Fate mapping and cell lineage analysis of Hensen's node in the chick
embryo
MARK A. J. SELLECK and CLAUDIO D. STERN
Department of Human Anatomy, South Parks Road, Oxford OX1 3QX, UK
Summary
Fate maps of chick Hensen's node were generated usingDil and the lineage of individual cells studied byintracellular injection of lysine-rhodamine-dextran(LRD). The cell types contained within the node areorganized both spatially and temporally. At the defini-tive primitive streak stage (Hamburger and Hamiltonstage 4), Hensen's node contains presumptive notochordcells mainly in its anterior midline and presumptivesomite cells in more lateral regions. Early in develop-ment it also contains presumptive endoderm cells. At all
stages studied (stages 3-9), some individual cellscontribute progeny to more than one of these tissues.
The somitic precursors in Hensen's node onlycontribute to the medial halves of the somites. Thelateral halves of the somites are derived from a separateregion in the primitive streak, caudal to Hensen's node.
At the rostral end of the primitive streak of the chickand other amniote embryos lies a bulbous mass of cellscalled Hensen's node, after Viktor Hensen who firstdescribed it in 1876. It is generally considered to be the'organizer' of the amniote embryo as it has propertiessimilar to those of the dorsal lip of the blastopore inamphibians. It can self-differentiate into a number ofembryonic tissues and can induce a second embryonicaxis when grafted into a host embryo (for reviews seeLeikola, 1976; Hara, 1978; Slack, 1983; Nieuwkoop etat. 1985).
Hensen's node in the chick embryo has been studiedby marking groups of cells with carbon particles or vitalstains (Spratt, 1955), and radiolabelled grafts (Rosen-quist, 1983), which have allowed the cell typesgenerated by the node to be identified. However, thesetechniques are not always reliable because, forexample, it cannot be assumed that carbon particles willalways remain attached to the cells on which they areplaced. Recently, some new techniques have becomeavailable that allow either small groups of cells, or evensingle cells to be labelled in situ, without transplan-tation.
Here, we have used these new techniques to producemore detailed fate maps of Hensen's node of the chickembryo at various stages of development. The resultsconfirm and extend earlier fate maps. We find that thenode exhibits considerable spatial and temporal organ-ization; however, single cells in certain regions of thenode can contribute to more than one tissue. Our fate
maps also reveal hitherto unknown organization withinthe mesoderm of both the somite and the notochord.
Materials and methods
Embryo techniquesFertile hens' eggs (Light Sussex or Rhode Island Red) wereincubated at 38 °C for 12-30 h to give embryos at stages 3 to 9(Hamburger and Hamilton, 1951).
For confocal microscopy, the embryos were explanted inTyrode's saline and fixed in buffered formol saline (pH7.0)for lh. They were then washed in phosphate-buffered saline(PBS) and labelled by immersion in a 10/igmP1 solution ofbisbenzimide (Hoechst 33258) in PBS. Following furtherwashing in PBS, they were mounted under a coverslip in PBSand viewed using a BioRad MRC500 Confocal ScanningLaser Microscope.
For fate mapping and cell lineage studies, embryos wereoperated in ovo. With the egg placed on its side, about 0.5 mlof thin egg albumen was withdrawn from the blunt end using ahypodermic syringe. A square window of shell and shellmembranes, measuring approximately lcm2, was made in theuppermost side of the egg, using a scalpel. Calcium- andmagnesium-free Tyrode's solution (CMF) was added to floatthe yolk to the level of the window. High-vacuum silicongrease (BDH) was applied to the edges of the window tocontain a bubble of CMF above the embryo. To improve thecontrast between the yolk and the embryo, 30-50/il of IndianInk (Pelikan Fount India) in CMF at a dilution of 1:10 wereinjected under the blastoderm using a hypodermic syringe. Asmall hole was then made in the vitelline membrane over thearea to be marked.
After the marking procedure (see below), the egg was
616 M. A. J. Selleck and C. D. Stern
sealed: 3 ml of thin albumen were withdrawn from the hole atthe blunt end of the egg and a few drops of antibiotic andantimycotic (Sigma) added. The silicone grease was removedand the hole sealed with PVC tape. The eggs were thenincubated for a further 24 h at 38°C in a humid environment.After this time, the embryos were explanted into phosphate-buffered saline (PBS), pinned out in Sylgard dishes and fixed(see below).
