initial trajectories of sensory axons toward laminar targets in the developing mouse spinal cord

Initial Trajectories of Sensory AxonsToward Laminar Targets in theDeveloping Mouse Spinal Cord

SHIGERU OZAKI AND WILLIAM D. SNIDER*

Center for the Study of Nervous System Injury, Department of Neurology,Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACTThe formation of laminar-specific projections is a key event in the development of

appropriate neuronal connections in many regions of the central nervous system. In order toprovide a framework for defining functions of molecules related to spinal laminar targeting ofdorsal root ganglion neurons in mice, we have characterized the initial trajectories of sensoryaxons in relation to the maturation of their target laminae in the spinal cord. We show thatmorphological and biochemical differentiation of distinct clusters of neurons in the dorsalregion of the spinal cord precedes initial collateral branching from sensory axons. Betweenembryonic day (E)12.5 and E13.5, sensory axons develop swellings (‘‘nodes’’) along their entireintraspinal extent and elaborate interstitial collateral branches from these nodes. Collateralsfrom the different classes of sensory axons then penetrate the gray matter of the spinal cordsequentially. Each class of sensory axons projects directly to its target lamina, neverbranching into inappropriate laminae en route. Some cutaneous afferents traverse the entirewidth of the spinal cord to reach superficial laminae on the contralateral side, strictly avoidingboth the ventral spinal cord and inappropriate laminae of the deep dorsal horn. The pathwaystaken by developing sensory afferents are compatible with the idea that cells in inappropriatelaminae exert inhibitory influences on sensory axons which regulate their laminar specificity.J. Comp. Neurol. 380:215–229, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: spinal cord development; dorsal horn maturation; primary afferents; collateral

branching; axon guidance

The segregation of afferent inputs into laminar-specificprojections is a key early event in the development ofspecific connections in many regions of the central nervoussystem (CNS) in vertebrates. Both diffusible factors andsurface and matrix molecules have been implicated inlaminar targeting. For example, semaphorin III (Sema IIIor Sema D), a diffusible member of a large family ofguidance molecules, semaphorins/collapsins, has beenshown to repel nerve growth factor (NGF)-dependentsensory axons in vitro, suggesting that this moleculeregulates sensory axon patterning in the spinal cord(Messersmith et al., 1995). Sema III and its numeroushomologues are expressed not only in the spinal cord butalso in other regions of the nervous system during develop-ment (Luo et al., 1995; Messersmith et al., 1995; Puschel etal., 1995; Wright et al., 1995; Zhou et al., 1995, 1996;Shepherd et al., 1996), suggesting a widespread role forthese molecules in regulating axon patterning. In theory,influences from the netrins, a family of molecules withboth chemotropic and chemorepellent activities whichguide spinal commissural axons to the floor plate (Kennedy

et al., 1994; Serafini et al., 1994; Colamarino and Tessier-Lavigne, 1995), may also be important. However, whetherthe netrins regulate projections to specific layers or onlyprovide gross directional guidance is as yet unknown.Surface and matrix molecules are also likely to be

important regulators of laminar targeting. In the chickoptic tectum, various classes of surface and matrix mol-ecules are distributed in laminar-specific patterns(Yamagata et al., 1995). Furthermore, retinal axons ap-pear to find appropriate laminae in the tectum even whenthe target tissue is fixed (Yamagata and Sanes, 1995).Thus, in this system, molecules associated with membrane

Contract grant sponsor: NINDS; Contract grant numbers: RO1NS31768,PO1 50757.*Correspondence to: William D. Snider, M.D., Center for the Study of

Nervous System Injury, Department of Neurology, Washington UniversitySchool of Medicine, Campus Box 8111, 660 S. Euclid Ave., St. Louis, MO63110-1093. E-mail: [email protected] 30 May 1996; Revised 7August 1996;Accepted 20August 1996

THE JOURNAL OF COMPARATIVE NEUROLOGY 380:215–229 (1997)

r 1997 WILEY-LISS, INC.

or matrix of the targets appear to regulate arborization ofafferents in appropriate laminae. Other surface moleculessuch as erythropoetin-producing hepatocellular (Eph) recep-tor tyrosine kinase ligands, which have been hypothesized toregulate topographic maps in the retinotectal system, mayalso be important for laminar targeting in some CNS regions(for a review, see Friedman and O’Leary, 1996).A favorable system in which to analyze the development

of laminar specificity is the dorsal root afferent projectionto the spinal cord. The overall time course of the develop-ment of this projection has been characterized in detail inseveral species including human (e.g., see Konstantinidouet al., 1995; Mirnics and Koerber, 1995, and referencestherein). A key feature of this projection is that the growthfactor survival requirements of dorsal root ganglion (DRG)neurons projecting to different laminae have recently beenelucidated (for reviews, see Klein, 1994; Snider, 1994;Snider and Wright, 1996). This feature has allowed thedemonstration that functionally distinct classes of sensoryneurons respond differently to the guidance molecule,Sema III (Messersmith et al., 1995), and points the way forstudies of the specificity of other classes of molecules.Moreover, a variety of molecules known to regulate axonalgrowth including several recently identified members ofthe semaphorin/collapsin family (Luo et al., 1995; Messer-smith et al., 1995; Puschel et al., 1995; Wright et al., 1995;Zhou et al., 1995, 1996; Shepherd et al., 1996), netrin-1and -2 (Kennedy et al., 1994; M. Tessier-Lavigne, personalcommunication) and several of the Eph-like receptor tyro-sine kinases (Mori et al., 1995) are known to be expressedin the embryonic spinal cord in discrete locations at stageswhen the dorsal root afferent projection is developing. Itshould be possible to study effects of these molecules onsensory neurons in vitro in paradigms similar to that usedfor Sema III. The opportunity to further clarify functions ofthese molecules in relation to axon growth and branchingin vivo will undoubtedly soon be available in transgenicand null mutant mice.In order to provide a framework for defining functions of

