjacent mesenchyme of the
Embryos were cultured in nialamide+L-cysteine (10 5M) 1 hprior to treatment with 5-HT (10"5M) for 3h. Figs 1, 2 illustratethe appearance of these sites. Abbreviations and rating scale asindicated in Table 1.
714 /. M. Lauder, H. Tamir and T. W. Sadler
ed
Serotonin and morphogenesis 715
were no longer present. However, prominent SBPimmunoreactivity was seen in regions of chondrogen-esis in the nasal septum, maxillary and mandibularprocesses (Fig. 3D-F). This finding suggests that atleast some of the SBP immunoreactive mesenchymalcells seen at day 12 may have differentiated intochondrocytes.
Discussion
Sites of 5-HT uptake or synthesis in epithelia andbrainUsing whole-embryo culture in the presence of5-HT or its precursors, together with immunocyto-chemistry, a number of non-neuronal sites of 5-HTuptake have been identified in the day-12 mouseembryo. All of these sites are located in epithelia ofthe head and neck or in non-neural midline structuresof the developing brain. The characteristics of theepithelial sites are different from those in brain, sincethey are seen after incubation of embryos withconcentrations of 5-HT in the high-affinity range (e.g.10~7 M) or with 5-HT precursors, and are blocked byfluoxetine (a specific inhibitor of 5-HT neuronaluptake; Wong et al. 191 A). Together, these obser-vations suggest that the epithelial sites are similar tosites of high-affinity uptake in serotonergic neurones.The brain sites, however, appear to be of the low-affinity type, since they are only seen after 5-HT isadministered at 10~5 M, even though their appearancecan be blocked by fluoxetine. They also differ in thesense that they are not seen after exposure of em-bryos to 5-HT precursors.
Both the epithelial and brain sites share the com-mon property of being visible only if the monoamineoxidase inhibitor nialamide is present in the medium,suggesting that 5-HT may be rapidly metabolized atthese sites, or elsewhere in the embryo, associated
Fig. 2. Sites of 5-HT uptake in epithelial structures of theeye, ear and brain (left panels) visualized using anti-5-HTimmunocytochemistry compared to the distribution ofimmunoreactivity for SBP in nearby mesenchyme onadjacent sections (right panels). (A,B) Eye (In, lensvesicle; nr, neural retina; arrow, 5-HT immunoreactivityin the surface epithelium of head). (C,D) Ear (otocyst);solid arrow, 5-HT immunoreactivity in the surfaceepithelium of head; clear arrow, 5-HT immunoreactivityin vestibular portion (v) of the otocyst (o); c, cochlearportion of otocyst; ed, endolymphatic duct. Note co-distribution of 5-HT immunoreactivity in otocyst and SBPimmunoreactivity in adjacent mesenchyme. (E-H) Brain.(E,F) roof of diencephalon/3rd ventricle (Hi); arrowindicates site of 5-HT immunoreactivity and hippocampalanlage (h); Iv, lateral ventricle. (G,H) Epiphysis (ep)with 5-HT immunoreactivity (arrow); Hi, 3rd ventricle.All sections are in the transverse plane. Bars, 180^m.
structures (e.g. the yolk sac, ectoplacental cone) orculture medium. This finding is consistent with theresults of previous studies demonstrating that mono-amine oxidase activity is especially high during em-bryonic development in the mouse (Baker et al.1974). Thus, nialamide could be acting to elevateendogenous levels of 5-HT in the embryo. However,since significant levels of 5-HT have been measured inthe heat-inactivated rat serum used in the culturemedium (Tamir, unpublished results), which has alsobeen shown for fetal calf serum, Agrez et al. (1984),we must consider the possibility that nialamide isacting to prevent breakdown of endogenous stores of5-HT in the culture medium.
The ability to visualize the epithelial sites of 5-HTuptake with precursors involved in 5-HT synthesis (orby raising endogenous levels of 5-HT by inhibitingmonoamine oxidase with nialamide) could suggestthat these are sites of 5-HT synthesis as well asuptake. However, the appearance of these sites aftertreatment with 5-HT precursors or nialamide can beblocked by fluoxetine (Table 1), suggesting thatalthough endogenous 5-HT synthesis can be stimu-lated by such precursors, synthesis does not takeplace at the sites of 5-HT immunoreactivity. Rather,5-HT is probably synthesized in excess elsewhere andtaken up into these sites, an event which can beblocked by fluoxetine. Possible sites of 5-HT syn-thesis in the cultured mouse embryo include the fetalcirculation, the visceral yolk sac, ectoplacental coneor mast cells (which are beginning to be numerous inthe mesenchyme at 12 days of gestation). The amni-otic fluid is not a possibility since the yolk sac was tornopen at the beginning of culture.
Embryological origins and fates of epithelia andmesenchyme with sites of 5-HT uptake or bindingThe distribution of epithelial sites of 5-HT uptake inthe craniofacial region of the day-12 mouse embryohas been delineated and compared to sites in themesenchyme that stain with an antiserum to the 45Kform of SBP. In general, all of the epithelial struc-tures where sites of 5-HT uptake are found (Tables 1,2) are derived from the non-neural ectoderm (thatpart of the ectoderm not induced by the chordameso-derm; Vogt, 1929). In contrast, the mesenchymecontaining SBP immunoreactivity is formed from theneural crest (Johnston & Hazelton, 1972; Johnston,1974; LeLievre & LeDouarin, 1975; Nichols, 1986;Noden, 1984, 1986; Tan & Morriss-Kay, 1985) whichis of neurectodermal origin.