Injection of DilThe method used has been described previously (Stern, 1990).Briefly, microelectrodes were made using 50^1 YankeeDisposable Micropet capillary tubes (Clay Adams), pulledwith an Ealing vertical microelectrode puller. The electrodeswere then back-filled with Dil (l,l'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate; MolecularProbes, Inc.), at 0.25% in ethanol containing 5% dimethyl-sulphoxide (DMSO). In a few experiments, a differentmethod was used: Dil was first dissolved at 0.5 % in absoluteethanol and this diluted 1:9 with 0.3 M sucrose in distilledwater at 40°C (see Serbedzija et al. 1990). By applying gentleair pressure, a small bolus of dye was applied to the desiredregion. Dil, a lipophilic carbocyanine dye, inserts into themembranes of the cells lying adjacent to the injection site (seeHonig and Hume, 1989). A small group of cells could thus belabelled. The injected dye could easily be seen under thedissecting microscope as a small pink dot. Occasionally, theexact position was checked under a microscope equipped withepifluorescence optics. The eggs were then sealed andincubated for 24-36 h. After this period of further incubation,the embryos were explanted and fixed in 0.25% glutaralde-hyde in 4% buffered formol saline (pH7.0).
Injection of LRD into single cellsThe methods used for injection of lysinated rhodaminedextran (LRD; Gimlich and Braun, 1985) were similar tothose described elsewhere (Kimmel and Warga, 1987;Bronner-Fraser and Fraser, 1988; Stern et al. 1988; Wetts andFraser, 1988). Injections were performed using a fine glassmicropipette made from aluminosilicate capillaries withinternal filament (1.2 mm outer diameter, 0.9 mm internaldiameter; A-M Systems Inc.), pulled with a verticalmicroelectrode puller (Ealing). The tip of the pipette was firstfilled with a lOrngmF1 solution of LRD (Mr 10000;Molecular Probes, Inc.) and then back-filled with 1.2M LiCl;filled electrode resistances ranged from 50 to 120 MQ. Thisarrangement allowed recording and injection through thesame electrode, which is necessary to determine when theelectrode has penetrated a cell.
Recording and injection were done using a NeurologNL102G preamplifier and headstage (Digitimer) and theoutput visualised through a digital storage adaptor (DSA511;Thurlby) and Hitachi oscilloscope V222, 20 MHz. Injection ofdye was achieved iontophoretically, using 2-8 nA pulses(lHz, 500 ms duration) of current generated by a periodgenerator (NL304; Digitimer) and digital width controller(NL401; Digitimer) fed into a current injection module of theNL102G preamplifier. The use of pulses of current allows themembrane resistance and resting potential to be monitoredduring the injection, which can be used to assess the state ofthe cell being injected.
Movement of the electrode for impalement was controlledwith a Significat SCAT-Ole computer-controlled steppermotor (Digitimer) mounted on a 3-axis micromanipulator.The Significat allowed movement of the electrode along itsaxis in steps of 2/an.
After injection, the electrode was withdrawn rapidly from
the cell, and the labelled cell observed using epi-fluorescenceoptics, to confirm that a single cell had been injected in thecorrect region. The egg was then sealed with PVC tape andplaced in an incubator at 38 °C to develop for a further24-36h. After this, the embryo was explanted and fixed for30-60 min in buffered formol saline (pH7.0) and washed inPBS.
Examination of the embryosInjected embryos were examined as whole mounts; inaddition, LRD-injected embryos were examined after paraf-fin wax embedding and sectioning at 30j/m. Sections weredewaxed, rehydrated down an alcohol gradient and mountedin Gelvatol (14% polyvinyl alcohol 20/30 [Fisons] containingS.Smgmn1 diazobicyclooctane [DABCO, Aldrich, the anti-quenching agent], 30% glycerol andSSO^gml"1 sodium azideas preservative in a PBS base, pH6.8).
Both whole mounts and sections were viewed through anOlympus Vanox-T microscope with epifluorescence optics(rhodamine filter set) and photographed with Kodak TMAX400, Ilford HP5 or Fuji 1600P film.