molecules related to laminar targeting of spinal sensoryneurons inmice, we have characterized the development oflaminar-specific projections of different functional classesof sensory axons in the spinal cord. We show here thatdifferentiation of morphologically and biochemically dis-tinct clusters of neurons in the dorsal region of thedeveloping spinal cord is apparent well before penetrationof the gray matter by sensory axons. We further demon-strate that different classes of sensory axons enter thedeveloping spinal cord in sequence and project directly totheir target laminae, strictly avoiding inappropriate lami-nae en route. These results suggest that cells in inappropri-ate laminae exert inhibitory influences on sensory axongrowth.

MATERIALS AND METHODS

Anesthesia and perfusion

CF1 mice ranging in age between embronic day (E) 10.5and postnatal day (P) 1 were used in this study (E10.5,n 5 13; E11.5, n 5 16; E12.5, n 5 23; E13.5, n 5 46; E14.5,n 5 25; E15.5, n 5 24; E17.5, n 5 13; P1, n 5 2). Timed-pregnant females were obtained from the Charles RiverLaboratories. The plug date was considered E0.5. Thedevelopmental stage of embryos was confirmed bymeasur-ing crown-rump length and by their appearance (Kauf-

man, 1992). The day of birth was considered P0. Pregnantmice were anesthetized with halothane and overdosedwith an intraperitoneal injection of sodium pentobarbital.The embryos were removed by Cesarean section andplaced in ice-cold phosphate buffered saline (PBS, pH 7.4).Animals were perfused transcardially with 4% paraformal-dehyde in 0.1 M phosphate buffer (pH 7.4). Animalsyounger than E13.5 were immersed in the same fixative.All animals were postfixed in fresh fixative for at least 12hours.

Dil labeling

The development of dorsal root projections in the tho-racic spinal cord was investigated using the lipophiliccarbocyanine dye 1,18-dioctadecyl-3,3,38,38-tetramethylin-docarbocyanine perchlorate (Dil, Molecular Probes, Eu-gene, OR). Following postfixation, crystals of Dil wereplaced within the proximal region of the thoracic nervesbetween 5th and 10th segments. In many cases, theventral roots were cut to prevent Dil from backfillingmotoneurons. Specimens were placed in a 38°C oven andwere periodically examined to determine the extent of dyelabeling. Following the appropriate time interval (24 hoursto 2 weeks), spinal cords were removed, embedded in 3.5%agar, and sectioned on a vibratome at 50–125 µm. Sectionswere then placed on microscope slides in a drop of 0.05 Mphosphate buffer (pH 7.4) and coverslipped. Whole mountand ‘‘flattened’’ (see below) thoracic spinal cords were alsoprepared.Preparations were viewed on a Nikon Microphot FXA

microscope under rhodamine epifluorescence and photo-graphed using Kodak Ektachrome color slide film (ISO400). Slides were digitized using a Polaroid Sprint Scan 35and down-loaded into an Apple Power Macintosh 9500.Images were sharpened, contrast adjusted, and assembledinto panels with Adobe Photoshop 3.0. Suitable prepara-tions were also observed using an Odyssey confocal laser-scanning microscope (Noran Instruments, Middleton, WI).A Z-series spanning the section was collected and recon-structed in a two-dimensional (2D) plane. Confocal imageswere converted to TIFF files and montaged using AdobePhotoshop 3.0.To convert Dil to a permanent label, sections were

photoconverted utilizing diaminobenzidine (DAB, Sigma,St. Louis, MO; Sandell and Masland, 1988). Sections wereplaced on a slide in a solution of 2.6 mg DAB/ml in 0.1 MTris buffer (pH 8.2). Under rhodamine epifluorescence anda 310 objective for 30–60 minutes, the DAB replaced theDil fluorescence with a permanent brown reaction product.Following photoconversion, the sections were rinsed in 0.1M Tris buffer (pH 8.2) and air-dried overnight on gelatin-coated slides. The sections were then stained with cresylviolet, dehydrated in a graded series of ethanols, cleared inxylene, and coverslipped with DPX mountant (ElectronMicroscopy Sciences, FT. Washington, PA).In order to characterize neurons in the spinal cord, tiny

crystals of Dil were placed in either the lateral funiculus,ventral commissure or ventral funiculus on one side of thethoracic spinal cord. Preparations were then processedusing the same procedures described above.In order to distinguish cutaneous and muscle afferents,

bothDil and 4-(4-(dihexadecylamino)styryl)-N-methylpyri-dinium iodide (DiA, Molecular Probes, Eugene, OR) wereapplied to E14.5 embryos. Dil was placed on the skin in thethoracic region and DiAwas placed in the proximal region

216 S. OZAKI AND W.D. SNIDER

of several thoracic nerves several days later. Although thethoracic nerve contains both cutaneous and muscle affer-ents, cutaneous axons preloaded with Dil rarely absorbedDiA applied later (see Balice-Gordon et al., 1993). In thismanner, cutaneous and muscle afferents were visualizedwith red and green colors, respectively, under the appropri-ate epifluorescence.

