Clarke JD et al. (1991), Neuroanatomical and functional analysis of neur...

XB-ART-24798

Development 1991 Jun 01;1122:499-516.

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Neuroanatomical and functional analysis of neural tube formation in notochordless Xenopus embryos; laterality of the ventral spinal cord is lost.

Clarke JD , Holder N , Soffe SR , Storm-Mathisen J .

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Notochordless Xenopus embryos were produced by u.v. irradiation of the uncleaved fertilized egg. The spinal cords were examined using intermediate filament staining for glial cells, retrograde HRP staining for neuronal morphology and an anti-glycinergic antibody to reveal commissural cells and axons. The floorplate cells of the normal cord appear to be absent and their position along the ventral midline of the cord is occupied by motor neurones, Kolmer-Agduhr cells, radial glial cells and a ventrally placed marginal zone containing the longitudinal axons. Motor neurone number is reduced to 15% of control values, and the sensory extramedullary cell number is increased twentyfold. Commissural axons are still able to cross the ventral cord but do so at abnormal angles and some commissural axons continue to grow circumferentially up the contralateral side of the cord rather than turning to grow longitudinally. Extracellular electrophysiological recordings from motor axons reveal that the normal alternation of locomotor activity on the left and right side of the embryo is lost in notochordless animals. These results suggest that the notochord and/or the normal floor plate structure are important for the development of the laterality of spinal cord connections and may influence motor neurone proliferation or differentiation.