Since Dil-labelled embryos cannot be sectioned directly onaccount of the dye being insoluble in water, any embryosrequiring sectioning had to be processed by photooxidation of3,3'-diaminobenzidine (DAB) (Maranto, 1982; Buhl andLiibke, 1989; Stern, 1990) prior to wax embedding andsectioning. Embryos were removed from the fixative andrinsed twice in 0.1M Tris at pH7.4, each for lh. They werethen changed into DAB in 0.1M Tris at 500 ^g m l . Thespecimens were then placed under epifluorescence optics andilluminated until no fluorescence remained visible. After this,the embryos were rinsed three times in 0.1 M Tris and rapidlydehydrated and embedded in Paraplast. Embryos were thensectioned at 15 fim and examined using bright-field optics.
Results
Morphology of Hensen's nodeHensen's node was examined in whole mounts with thedissecting microscope the confocal microscope, and inparaffin sections. A stereo-pair image of a stage 4Hensen's node obtained by confocal microscopy isshown in Fig. 1. At this stage, the node is a bulbousmass of cells lying at the rostral end of the primitivestreak immediately anterior to the primitive pit. It isapproximately 80 fan in width. The anterior, lateral andposterior boundaries of Hensen's node were easilyvisible in the whole mount both with the confocalmicroscope and with the dissecting microscope at stage4. The limits of the node become less clear at bothearlier and later stages, so that it was difficult to definethe node in experiments performed before stage 3+ orafter stage 6. A posterior boundary to the node appearsbetween stages 3+ and 4 (Fig. 2). Paraffin sectionsthrough Hensen's node showed that it is not histologi-cally separate from other tissues; its mesoderm iscontinuous with that of surrounding parts of theembryo.
Fate maps produced using the carbocyanine dye, DilIn a total of 180 embryos, a small group of cells inHensen's node was labelled with Dil. Of these, 61yielded results. The results obtained are summarised in
Cell lineage in chick Hensen's node 617
Fig. 1. Stereo-pair showing the morphology of a stage 4 Hensen's node, stained with bisbenzimide (Hoechst) and obtainedwith the confocal microscope. Scale bar, 10 ^m.
Figs 3-6. The node at the definitive primitive streakstage (stage 4) was studied in most detail (n=31). Fig. 3summarises the results obtained after incubation interms of the fates of labelled cells in different regions ofthe node. Briefly, deep injections of Dil in the medialV-shaped part of the node label cells contributing tothe notochord only. Injections into the epiblast of thisregion label both neural tube and notochord. Groups ofcells in the lateral parts of the node contribute both tonotochord and to somite tissue.
Earlier in development, at stage 3 to 3+ (n=9embryos), the lateral portions of the node contain cells
st. 3 st. 3+ st. 4 st. 5 st. 6
Fig. 2. Diagram showing the changes in the shape ofHensen's node between stages 3 and 6. Arrows mark theanterior and posterior limits of the node. At stage 6 and beyond,the posterior limit of the node becomes less distinct (?).
destined mainly for the endoderm (Fig. 3A). Later indevelopment, between stages 5 and 9 (n=21;Figs3C,D), the V-shaped presumptive notochordregion has narrowed, and presumptive somite regionsextend more medially than in the node at stage 4.
The contribution of Hensen's node cells to variousparts of the notochord was analysed separately, and theresults summarised in Fig. 6. At stage 4, the lateralparts of the node contribute cells to the whole length ofthe notochord, including the head process. Moreposterior regions populate the notochord caudal to theheart. This pattern changes with time: at stages 3 and3+, cells able to populate the entire length of thenotochord lie in the rostral midline of the node. Thepresumptive posterior regions of the notochord liemore posteriorly. Later in development (stages 5-9),the prospective notochord material remaining in thenode contributes only to the posterior parts of thenotochord.
A striking finding is that whenever cells labelled withDil in the node contributed to the somitic mesoderm,the marked cells were confined to the medial half of thesomites (Figs 4A and 5C). We therefore undertook asearch for the cells that contribute to the lateral half. Itwas found that a region of primitive streak, some200 jum behind the node, contains the precursors of thelateral halves of the somites (Figs 4B and 5D).
618 M. A. J. Selleck and C. D. Stern
St. 3-3+ St. 4
Notochord (N)
Neural tube (NT)
Somite (S)
Endoderm (E)
Notochord + S
Notochord + E
Notochord + NT
Somite + E
» ^ A
B
DFig. 3. Summary of results of cell marking experiments with Dil in different regions of Hensen's node at various stages.The diagrams represent the node and the rostral end of the streak, the primitive pit and groove. Each shaded blocksymbolises the results of a single injection of Dil in one embryo, in terms of the fates of the descendants of the labelledcells. Different shadings (shown in the key) represent different tissue types.