Immunohistochemistry

After anesthesia described above, mouse embryos wereremoved and perfused transcardially with PBS, followedby fixative consisting of 4% paraformaldehyde in 0.1 Mphosphate buffer (pH 7.4). Following postfixation in freshfixative overnight, embryos were immersed in 30% sucrosein 0.1 M phosphate buffer for 12–24 hours, then frozen inOCT embedding medium (Tissue Tek, Miles, Elkhart, IN).Transverse sections were cut at 12 µm on a cryostat,mounted on SuperFrost/Plus slides (Fisher Scientific, Pitts-burg, PA), and allowed to air-dry.Mounted sections were encircled by using a Pap Pen

(Kyota International Inc., Elk Grove Village, IL), andincubated for 1 hour in a blocking solution consisting ofSuperblock buffer (Pierce Chemical Company, Rockford,IL), 0.3% Triton X-100 (Sigma, St. Louis, MO), and 1.5%normal serum (Vector, Burlingame, CA) at room tempera-ture. Sections were incubated in the primary antibody for12–16 hours at 4°C. Slides were then washed 3 times (5minutes each) with PBS and incubated in the secondaryantibody for 30 minutes at room temperature, washedagain three times (5 minutes each) in PBS and cover-slipped in PBS.A monoclonal antibody, 4D5, to Islet-1 and Islet-2 was

generously provided by Dr. T.M. Jessell and used at 1:100dilution. Amonoclonal antibody to microtubule-associatedprotein 2 (MAP2, Sigma, St. Louis, MO) was used at1:1,000 dilution. Secondary antibodies conjugated to CY3( Jackson Immunochemicals, West Grove, PA) were used at1:200. Sections were examined under fluorescence opticsand using a confocal laser-scanning microscope as de-scribed above.

Axon projections in relationto laminar morphology

Following postfixation, spinal cordswere dissected, osmi-cated, dehydrated through a graded ethanol series, andembedded in Spurr resin (Electron Microscopy Sciences,FT. Washington, PA). Semithin sections (1 µm) were cutand stainedwith toluidine blue. Dil-labeled, DAB-photocon-verted sections at 125 µm thickness were also embedded inSpurr resin, sectioned at 1 µm, and stained with toluidineblue in this same manner.Primary afferent axons and spinal neurons in fluorescent-

labeled or photoconverted materials were reconstructed inthe transverse plane by using a camera lucida drawingtube with a 320 objective. Cellular profiles labeled withthe 4D5 monoclonal antibody were photographed andtraced by using a transparent sheet overlying the photomi-crograph. Tracings were scanned and down-loaded to thePower Macintosh and then superimposed on either photo-micrographs of semithin sections or schematic drawings ofthe corresponding spinal cord with Adobe Photoshop 3.0.Composite drawings of tracings of cellular profiles weremade with their location maintained.In order to show trajectories of primary afferent axons in

semithin sections in detail, semithin sections of Dil-

labeled, DAB-photoconverted preparations were photo-graphed using both bright- and darkfield optics. Primaryafferent axons in the darkfield photomicrograph weretraced using a transparent sheet overlaying the photomi-crograph. Tracings were scanned and down-loaded to thePower Macintosh. Brightfield photomicrographs were alsodigitized and stored. Tracings were then pseudocoloredand superimposed on the brightfield photomicrograph ofthe same sections by usingAdobe Photoshop 3.0.

RESULTS

Time course of the development of themurine dorsal root afferent projection

Figure 1 shows the primary afferent projection into thethoracic spinal cord in transverse and sagittal orientationsat different stages of embryonic development. At E13.5, afew primary afferent axons (arrowheads) have entered thedorsal gray matter. These axons project along the midlinetoward to the ventral spinal cord, strictly avoiding thelateral edge of the ventricular zone and virtually all of thedifferentiating dorsal horn. By E15.5, an age at which thelaminar architecture of the spinal cord is apparent (seebelow), axon projections to both the superficial (asterisks)and deep dorsal horn (small arrows) have developed.Axons projecting to the ventral horn (arrowheads) arefasciculated near the midline and ramify ventrally in thevicinity of the medial and lateral motor pools. By E17.5,more axon fascicles have reached all laminar targets in thespinal cord and terminal arborizations in the intermediatezone, the motor nuclei and the dorsal horn are apparent.Axons projecting to the superficial and deep dorsal horn inboth medial and lateral locations traverse the midline(large arrows) to reach the corresponding laminae in thecontralateral spinal cord. Laminar-specific patterns arealso apparent in the sagittal orientation by E17.5 (bottompanels).

Growth of primary afferents prior topenetration of the spinal gray matter

Early axon growth into the spinal cord in relation to thedeveloping spinal architecture is shown in Figure 2 (leftpanels, semithin sections; middle panels, Nomarski im-ages of photoconverted Dil labeling) and Figure 3 (high-power views of the dorsolateral spinal cord). At E10.5, themouse spinal cord consists almost exclusively of undifferen-tiated cells of the ventricular zone except for the ventrolat-eral region where motoneurons are differentiating. Thecentral processes of the early-born DRG neurons extendinto the dorsal root and reach the dorsolateral wall of thespinal cord, the dorsal root entry zone, (Fig. 2B, arrow-head; Fig. 3A, arrows) by this stage.At E11.5, a thin layer of laterally located neurons has

differentiated. At this stage, more DRG neurons extendaxons into the dorsolateral margin of the spinal cord,forming a clearly defined primordium of the dorsal funicu-lus (commonly referred to as the oval bundle of His)immediately adjacent to differentiating neurons (Fig. 2E,arrowhead; Fig. 3B, arrows). However, no primary afferentaxons penetrate the gray matter. At E12.5, many moredifferentiated neurons are present and form a thickerlayer which extends to the dorsal surface of the spinalcord. The primordium of the dorsal funiculus is flattenedand contains more axons (Fig. 2H, arrowheads; Fig. 3C,