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Species referenced: Xenopus laevis
Genes referenced: grap2 vim
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	Fig. 1. Xenopus embryos at stage 35/36 (A) and stage 29/30 (B) or their u.v.-treated equivalent. From top to bottom; normal embryo, IAD Type 1/2, IAD Type 3, IAD Type 4. -Lines en background grid are 1 mm apart.
	Fig. 2. (A) Photomicrograph of an unstained transverse section of a normal stage 35/36 spinal cord. Note lateral marginal zones and ventrally placed central canal. (B) Toluidine-blue-stained transverse section of a normal stage 35/36 IAD Type 1 spinal cord. Note central canal is now placed dorsally and the pale-staining marginal zone (*) lines the ventral half of the cord. (C,D) Electron micrographs of the ventral midline of Type 1 spinal cord shown in B. Note the characteristic radial, glia-like endfeet, normally only seen in the lateral marginal zones, the large extracellular spaces and, in D, several axonal profiles lying immediately adjacent to the basal lamina (arrows). (E,F) Transverse sections of normal (E) and Type 1 spinal cord (F) stained with the anti-vimentin antibody Z9. Vimentin-positive radial processes and endfeet are present only in the lateral marginal zones of normal cords, but are present also across the ventral midline of the uv-treated cord. My, myotomes; not, notochord; fp, floor plate; c, central canal; mz, marginal zone; ef, endfoot. Scale bar in A is 50 fan and applies to A, B, E and F, and is 1um in C and D.
	Fig. 3. Camera-lucida drawings of motoneurones. (A) Side-view whole-mounted spinal cord from a normal embryo. Neurons filled from a myotome HRP application. Large black cell is a primary motor neurone and small black cell is a secondary motor neurone. Positions and sizes of other motor neurone cell bodies are shown as unfilled outlines. Rohon- Beard cells are shown stippled along the dorsal cord. Rostral is to the left. (B) Transverse sections from a normal embryo showing two large primary motor neurones and three smaller secondary motor neurones. Note dendrites are restricted to one side only. No motor neurones are found ventral to the neurocoel. (C,D,E) Ventral views of whole-mounted spinal cords from u.v.-treated embryos. Motor neurone.s and their caudally running axons lie on or close to the ventral midline. In C both cells have dendrites that clearly extend in both left and right halves of the marginal zone. Arrows indicate positions where axons exit the cord. The most rostral cell in C is unusual in that the motor dendrites overlap with the Rohon-Beard axons (shown dashed). One Rohon-Beard axon is shown crossing the cord ventrally; this was only seen once. (F,G) Transverse sections from u.v.-treated embryos showing motor neurones with clear bilateral dendrites. Rohon-Beard axons are shown as short wavy lines dorsolateral to the motor cells. Rohon-Beard and extramedullary cells are stippled. (H,I) The position of motor neurones (ventrally) and Rohon-Beard and extramedullary cells (dorsally) plotted onto standardised section outlines. H is from a normal embryo and I from a u.v.-treated embryo. Note the large number of cells outside the spinal cord in I. My, myotome; not, notochord. Scale bar is 50um for B, C, H, I and 30 um for A, D, E, F, G.
	Fig. 4. (A.B) Transverse sections of normal cords containing HRP-filled motor neurones. A shows two large, heavily filled primary motor neurones and one small, pale secondary (arrowed). B shows a large primary and its dendrites confined to the ipsilateral marginal zone. (C,D) Transverse sections of u.v.-treated cords containing large HRP-filled motor neurones along the ventral midline. In C a labelled Rohon-Beard cell is present in the dorsolateral cord. (E,F) Transverse sections of u.v.-treated cords containing HRP-labelled sensory and motor axons and several extrameduUary cells (curved arrows) attached in lines or bunches to the dorsal cord surface. In E note that the sensory axons (filled straight arrows) occupy the dorsolateralmost region of the ventral marginal zone and the motor axon (open arrow) lies midventrally. My, myotomes; not, notochord; asterisk, melanocyte. Scale bar is 50j/m.
	Fig. 5. Camera-lucida drawings of neurones with commissural axons. (A) Side view, and (B) ventral view of whole-mounted spinal cords from normal embryos. Cells labelled from HRP applied to the contralateral rostral spinal cord. A shows the two morphological classes seen. Dorsolateral commissural interneurones have multipolar cell bodies and a thin ventrally directed axon, while commissural interneurones (stippled) have unipolar cell bodies and dendrites that arise from a thicker ventrally directed process. B shows that the axons from these cells cross the ventral surface at angles close to 90Â° to the long axis. (C) Side view and (D) ventral view of whole-mounted spinal cords from u.v.-treated embryos. Cells labelled from HRP applied to the contralateral rostral cord. C shows that both morphological classes of commissural neurone are present (compare with A). D shows that the angle at which their axon crosses the ventral midline (shown dotted) is more variable than in control spinal cords. Scale bar is 50/an.
	Fig. 6. Camera-lucida drawings of Kolmer-Agduhr cells. (A) Transverse section containing a Kolmer-Agduhr cell filled caudal to a rostral cord HRP application in a normal embryo. This cell has a typically simple morphology, contacts the cerebrospinal fluid and occupies a position adjacent or close to the floor plate cells. (B) Transverse section containing a Kohner-Agduhr cell filled caudal to a rostral cord HRP application in a u.v.-treated embryo. This cerebrospinal fluid contacting cell has characteristic simple morphology but now occupies a position close to the midline beneath the neurocoel. (C) The positions of seven Kolmer-Agduhr cells from a u.v.-treated embryo have been superimposed on a typical transverse section. All but one occupy a position close to the midline. (D) Side view of whole-mounted spinal cord. In this u.v.-treated case, four Kohner-Agduhr cells have been filled rostral to a caudal HRP application. This is never seen in normal embryos. The outline of the neurocoel is indicated, caudal is to the left and ventral at the bottom. Scale bar is 50 um.
	Fig. 7. Histograms to show the variability in motor neurone size in control and u.v.-treated embryos. The large numbers of small cells (less than 100 /xm2) are absent from u.v.-treated embryos. Data collected from 5 control animals and 4 experimental animals. The cross-sectional area of cell bodies was measured from enlarged cameralucida drawings of HRP stained motor neurones.
	Fig. 8. Interneurones. (A,B) Camera-lucida drawings of transverse sections containing neurones filled from rostral cord (in B) and caudal cord (in A) in u.v.-treated embryos. Notice how many cells have a clear dorsal to ventral polarity of their main process. Stippled cells are Rohon-Beard cells and extramedullary cells. Unfilled cell profiles are from cells with no clear processes and which are thus probably oriented in a rostrocaudal direction. (C) Photomicrograph of the side view of a whole-mounted u.v.-treated cord. HRP was applied to the rostral cord (to the left). Note the predominant dorsal-to-ventral orientation of most of the labelled cells. Scale bar is 50 fim for A and B, and 100 um for C.
	Fig. 9. Photomicrographs of the ventral surface of whole-mounted cords stained for glycinergic axons. A and B are from control embryos, A from the rostral cord/caudal hindbrain area and B from midtrunk cord. Note the stained axons cross the ventral cord in a fairly direct manner. C and D are from equivalent regions of a u.v. type 1 embryo. Note now how the angle of crossing is highly variable. Scale bar is 50um and applies to all.
	Fig. 10. Histograms to show the variability of angle of crossing of glycinergic commissural axons in control and u.v.-treated embryos. Angles were measured relative to the ventral midline of the spinal cord, those directed rostrally have values less than 90Â° and those directed caudally have values greater than 90Â°. Angles were allocated into bin widths of 10Â°. Note the increased variability in the experimental cords and the increased proportion that are directed caudally (hatched) rather than rostrally.
	Fig. 11. Camera-lucida drawings of glycinergic cells and axons. (A,B) Ventral views of whole-mounted spinal cords from u.v.-treated embryos. The positions of all glycinergic cell bodies within the drawn segments are indicated, but only some of the axons are included for clarity. Most of the undrawn axons run longitudinally along the ventral surface of the cord and would thus obscure other details. The arrowed axons have all grown dorsally and probably all arise from contralateral cell bodies. This is clearly seen in three of the filled cells in A and in all four of the filled cells in B. The remaining two filled cells in A have caudally directed axons which remain ipsilateral for unusually long distances before eventually crossing to the other side of the cord. The growth cones of these cells were clear. Dotted lines represent axons growing on dorsal surface of cord. (C,D) Side views of whole-mounted spinal cords from u.v.-treated embryos. Arrows indicate trajectories of dorsally positioned glycinergic axons. In some cases (C) axons turn to grow back down ventrally on the same side of the cord, while in others they continue circumferentially and grow over the dorsal surface and down ventrally on the contralateral side of the cord (dotted in D). Stipple in C indicates position of large numbers of stained longitudinal axons. Scale bar is 50 um.
	Fig. 12. Motor neurone activity in paralysed embryos. (A) Control embryo. Extracellular action potentials recorded from one left (L) and one right (R) intermyotome cleft region. Activity was evoked by a single electrical stimulus to the skin (not shown). Note that the activity in left and right usually alternates. Occasionally short periods of synchronous activity occurs, usually towards the start of an episode. Phase relations are indicated by dotted lines. (B) U.v.-treated embryo. Extracellular potentials recorded from left and right intermyotome regions now show no periods of alternating motor activity, rather the activity is always synchronous. Scale bar is 50um. syn, synchrony.