Fate maps produced by injection of LRD into singlecellsA total of 339 injections of LRD into individual cells ofHensen's node were performed, of which 96 producedclones. The results obtained are summarised inFigs 7-10.
Fig. 7B summarises the results obtained from injec-tions of LRD into single cells in the node of stage 4embryos (n=63 clones recovered from 171 injections).As seen with Dil, there is a V-shaped region in theanterior midline of the node, where cells contributeonly to notochord. In lateral and posterior parts of thenode, cells contribute only to somitic tissue (somitesand segmental plate). However, in a band stretching
antero-laterally from the primitive pit (between thepresumptive somite and notochord regions), theprogeny of single cells populate both somite andnotochord (18 clones out of 35 were mixed; 51%).Epiblast cells in the antero-medial V-zone maycontribute to both notochord and floor plate of theneural tube (3 clones out of 7 derived from epiblast cellsproduced mixed progeny).
Earlier in development, at stage 3 to 3+ (n=5 clonesrecovered from 17 injections) (Fig. 7A), the nodecontains prospective definitive endoderm cells, as wellas somite and notochord cells. Later, at stages 5-9(«=28 clones from 151 injections; Fig. 7C,D), thesomite regions of the node extend further towards the
Fig. 4. Somite descendants of Dil-labelled cells. (A) Injection of Dil into a lateral portion of the node results in themedial portion of the somites being labelled. This particular group of labelled cells also contributed progeny to thenotochord posteriorly (bottom of photograph) and to endoderm underlying the more posterior somites (out of focusfluorescence underlying last 5 somites). (B) Dil labelling of cells in the rostral primitive streak behind Hensen's noderesults in the lateral portion of the somites being labelled. The midline endoderm and more lateral mesoderm also containsome labelled cells. Scale bars, 100 j«n.
Cell lineage in chick Hensen's node 619
n
B
V s .
Fig. 5. Descendants of Dil-labelled cells in notochord and somite. (A) Labelling cells with Dil in the anteromedial V-shaped part of the node results in a length of notochord containing labelled cells. The photograph shows this pattern inwhole-mount. After photo-oxidation of the dye with DAB, histological sections can be obtained, as shown in B-D.(B) Section through the embryo shown in A, following photo-oxidation of the Dil. Labelled cells are seen in thenotochord. (C) After marking cells in a more lateral region of the node, descendants are found in both the notochord andthe medial part of both the left and right somites at this level. (D) Injection of Dil into the rostral portion of the primitivestreak behind the node labels cells in the lateral part of the somite and includes descendants in both dermomyotome (d)and sclerotome (s). (n) notochord. Scale bars, 100^m (A), 50/m\ (B,C,D).
anterior midline, narrowing the V-zone. Single cells inregions between the prospective somite and notochordareas can contribute to both structures (6/11, 55%).
Injections into a cell in the midline of the node regionoften contributed labelled progeny to both left and rightsides of the embryo. Injections into more lateral cellsgave rise to progeny that were confined to the ipsilateralside of the embryo.
The spatial distribution of labelled cells that contribu-ted to the notochord confirms the results obtained withDil. Cells marked in older embryos (stages 5-6)contributed only to more caudal portions of thenotochord (Fig. 10), while some presumptive noto-chord cells in younger embryos produced progeny thatwere distributed widely over the entire length of thenotochord. It is perhaps interesting that the labelleddescendants found in the notochord were sometimesarranged into regularly spaced groups of cells along itslength, separated by an interval equivalent to about twosomite lengths (e.g. Fig. 8A). The number of cells in
each group appeared to decrease caudally, whilst theintensity of fluorescence increased in the same direc-tion.
As found with Dil, whenever node cells contributedprogeny to the somitic mesoderm, the labelled descend-ants were confined to the medial halves of the somites(Fig. 8B,D). Labelled cells were not confined to rostralor caudal halves of the somites and in many cases two orthree consecutive somites contained labelled cells(Figs 8B and 9A).