TRAJECTORIES OF DEVELOPING SPINAL SENSORY AXONS 217

arrows). The first axons projecting directly medially pen-etrate the gray matter at this age (Fig. 3D, arrows). Thus,there is a delay of at least 48 hours from the time axonsenter the spinal cord prior to elaboration of any primaryafferent axons into the gray matter.Rostrocaudal extension of primary afferents was exam-

ined using longitudinal whole mount spinal cord prepara-tions. At E10.5, the central axons of DRG neurons bifur-

cate rostrocaudally for a short distance within a singlesegment (Fig. 2C, asterisk). At E11.5, afferent axonsextended two segments away from the entry point (Fig. 2F,asterisks). By E12.5, the axons extend more than threesegments rostrocaudally (Fig. 2I, asterisks). By E13.5,some primary afferent axons reach more than four seg-ments rostral and three segments caudal from the site ofentry (not shown).

Fig. 1. Dorsal root axon projections to the mouse thoracic spinal cord at different embryonic ages. Darkfield photomicrographs of Dil-labeled,photoconverted preparations in transverse (top) and sagittal orientations (bottom). A,B: At E13.5, a few primary afferent axons (arrowheads)enter the dorsal gray matter and take a parabolic trajectory avoiding the ventricular zone and the developing dorsal horn. The motoneuron pool(MN) is backfilled. C,D: By E15.5, axon projections to the superficial (asterisks) and deep dorsal horn (small arrows) have developed. Axonsprojecting to the ventral horn (arrowheads) have defasciculated and reached the gray-white border. E,F: By E17.5, many more axons of allclasses have reached their laminar targets in the spinal cord (the superficial [asterisks], deep dorsal horn [small arrows] and ventral horn[arrowheads]), and terminal axon arborization has begun. Some axons projecting to the medial and lateral dorsal horn project to thecorresponding laminae in the contralateral spinal cord (large arrows). Scale bars 5 50 µm.


Fig. 2. Earliest stages of dorsal root axon growth in the spinal cord.Left: Semithin sections of the thoracic spinal cord. Middle: Transversesections of Dil-labeled, photoconverted preparations photographedusing Nomarski optics. Right: Photoconverted preparations viewed aswhole mounts.A–C:E10.5. The spinal cord consists almost exclusivelyof proliferating cells of the ventricular zone (A). The first sensoryaxons penetrate the spinal cord at the dorsal root entry zone by thisage (arrowhead, B). Lateral view of a whole mount preparation showsthat sensory axons extend less than one segment (C). D–F: E11.5.Although the ventricular zone is still prominent, a layer of neurons

has differentiated laterally adjacent to entering sensory axons (D).More sensory axons have entered the spinal cord forming the primor-dium of the dorsal funiculus (arrowhead, D and E). Lateral view of awhole mount preparation shows axons extending less than twosegments (F). G–I: E12.5. Differentiated neurons form a prominentmantle layer (G). The primordium of the dorsal funiculus becomesflattened (arrowheads, G and H). Lateral view of a whole mountpreparation shows axons extendingmore than three segments (I). MN:motor neurons. Asterisks indicate the dorsal root ganglion (DRG).Scale bars 5 50 µm (A,B), 100 µm (C–I).

Fig. 3. High-power views of the developing dorsal funiculus. A: AtE10.5, ventricular zone cells occupy the region immediately adjacentto the points where the first sensory axons enter the spinal cord, thedorsal root entry zone (arrows). B: At E11.5, a thin layer of neuronshas differentiated (demarcated by small squares) and the primordiumof the dorsal funiculus has formed (arrows). C,D: At E12.5, differenti-

ated neurons form a mantle layer. The primordium of the dorsalfuniculus becomes flattened (arrows, C). The first primary afferentaxons penetrate the gray matter at this age (arrows, D). Scale bar 520 µm.


Nodal branching and fasciculation

In order to examine the details of initial collateralbranching, we utilized confocal microscopy in conjunctionwith Dil staining. Following Dil labeling of afferent axons,the spinal cord was flattened along mediolateral or dorso-ventral axes. Figure 4 shows montages of 2D-recon-structed confocal images from 10 µm optical sections.Lateral views of E12.5 preparations are shown in Figure4A and B. Primary afferent axons are relatively smoothand swellings or ‘‘nodes’’ are infrequent. Only an occa-sional branch is noted at this age (Fig. 4B, arrows).Dorsal views of E13.5 preparations are shown in Figure

4C and D. The appearance of axons has changed dramati-cally. At this stage, primary afferents growing in themedial portion of the dorsal funiculus exhibit many clearswellings, ‘‘nodes’’ and elaborate collateral branches nearthe bifurcation point (Fig. 4D, arrows). Interestingly, pri-mary afferent axons growing in the lateral portion of thedorsal funiculus are shorter than the medial ones and arestill relatively smooth with prominent growth cones (Fig.4C, arrowheads). These axons almost certainly represent adifferent functional class of later-born neurons than thosethat occupy a medial position (see below).Development of collateral branches and fascicles are

shown in the sagittal orientation in Figure 5A–D. Rela-tively smooth afferent axons with prominent growth conesare seen at E12.5. At E13.5, axons have many nodes andthe collaterals originating from nodes of parent axonsenter the gray matter. At E15.5, collateral branches fromneighboring axons are fasciculated. Between E15.5 andE17.5, the number of axons per fascicle appears to increasesubstantially. Note that collateral branches forming fas-cicles tend to originate from similar locations along therostrocaudal axis, suggesting tight spatial controls ofcollateral branching. It appears that there are many morenodes than collateral branches at every developmentalstage.