Despite these different origins, the two tissues maybe linked in important interactions in which 5-HT inepithelia and SBP in adjacent mesenchyme playimportant roles. This interaction may include theextracellular matrix, since previous studies suggest
716 J. M. Lauder, H. Tamir and T. W. Sadler
B
ns
n
cc
n
me
Fig. 3. Sites of SBP immunoreactivity in the day-12 (A-C) and day-14 (D-F) mouse embryo not associated with sites of5-HT uptake. (A) Region of the caudal neuropore (en). Arrows indicate SBP immunoreactivity surrounding thenotochord (no) and adjacent to the somites (s). Bar, 180nm. (B) Notochord adjacent to the brainstem showing SBPimmunoreactivity in the pennotochordal sheath (p) and mesenchyme (m). Bar, 120^m. (C) Region of the 4th ventricle(iv); arrow, SBP immunoreactivity in the meninges. Note immunoreactive floccular material in ventricle. Bar, 280^m.(D-F) SBP immunoreactive chondrogenesis sites in the nasal septum (ns), nasal capsule (nc), chondrocranium (cc) andjaw (me, Meckel's cartilage); n, nasal cavity; b, brain; f, tongue. Bars, 180\im. All sections are in the transverse plane.
Serotonin and morphogenesis 111
that binding of 5-HT to SBP is modulated by suchmolecules as gangliosides (Tamir et al. 1980). More-over, although SBP is primarily associated with 5-HTneurones in the adult brain, where it appears to serveas a storage protein for 5-HT in synaptic vesicles(Gershon et al. 1983; Tamir & Gershon, 1979), it isalso released into the extracellular space duringexocytosis. Indeed, the appearance of the SBP immu-noreactivity observed at the light microscopic level inthe present study suggests that it may be associatedwith the extracellular milieu of the craniofacial mes-enchyme, although this distribution must be exam-ined at the electron microscopic level before clearcutconclusions can be drawn.
Possible functional significance of adjacent 5-HTuptake and binding sitesThe importance of epithelial-mesenchymal interac-tions in development is well documented, especiallyin the craniofacial region where reciprocal interac-tions between mesenchyme and ectodermally derivedepithelia (Grobstein, 1967) appear to play importantroles in various aspects of morphogenesis (Benoit &Schowing, 1970; Holtfreter, 1968; Holtfreter &Hamburger, 1955; Horstadius, 1950; Noden, 1983).Specific examples of these relationships include(1) interactions between cranial neural crest mesen-chyme and surface epithelia of the head to initiatechondrogenesis leading to formation of the membra-nous bones of the skull and jaw (Bee & Thorogood,1980; Benoit & Schowing, 1970; Hall, 1980, 1981;Thorogood, 1981; Tyler & Hall, 1977); (2) inductionof the nasal placode by the mesenchyme to form thenasal rudiment (Yntema, 1955); (3) induction of themesenchyme surrounding the otocyst by the oticvesicle to form the otic capsule (McPhee & Van deWater, 1986; Holtfreter, 1968; Yntema, 1955);(4) interaction between mesenchyme and the eyevesicle leading to subsequent development of theoptic cup (Holtfreter, 1939) and (5) formation of thecorneal epithelium and stroma during interaction ofthe lens, mesenchyme and overlying epithelium(Holtfreter, 1968; Twitty, 1955).
In considering the possible significance of theresults of the present study, all of the above examplesof epithelial-mesenchymal interactions are relevant,since adjacently located sites of 5-HT uptake and SBPimmunoreactivity have been identified in all of theseregions. Moreover, in the case of formation of themembraneous bones of the skull and jaw, SBPimmunoreactivity has been observed in day-14 em-bryos at chondrogenesis sites involved in the forma-tion of these structures, as shown in Fig. 3D-F. Thisobservation provides support for the view that mesen-chyme cells expressing SBP are part of the contingentof neural crest cells that are induced by the overlying
epithelium to form specific skeletal structures in thecraniofacial region.
An important aspect of epithelial-mesenchymalinteractions appears to be the viability and prolifera-tive capacity of the epithelium, as demonstrated byHall (1980), for the initiation of chondrogenesis bymesenchyme through interactions with the mandibu-lar epithelium. This raises the issue of whether theuptake of 5-HT, as demonstrated in the presentstudy, may play some role in regulating cell prolifer-ation in this tissue. Such a possibility is of specialinterest, since 5-HT has been implicated in theregulation of cell proliferation during brain develop-ment (Lauder & Krebs, 1978; Lauder et al. 1982;reviewed by Lauder & Krebs, 1986).
The results of this study raise the possibility that5-HT uptake by epithelia and the expression of thebinding protein for this neurotransmitter in adjacentmesenchyme could play a role in the reciprocalrelationships between epithelia and mesenchymeknown to be required for normal development of thecraniofacial region.
This work was supported by NIK grant HD 22052. Theauthors would like to thank Robin Thomas and RobinWynn for technical assistance; and Drs Malcolm Johnstonand Kathleen Sulik for critical reading of the manuscript.
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