Discussion
We have constructed fate maps of Hensen's node of thechick embryo using Dil to label small groups of cells,and injection of lysine-rhodamine-dextran (LRD) tofollow the descendants of individual cells. The resultsobtained confirm and extend earlier fate maps of thisregion such as those of Wetzel (1929), Rudnick (1935),Spratt (1952, 1955), Rosenquist (1966, 1983) and
620 M. A. J. Selleck and C. D. Stern
St. 3-3+ st. 4
DFig. 6. Diagram summarising the contribution of different regions of the node to different rostrocaudal portions of thenotochord. As in Fig. 3, each shaded block represents one group of labelled cells in one embryo, and the rostrocaudalposition of their descendants is symbolised by the shading, which is explained in the key. The position of progenitor cellsthat contribute progeny to the entire length of the notochord (including the head process) changes between stages 3 and 4.By stage 5, cells no longer can contribute to the entire notochord and head process. Otherwise, there appears to be noobvious correlation between position in the node and distribution of labelled descendants in the notochord and headprocess.
Schoenwolf and Sheard (1990). At the definitive streakstage (stage 4), the node contains presumptive noto-chord cells mainly in a V-shaped region at its anteriorend, and presumptive somite cells in more lateralregions. The zone between these two regions containscells that contribute progeny to both tissues. Earlier indevelopment, the node also contains endoderm pro-genitor cells. Fate maps for various stages of develop-ment, summarising our findings with the two tech-niques, are presented in Fig. 11.
Our investigations also reveal a hitherto unsuspectedorganization within the somitic mesoderm. The somite
progenitor cells of the node only contribute to themedial halves of the somites; the lateral halves of thesestructures arise from a separate region in the primitivestreak, lying some 200pm caudal to the node.
Assessment of the techniques usedTo label small groups of cells within Hensen's nodewithout using transplantation or particulate markers,we have made use of the recently introduced carbocya-nine dye, Dil (see Honig and Hume, 1989; Wetts andFraser, 1989; Serbedzija et al. 1990; Stern, 1990). Thislipophilic molecule inserts into cell membranes. Be-
Cell lineage in chick Hensen's node 621St. 3-3+ st. 4
•
D
cause of its insolubility in water, the dye does notspread far from the injection site and it is thereforepossible to label a localised group of cells (see Honigand Hume, 1989). The same property prevents the dyefrom being passed from cell to cell except during celldivision. These properties of the dye make it an idealmarker to study cell fates. One disadvantage, however,is that its lipophilic character precludes the use ofconventional histological techniques. However, thefluorescence in Dil labelled cells can be converted to aninsoluble precipitate of oxidised diaminobenzidine byexposure to the excitation wavelength, and the speci-mens then processed conventionally for wax sectioning(Maranto, 1982; Buhl and Lubke, 1989; see Stern,1990).
The second technique we have used is also estab-lished as a routine technique (e.g. Gimlich and Cooke,1983; Kimmel and Warga, 1987; Bronner-Fraser andFraser, 1988; Stern etal. 1988; Wetts and Fraser, 1988);unlike carbocyanine dyes, it can be used for mapping
Fig. 7. Diagrams summarising the resultsof injection of LRD into individual cells ofHensen's node at different stages. Thediagrams represent the node and anteriorportion of the primitive streak of embryosof different stages. Each shaded blockrepresents one injection into a single cell,in one embryo. The shading (see key inFig. 3) denotes the fate of the progeny ofthe injected cell.
the descendants of single labelled cells. It makes use ofthe fluorescent tracer lysinated rhodamine dextran(LRD; Gimlich and Braun, 1985). The rhodamine is thefluorescent part of the molecule, the lysine makes thedye fixable within cells using aldehyde fixatives, and thehigh molecular mass dextran ensures that the moleculeis too large to pass from one cell to another through gapjunctions (Gimlich and Braun, 1985). LRD is injectedinto individual cells by iontophoresis, which results inno volume increase of the cells; the procedure cantherefore be applied to very small cells such as those ofHensen's node, which are only about 5-8/zm indiameter.