Initial trajectories of sensory afferentsin relation to laminar targets

In order to correlate the development of primary afferentprojections with the maturation of laminar targets, sec-tions containing Dil-labeled, photoconverted axons wereembedded in plastic and sectioned at 1 µm (Fig. 6). ByE13.5 (Fig. 6A, B), the dorsal funiculus and the dorsal rootentry zone have shifted dorsomedially in relation to theirposition at E12.5. The future dorsal horn is morphologi-cally distinct (Fig. 6B). Primary afferent axons (arrow-heads) penetrate the dorsal gray matter from the dorsome-dial portion of the dorsal funiculus at this stage. Theseaxons project parabolically toward the ventral spinal cordbetween the lateral edge of the ventricular zone and thedifferentiating dorsal horn. These axons traverse a regionoccupied by morphologically and biochemically distinctclusters of neurons (see below) along themidline and nevertraverse or branch into the developing dorsal horn.In order to compare the initial trajectories of cutaneous

and muscle afferents, we stained cutaneous and muscleafferents separately using Dil (red) and DiA (green),respectively (Fig. 6C). The first cutaneous afferent axons(large arrows) penetrate the spinal cord at E14.5 andproject directly ventrally and laterally to a distinct regionin the ventral aspect of the developing dorsal horn (Fig.6D). These axons do not branch into more superficial

regions. This distinctive target field suggests that theseaxons are large-caliber sensory afferents from low-threshold mechanoreceptors (Snider et al., 1992; Ruit etal., 1992; for a review, see Fyffe, 1992). In some prepara-tions, a few cutaneous axons also entered the spinal cordfrom the most lateral region of the dorsal funiculus atE14.5. In contrast to the large-caliber cutaneous afferents,these afferents project ventrally and medially and remainconfined to the superficial dorsal horn. These axons arepresumably NGF-dependent fine cutaneous afferents (Ruitet al., 1992, for a review, see Fyffe, 1992). At E15.5, finecutaneous afferents enter the superficial dorsal horn in fargreater numbers but do not project into ventral andmedialregions of the dorsal horn as shown in Figure 6E. Thus,each major class of sensory afferents exhibits a differentinitial trajectory toward its distinctive target field anddoes not elaborate branches into inappropriate regions.

Biochemical and morphologicalcharacteristics of neuronsin laminar target fields

We used several methods to assess the differentiation ofneurons along the pathways and in the target fields ofsensory afferents. A monoclonal antibody to microtubule-associated protein 2 (MAP2) was used to selectively labeldendritic arborizations of developing neurons in the dorsalhorn. Montages of 2D-reconstructed confocal images from10 µm optical sections are shown in Figure 7. At E13.5,MAP2-immunoreactive dendritic arborizations are abun-dant in the developing gray matter, although only few, ifany, dendrites are found in the region of the future dorsalfuniculus (arrowheads). This situation changes strikinglyat E14.5. ManyMAP2-immunoreactive dendrites are dem-onstrated in the lateral region of the developing dorsalfuniculus at this age (arrowheads). Interestingly, matura-tion of dendritic morphology occurs prior to, or concurrentwith, entrace of the fine cutaneous afferent projections (seeabove). Note, however, that dendrites in the white mattercannot account for the entry of all classes of afferents asthe medial region of the dorsal funiculus where muscleafferents penetrate the gray matter is MAP2-negative atE13.5.Monoclonal antibody 4D5 against members of LIM

homeobox gene proteins, Islet-1 and Islet-2, was used as amarker to define subclasses of biochemically distinct neu-rons in the developing spinal cord. At E13.5, in addition tothe lateral motor pool, immunoreactive neurons werealigned in the band along the lateral edge of the ventricu-lar zone (Fig. 8A, red dots). Since it has been observed inthe chick embryo that groups of dorsal horn neuronstransiently express Islet-1 but not Islet-2 (Ericson et al.,1992; Tsuchida et al., 1994; Shiga and Yaginuma, 1995),these neurons are likely to express Islet-1. At E14.5,although the immunoreactivity is down-regulated, labeledneurons are located in the medial gray (presumablyClarke’s column) and the intermediolateral nucleus aswell as in the lateral motor pool (Fig. 8B, red dots). Thepathways of sensory afferents at E13.5 and E14.5 arerendered in purple. The obvious correspondence betweenthe muscle afferent pathway and the pattern of Islet-1staining raises the possibility that muscle afferents taketheir ventral trajectories along Islet-1-positive neurons.We further characterized the differentiation of spinal

neurons related to sensory axons by staining their den-dritic arbors with Dil (Fig. 8). We focused on neurons


Fig. 4. Initial formation of nodes and collateral branches. A,B:Montages of 2D-reconstructed confocal images of Dil-labeled sensoryaxons at E12.5 viewed longitudinally from the lateral surface of thespinal cord. Single primary afferents are labeled in B. Axons arerelatively smooth exhibiting only a few swellings (‘‘nodes’’). Occasionalshort collateral branches are observed (arrows, B). C,D: Montages of2D-reconstructed confocal images from E13.5 preparations viewedlongitudinally from the dorsal surface of the spinal cord. Primary

afferents extending in the lateral portion of the spinal cord are muchshorter than medially-located axons and are relatively smooth withprominent growth cones (arrowheads, C). Single primary afferents arelabeled in D. Sensory axons at E13.5 have many clear swellings‘‘nodes’’ and elaborate collateral branches near the bifurcation point(arrows, D). D, dorsal; V, ventral; L, lateral; M, medial. Scale bars 5100 µm (A,C), 50 µm (B,D).

extending their axons to either the lateral funiculus(black) or ventral commissure (red). As reported in the ratembryo (Silos-Santiago and Snider, 1992, 1994), largenumbers of morphologically distinct classes of neurons areapparent. Notably in relation to sensory axon pathways,many of the midline neurons project across the ventralcommissure. In contrast, many deep and superficial dorsalhorn neurons project via the lateral funiculus. Thus, targetfields of muscle and cutaneous afferents are distinguishedby biochemical markers, dendritic morphology and axonprojections of spinal neurons prior to the penetration of thegray matter by sensory axons. We were unsuccessful inlabeling neurons in superficial lateral dorsal horn with

either commissural or lateral crystal placement. Thus, de-tailed morphological analysis of dendritic arbors and axonprojections of these neuronswill require further study.

DISCUSSION

Development of primary afferentcollateral branching

The present study shows that murine primary afferentaxons reach the thoracic spinal cord at E10.5 and growrostrocaudally for at least 48 hours prior to extendingcollateral branches into the spinal gray matter. Such a

Fig. 5. Collateral branching and fasciculation of primary afferentaxons in the developing spinal cord. A–D: 2D-reconstructed confocalimages of Dil-labeled afferents in the sagittal orientation at indicatedages. At E12.5, afferent axons tipped with growth cones are relativelysmooth (A). At E13.5, afferent axons are covered with nodes and

elaborate collateral branches from the nodes (B). At E15.5, collateralbranches originating from different axons fasciculate (C). By E17.5,fascicles contain many more collateral branches (D). Scale bar 5 20µm.


Fig. 6. Initial trajectories of primary afferent axons in relation tolaminar targets at E13.5–E15.5. A:Darkfield photomicrograph of a trans-verse semithin section of a Dil-labeled, photoconverted preparation. B:Tracing of the Dil-labeled axons shown inA superimposed on a brightfieldphotomicrograph of the same section. At E13.5, muscle afferents (arrow-heads, A) penetrate the gray matter and project parabolically toward theventral spinal cord avoiding the ventricular zone and the developing dorsalhorn. The motoneuron pool (MN) is backfilled in A. C: Double labeling ofmuscle afferents with DiA and cutaneous afferents with Dil at E14.5.Muscle afferents (green) penetrate from medial portion of the dorsalfuniculus andproject to the ventral horn in fascicles (arrowheads). Cutane-ousafferents (red) penetrate fromthe lateral portion of thedorsal funiculus

and project laterally toward the deep dorsal horn, sometimes crossing overmuscle afferents (large arrows). A few cutaneous afferents penetrate fromthe most lateral portion of the dorsal funiculus at this age and projectmedially to enter the superficial dorsal horn (small arrows). Themotoneu-ron pool (MN) is backfilled with DiA.D,E: Tracings of dorsal root afferentsfrom semithin sections superimposed on brightfield photomicrographs ofthe same sections. At E14.5, different populations of cutaneous afferentsproject to the superficial versus deep dorsal horn (yellow dots demarcatemorphologically distinct group neurons in the deep versus superficialdorsal horn). At E15.5, many more cutaneous afferents have entered thespinal cord but the demarcation between the two classes remains apparent(E). Scale bars5 100 µm (A,C), 50 µm (B,D,E).

‘‘waiting period’’ has been documented in the spinal cord ofall species so far examined (frog: Smith and Frank, 1988,chick: Lee et al., 1988; Davis et al., 1989, rat: Smith, 1983;Fitzgerald et al., 1991; Mirnics and Koerber, 1995). Themajor correlation we note here is that the onset of collat-eral branching does not occur until well after differentia-

tion of certain cell groups in the developing dorsal part ofthe spinal cord (see below).Aclear-cut finding of this study is thatmajormorphologi-

cal changes occur along the axons at the time collateralbranches are forming. For example, at E12.5, axons arerelatively smooth in the dorsal root as well as in the dorsal

Fig. 7. Extension of dendrites into the dorsal funiculus. Montagesof 2D-reconstructed confocal images of microtubule-associated protein2 (MAP2)-immunoreactive profiles in transverse sections of the dorsalhorn. A: At E13.5, few MAP2-immunoreactive dendrites are found inthe dorsal funiculus (border of the dorsal funiculus outlined with

arrowheads). B: At E14.5, MAP2-immunoreactive dendrites are dem-onstrated in the lateral region of the dorsal funiculus (arrowheads).Note that dendrites are absent more medially. High-power view of Baoutlined with lines is shown in Bb. Arrow in both A and Ba indicatesthe midline. Scale bars 5 50 µm (A,Ba), 20 µm (Bb).

Fig. 8. Biochemical and morphological characteristics of neuronsin laminar target fields. Composite drawings of camera lucida tracingsof Dil-labeled afferents (purple), Dil-labeled neurons extending axonsto either the lateral funiculus (black cells) or ventral commissure (redcells), and Islet-1 and Islet-2-immunoreactive cells (red dots). A:E13.5. B: E14.5. Note that spinal neurons have elaborated dendrites

before sensory afferents penetrate the spinal cord. Muscle afferentprojections are related to the Islet-1/-2-immunoreactive cells, most ofwhich send axons into the ventral commissure. Cutaneous afferentprojections are related to neurons lacking Islet-1/-2, most of which extendaxons into the ipsilateral lateral funiculus. Scale bars5 100 µm.