These new techniques overcome some of the difficul-ties of earlier methods. Transplantation of marked cellsmight have disturbed the spatial, and perhaps temporal,organization of the tissues to be mapped, while carbonor carmine particles may not always follow the cells intheir movements. It has also been shown that neither ofthe modern methods interferes with the development of
622 M. A. J. Selleck and C. D. Stern
Fig. 8. Clones derived from the injection of LRD into individual Hensen's node cells. (A) Whole-mount view of a clone offluorescent cells in the notochord produced by the injection of LRD into a single cell in the anteromedial portion of thenode. Note the widspread distribution of labelled progeny in the rostrocaudal axis. The labelled descendants appear to beorganised into clusters separated by unlabelled cells with a periodicity of about 2 somite-lengths. More anterior (rostral,top in the photograph) cells are more faintly labelled and the number of cells in each cluster decreases caudally. (B) Cloneof fluorescent cells derived from an injection of LRD into a cell in the lateral part of the node. Labelled cells occupy themedial halves of two consecutive somites. (C,D) Sections through embryos similar to those in A and B, respectively,showing labelled cells in the notochord derived from the anteromedial node (C) and medial half-somite derived from thelateral node (D). Scale bars, 100^m (A), 50fim (B,C,D).
Cell lineage in chick Hensen's node 623
Fig. 9. Injection of LRD into a cell situated between the presumptive notochord and somite regions of the node: bothnotochord and somite cells are labelled. (A) Whole-mount view of one such embryo. The labelled somites always lie rostralto the labelled notochord cells. (B,C) Sections through embryos similar to that in A, showing LRD-labelled cells in thenotochord (B) and medial somite (C). Scale bars, 100^m (A), 50(xm (B,C).
B
Fig. 10. Diagram summarising therostrocaudal extent of the contribution ofindividual cells to different rostrocaudallevels of the notochord. The shading ofthe blocks is explained in Fig. 6.
624 M. A. J. Selleck and C. D. Stern
St. 3-3+
st 3-9
m•mm
KEYNOTOCHOflD
MEDIAL SOMCTE
LATERAL SOWTE
ENOOOEHM
NEURAL TUBE
Fig. 11. Summary fate map of Hensen's node, compiledfrom the experiments in the present study. The three upperdiagrams show the mesodermal and endodermal derivativesof the node, and the lower diagram shows the contributionof labelled Hensen's node cells to the neural tube.Although the fate map is bilaterally symmetrical, the areagiving rise to notochord is only shown on the left and thosegiving rise to somite and endoderm on the right of eachdiagram, for clarity. The boundaries of the presumptivenotochord region shown represent the approximate limitsof the left half of the V-region.
the labelled structures (for Dil see Honig and Hume,1989; Wetts and Fraser, 1989; Serbedzija et al. 1990;Stern, 1990; for LRD see Kimmel and Warga, 1987;Bronner-Fraser and Fraser, 1988; Stern et al. 1988;Wetts and Fraser, 1988).
Fate maps of the nodeThe fate maps generated by the Dil experiments andsingle cell injections reveal that the node is not ahomogeneous structure, but organized spatially. Inaddition, the fates of cells present in the node changewith time. The results obtained confirm the pioneeringwork of investigators such as Wetzel (1929), Rudnick(1935), Spratt (1952,1955) and Rosenquist (1966,1983),who used carbon or carmine particles or autoradio-graphy of labelled grafts. The accuracy of the fate mapsproduced by these workers using such crude methods isremarkable.
In the medial part of the node of a stage 4 embryo, wefind that mesoderm cells contribute only to thenotochord of the embryo, whereas in the lateral andposterior parts, the cells are destined only to contributeto the somites. Single cells in the region between thetwo contribute to both notochord and somite. Theslight differences between the maps generated by thetwo different methods is probably due to the fact thatwhen Dil is used to label the mesoderm, some cells ofthe epiblast are also labelled as the injection electrode isremoved through the epiblast.
Whenever descendants of a single cell are found in
two or more tissue types, it follows that at the time ofinjection the parent cell cannot have been committed toeither of these fates. However, restriction of theprogeny of a single cell to one tissue type does notnecessarily imply that the cell was committed to thatone fate at the time of labelling. It could be that the cellhas the potential to follow two or more developmentalpathways, but that this potential is not realised becauseits progeny never move into appropriate environmentsto affect their fate (see Slack, 1983).
One important result that emerges from the presentinvestigation is that, at least in lateral regions of thenode, single cells can give rise to progeny located inmore than one tissue type. Therefore, the node cannotbe made up entirely of committed cells. However, it isimportant to exclude the possibility that more than onecell was labelled by the injection. While we cannot saywith confidence that in every case injection wasconfined to a single cell, the following observationmakes it unlikely that mixed clones were always due toinjection of dye into more than one cell: labelled cellscontributed progeny to more than one cell type only inone confined region, situated between the presumptivenotochord and medial somite areas. In this region,24/46 clones (52 %) at stages 4-6 were mixed, althoughcontrol experiments in' which embryos were fixedimmediately after injection only revealed that morethan one cell was labelled in small proportion of cases.It therefore seems most likely that the mixed clonesobtained have arisen from uncommitted progenitorcells.
Although cells contributing to both mesoderm andendoderm were never seen at stages 4-6, single cellscan indeed give rise to progeny in more than one germlayer. In 3 cases at stage 4, cells in the epiblast portionof the V-shaped anteromedial region of the nodecontributed both to the notochord and to the medianhinge plate (MHP;=presumptive floor pate of theneural tube; see Schoenwolf and Smith, 1990). Thisindicates that the cells of the epiblast of this region arenot yet committed to form either ectodermal or meso-dermal derivatives. However, once cells have ingressedinto the mesodermal portion of the node, descendantsare no longer found in both mesoderm and ectoderm.
The finding that single mesoderm cells within thenode do not contribute to both floor plate andnotochord provides strong direct evidence that thenotochord itself does not contribute cells to the formerstructure, as has been suggested recently (Jessell et al.1989). However, it is also clear that, at an earlier stagein development, ectodermal cells do contribute to bothnotochord and floor plate. It has been suggested (Jessellet al. 1989) that the existence of common progenitorsmight be related to the ability of the notochord toinduce a floor plate in other regions of the neural tube(van Straaten et al. 1985; van Straaten and Drukker,1987; Smith and Schoenwolf, 1989; Jessell et al. 1989;Schoenwolf and Smith, 1990).
Mediolateral subdivision of the somitesOur studies reveal, unexpectedly, that somites are
Cell lineage in chick Hensen's node 625
subdivided into medial and lateral halves. The lineseparating the two halves does not correspond to thatseparating dermomyotome from sclerotome(Figs 5C,D), and the anatomical and functional signifi-cance of such a subdivision is therefore unclear.
Other workers have obtained comparable resultsfrom transplantation experiments. For example, Bel-lairs (1986; see also Ooi et al. 1986) homotopicallytransplanted pieces of quail primitive streak into chickembryos and found that quail cells were restricted to thedorsolateral part of the chimaeric somites that formed(see for example, Fig. 5 of Bellairs, 1986). In thepresent study, a contribution to the dermomyotomewas only seen in cases when the lateral part of thesomite was labelled; no cells from clones including themedial portion of the somite extend into the dermo-myotome. Ooi et al. (1986) found that in some cases,cells derived from the grafted primitive streak areconfined to the dorsal aspect of the somite andsuggested that the cells migrating out of the streak moveon the basal lamina underlying the ectoderm and thuscome to occupy a dorsal position in the somite.
One possible implication of these findings, consider-ing the lack of anatomical evidence for later develop-mental differences in fate between the medial andlateral halves of the somite, is that the two halves areinvolved in different aspects of somite formation. Forexample, Bellairs and Veini (1984) suggested thatsomites are laid down as a series of small 'somitogenicclusters' of founder cells, which later recruit other cellsto make up sufficient numbers to allow them toparticipate in the epithelialisation that accompaniessegmentation. It is possible, for example, that thepresumptive medial half somite cells contained withinthe node represent the precursors of Bellairs andVeini's somitogenic clusters.
Implications of the findings for regulation and neuralinductionThe finding that Hensen's node in the chick embryo issubdivided into regions with different fates begs thequestion of whether the cells in each of these regionsare committed to their fates or whether they can beregulated if transplanted. For example, does extir-pation of one region, such as the anteromedial V-shaped zone, result in loss of all or part of thenotochord? Does transplantation of cells from thelateral, somitic region of the node into the anteromedialV-region lead to the transplanted cells now giving riseto notochord? Moreover, could this complexity bearsome relationship to the inducing ability of the node? Isinducing ability or capacity to regionalize restricted to asingle region as revealed in our fate maps? Experimentsare in progress to attempt to answer these questions.
M. A.J.S. is funded by a Wellcome Trust Prize Studentship.The confocal microscope was purchased with a grant from theMedical Research Council. We are grateful to Geoff Carlsonfor his skilled technical assistance and to Brian Archer andColin Beesley for their help with photography.
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