columns of the spinal cord. Elongating axon tips haveprominent growth cones at this stage and have clearly notreached targets in the medulla. Between E12.5 and E13.5,the morphological appearance of the axons changes dra-matically. Axons become studded with swellings (‘‘nodes’’)which decorate essentially the entire length of the axon inthe spinal white matter. Interestingly, nodes are lessprominent in the dorsal root outside of the spinal cord,which suggests that node development is induced by thespinal environment. These nodes are almost certainly thesite of future interstitial collateral sprouting. In fact,examples of collateral branching from nodes are clearlyapparent at E13.5. In vitro, axons of cortical neuronsappear to elaborate many collateral branches, only afraction of which are ever stabilized (Sato et al., 1994). Wecould not determine using these methods whether morebranches are elaborated initially along sensory axons thanpersist into maturity. However, at every stage in thisstudy, there were many more nodes than collateralbranches at least until postnatal day 1, the latest stageexamined.A similar ‘‘waiting period’’ due to a delay in the elabora-

tion of collateral branches has been intensively studied inthe corticopontine projection (O’Leary and Terashima,1988). O’Leary and collaborators have provided severallines of evidence that nodal branching from cortical axonsis triggered by the action of a diffusible chemotropicinfluence (Heffner et al., 1990; Sato et al., 1994). Althoughthe nature of this chemotropic influence is still unknown,candidate molecules include at least one member of theneurotrophin family, neurotrophin-3 (NT-3), which hasbeen shown to exert chemotropic influences on corticalaxons in explant cultures (O’Leary and Daston, 1994) andto induce collateral branches of corticospinal neuronswhen exogenously applied in vivo (Schnell et al., 1994).Neurotrophin-3 has been considered as a primary candi-

date to regulate collateral branching of spinal sensoryaxons, since it is intensely expressed in the ventral hornprior to the onset of collateral branching (Ernfors andPersson, 1991; Schecterson and Bothwell, 1992; Ozaki andSnider, unpublished observations). Indeed, it is knownthat proprioceptive sensory neurons require NT-3 forsurvival and express its signaling receptor TrkC (Ernforset al., 1994; Klein et al., 1994). However, in chick organo-typic cultures, muscle afferents can enter the gray matterexhibiting appropriate initial trajectories even when theventral spinal cord, the main source of NT-3, has beenremoved (Sharma et al., 1995). Furthermore, chronic NT-3treatment following early limb bud deletion in the chick inovo resulted in normal muscle afferent projections on thedeleted side which exhibit loss of limb motoneurons andabsence of NT-3 synthesis within the spinal cord (Oakleyet al., 1995). Finally, neither overexpression nor underex-pression of NT-3 in the spinal cord of transgenic miceinfluence proprioceptive axon projections to the ventralhorn (Ozaki et al., 1996). Thus, it now seems unlikely thatNT-3 triggers collateral branching of proprioceptive affer-ents.Another mechanism long been considered as likely to

regulate sensory axon collateral branching into the graymatter is the presence of inhibitory molecules in the dorsalroot entry zone (see Pindzola et al., 1993 and referencestherein). Indeed, Sema III, a member of the semaphorin/collapsin gene family, is transiently expressed by cells nearthe dorsal root entry zone of the rat embryo (Wright et al.,

1995). However, actions of Sema III appear to be specific toonly certain classes of sensory afferents (Messersmith etal., 1995) and thus cannot fully explain the ‘‘waitingperiod’’ unless its actions are developmentally regulated.However, Sema III is a member of a large family whichmay include as many as 17 members (see below). The rolesof other members related to sensory afferent projectionsremain to be elucidated. An additional group of moleculeswhich may have inhibitory effects on sensory afferentcollateral branching is the netrins, which are expressed inthe ventral region of the chick spinal cord at early develop-mental stages (Kennedy et al., 1994). Surprisingly, in viewof its expression pattern in the chick, netrin-1 is expressedin the dorsal root entry zone in the developing mousespinal cord prior to penetration of the gray matter bysensory axons (M. Tessier-Lavigne, personal communica-tion). Whether DRG neurons express receptors for thenetrins is unknown at present. Potential functions for bothclasses of molecules in regulating collateral branching ofsensory axons will soon be elucidated inmice with targetedgene mutations.

Collateral branching is correlated withdifferentiation of neurons in laminar targets

The present study shows, in apparent contrast to thesituation in the chick (Mendelson et al., 1992), thatdifferent classes of murine primary afferents enter thespinal cord in sequence. Muscle afferents penetrate thegray matter as early as E13.5, whereas large-calibersensory afferents first penetrate 24 hours later at E14.5. Afew fine cutaneous afferents enter at E14.5, but most enter24 hours latter at E15.5. Sequential entry of differentclasses of afferents in the rat lumbar spinal cord was alsodescribed by Mirnics and Koerber (1995). The reason forthis sequential projection of different classes of afferents isunknown but may be related to the fact that, in the mouse,large DRG neurons are generated in peak numbers onE10.5, while small DRG neurons arise in the greatestnumbers on E12 (Sims and Vaughn, 1979).Interestingly, considerable maturation of laminar target

neurons was observed prior to sensory axon projections(Fig. 8). Thus, collateral branching of muscle afferents andprojections toward the ventral horn does not occur untilafter differentiation of neurons that express Islet-1, manyof which project axons across the ventral commissure.Similarly, large-caliber cutaneous afferents do not projectto deep dorsal horn until after maturation of morphologi-cally heterogeneous populations of interneurons in thisregion, many of which project to the ipsilateral lateralfuniculus at the level of the midline. These cells do notexpress the homeotic protein Islet-1. Finally, superficialcutaneous afferents do not penetrate the gray matter untilafter the differentiation of neurons in superficial dorsalhorn by E14.5, which elaborate dendrites into the dorsolat-eral white matter.These findings are fully consistent with the results in a

chick organotypic culture system where maturation of thedorsal spinal cord is a key determinant of sensory axonprojections (Sharma et al., 1994). Thus, explants culturedat stage 28 (E6.0) show no dorsal horn maturation andappropriate central projections of sensory afferents do notdevelop. In contrast, explants cultured at stage 30 (E6.5)exhibit dorsal horn differentiation and appropriate region-specific sensory projections. Our findings are also consis-


tent with the demonstration that the morphology of rattrigeminal afferents entering the brain stem in explantcocultures is heavily dependent on the age of the brainstem explant (Erzurumlu and Jhaveri, 1995). At youngembryonic stages, axons are long and unbranched, whereasin older brain stem explants, increasing branching isobserved. The developmental sequence we describe here isfully consistent with the idea that spinal neuronal differen-tiation is a prerequisite to appropriate targeting andbranching of sensory axons.

Initial trajectories of afferent axons areconsistent with inhibitory influences

A major finding in the present study is that differentclasses of sensory axons avoid inappropriate laminae enroute to their specific target regions. This finding raisesthe question of whether inappropriate laminae exert inhibi-tory influences on sensory axon growth. Potential inhibi-tory domains for the different classes of sensory axons aresummarized schematically in Figure 9. At E13.5, muscle

Fig. 9. Schematics of primary afferent projections indicating poten-tial inhibitory regions. A–C: Camera lucida tracings of Dil (or DiA)-labeled afferents superimposed on photomicrographs of semithinsections at the indicated ages. Different classes of afferents arearbitrarily rendered in different colors: muscle afferents (green),large-caliber cutaneous afferents (red), fine cutaneous afferents(purple). Symbols (¢) of putative inhibitory domains for each class ofaxons are shown by using the color for that class. D: Brightfield

photomicrograph of Dil-labeled afferents after photoconversion atE17.5. Afferent axons projecting from the medial and lateral dorsalhorn (arrows) reach the corresponding laminae in the contralateralspinal cord (arrowheads). Note that sensory axons do not branch intoinappropriate laminae while projecting toward their targets. Scalebars 5 100 µm (A–C), 50 µm (D).


afferents projecting to the ventral horn are restricted in adomain bounded by differentiating neurons of the dorsalhorn laterally and the lateral edge of the ventricular zonemedially. Muscle afferents never contact the somata ofproliferating cells and never branch into the developingdorsal horn (Fig. 9A). The striking restriction of muscleafferent pathways shown here is entirely consistent withthe findings of Mirnics and Koerber (1995) in the rat thatthe first dorsal root afferents penetrated the lumbar cordat E15 in a region between the neuroepithelial zone anddeveloping dorsal horn. The findings of both studies thussuggest that neither of these regions is permissive forgrowth of muscle afferent axons. Of course, there are otherpossible explanations, e.g., that muscle afferents taketheir medial trajectory because of preferential fascicula-tion rather than inhibition (see Tang et al., 1992, 1994). Infact, a molecule with putative inhibitory influences onsensory axons, netrin-1, is expressed in these regions (M.Tessier-Lavigne, personal communication) and techniquesare available for a definitive analysis of its role in shapingthe developing pathways of muscle afferents.Cutaneous afferents also appear to avoid inappropriate

laminae. At E14.5, large-caliber cutaneous afferents pen-etrate the gray matter, project directly toward a group ofdeep dorsal horn neurons and do not arborize amongsuperficial cell groups located more laterally (Fig. 9B). AtE15.5, fine cutaneous axons enter the superficial layers ofthe dorsal horn in large numbers, but do not enter deeperlayers. Neither large-caliber nor fine cutaneous afferentsproject into the ventral region of the spinal cord (Fig. 9C).Indeed, many cutaneous afferents make sharp right angleturns at E15.5 to project medially toward the contralateralspinal cord. Interestingly, at E17.5, some cutaneous affer-ents traverse the entire width of the spinal cord to reach thecontralateral superficial targets, strictly avoiding both theventral spinal cord and deeper layers of the dorsal horn enroute (Fig. 9D). This latter finding again raises the possibilitythat not only the ventral spinal cord but also deeper layers ofthe dorsal horn are inhibitory for these cutaneous afferents.Inhibitory influences of the ventral spinal cord on DRG

neuron axon outgrowth have been demonstrated in vitro(Fitzgerald et al., 1993). Such inhibitory influences onsensory axon growth in the spinal cord are now thought tobe mediated by the recently identified member of thesemaphorin/collapsin gene family, Sema III (Messersmithet al., 1995). Consistent with functions in inhibiting axonsof cutaneous afferents in vitro, Sema III is localized to theventral spinal cord at appropriate developmental stages(Messersmith et al., 1995; Wright et al., 1995). Impor-tantly, at least four other members of the semaphorin genefamily are differentially expressed in the developingmousespinal cord. Thus, Sema B, C, and E (Puschel et al., 1995)as well as a novel family member (Zhou et al., 1995, 1996),are expressed in distinct laminae and down-regulatedafter E17 in the murine spinal cord. Although functions ofthese proteins in vivo remain to be defined, our findingsare fully consistent with the idea that molecules whichinhibit axon growth of primary afferents are likely tocontribute to the generation of laminar-specific patterns inthe developing spinal cord.

ACKNOWLEDGMENTS

We thank Dr. T.M. Jessell for the generous gift of themonoclonal antibody 4D5. We thank Mr. J.C. Harding and

S. Plurab for their expert technical assistance. We thankDrs. C. Keller-Peck, S.I. Lentz, and D.E. Wright for helpfuldiscussions and a critical reading of the manuscript. Thiswork was supported by R01 NS31768 and P01 50757 fromthe NINDS.

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initial trajectories of sensory axons toward laminar targets in the developing mouse spinal cord

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