early appearance of tyrosine hydroxylase immunoreactivity in the retina of human embryos

5
Developmental Brain Research, 69 (1992) 283-287 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00 283 BRESD 60473 Early appearance of tyrosine hydroxylase immunoreactivity in the retina of human embryos Claudine Versaux-Botteri a,b, Catherine Verney c, Nada Zecevic d and Jeanine Nguyen-Legros a a INSERM U-86, Laboratoire de Neurocytologie Oculaire, Paris (France), t, Museum National d'Histoire Natureile, Laboratoire d'Anatomie Compar~e, Paris (France), c INSERM U-106, H~pital de La Salp~tri~re, Paris (France) and d Institute for Biological Research, Beograd (Yugoslavia) (Accepted 23 June 1992) Key words: Human; Retina; Development; Dopamine; lmmunocytochemistry; Tyrosine hydroxylase In the retina of 6 post-ovulatory week old human embryos, tyrosine hydroxylase (TH) immunoreactivity is expressed in retinoblasts in the peripheral retina, and ganglion-like cells with an axon in the optic nerve in the posterior retina. This is the first report on the expression of a catecholamine marker in a sub-population of migrating retinoblasts. Since in the adult retina the only TH + cells are a sub-population of amacrine cells, the expression of this enzyme in some ganglion-like cells must represent either a transient developmental event, or indicate that these cells subsequently undergo transformation through axonal degeneration. The establishment of neurotransmitter systems dur- ing retinal development has been studied in a number of mammalian species, including rodents, cat and man 6. Except for 7-aminobutyric acid (GABA), which is de- tected at early embryonic stages (probably related to its neurotrophic activity) 2t, it is currently assumed that neurotransmitters are synthesized after the cessation of mitoses and migrations, during retinal differentiation. The dopamine (DA) system was thought to follow this general plan of maturation 4'5'12'14'15'27'2s'29. In rats, which are born with closed eyes, the emer- gence of the DA system in the retina is mainly postna- tal, because of its immaturity at birth. DA is first detected biochemically at PN day 7 and reaches a concentration sufficient for its cellular localization by fluorescence histochemistry a few days later s. The ex- pression of tyrosine hydroxylase (TH, the biosynthetic enzyme of DA) immunoreactivity, a marker of DA cells in the retina, is detected earlier, about the day of birth n'~4'28. At this stage, the DA cells have clearly emerged from the neuroblastic layer to occupy the amacrine cell layer, their [3H]thymidine birthdate indi- cating that they were born at E18-E19 during the period of amacrine cell formation (E14-E20, full term being E21) 3. In rats, the DA system of the retina reaches its full maturity a week after eye opening (PN day 14-15). In contrast, in guinea-pigs which are born with their eyes open, the DA system of the retina is mature at birth 2°. The DA cells can already be visualized by fluorescence histochemistry during fetal life (E47, full term being E68) and it is highly probable that TH labeling would visualize DA cells earlier in embryonic development. In the human, which resembles more closely the guinea-pig with respect to retinal development, TH-im- munoreactive (THI) cells have been observed in flat mounted small pieces of retina at 24 post.ovulatory weeks. By this date they exhibit an immature appear- ance, with short thick dendrites terminating in growth cones Is. However, since younger retinas were not ob- served, the date of their initial appearance was un- known. Human embryos were obtained in conditions suit- able for a rapid fixation in toto which enabled us to observe the early appearance of TH immunoreactivity (THIR) in the neural retina. Nine human embryos, 5-7 post-ovulatory weeks old Correspondence: J. Nguyen-Legros, Laboratoire de Neurocytologie Oculaire, 15 rue de I'Ecole de M~decine, 75270 Paris Cedex 06, France. Fax: (33) 40 46 87 77.

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Page 1: Early appearance of tyrosine hydroxylase immunoreactivity in the retina of human embryos

Developmental Brain Research, 69 (1992) 283-287 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00

283

BRESD 60473

Early appearance of tyrosine hydroxylase immunoreactivity in the retina of human embryos

Claudine Versaux-Bot te r i a,b, Cathe r ine Verney c, N a d a Zecevic d and Jean ine Nguyen-Legros a

a INSERM U-86, Laboratoire de Neurocytologie Oculaire, Paris (France), t, Museum National d'Histoire Natureile, Laboratoire d'Anatomie Compar~e, Paris (France), c INSERM U-106, H~pital de La Salp~tri~re, Paris (France) and d Institute for Biological Research, Beograd (Yugoslavia)

(Accepted 23 June 1992)

Key words: Human; Retina; Development; Dopamine; lmmunocytochemistry; Tyrosine hydroxylase

In the retina of 6 post-ovulatory week old human embryos, tyrosine hydroxylase (TH) immunoreactivity is expressed in retinoblasts in the peripheral retina, and ganglion-like cells with an axon in the optic nerve in the posterior retina. This is the first report on the expression of a catecholamine marker in a sub-population of migrating retinoblasts. Since in the adult retina the only TH + cells are a sub-population of amacrine cells, the expression of this enzyme in some ganglion-like cells must represent either a transient developmental event, or indicate that these cells subsequently undergo transformation through axonal degeneration.

The establishment of neurotransmitter systems dur- ing retinal development has been studied in a number of mammalian species, including rodents, cat and man 6. Except for 7-aminobutyric acid (GABA), which is de- tected at early embryonic stages (probably related to its neurotrophic activity) 2t, it is currently assumed that neurotransmitters are synthesized after the cessation of mitoses and migrations, during retinal differentiation. The dopamine (DA) system was thought to follow this general plan of maturation 4'5'12'14'15'27'2s'29.

In rats, which are born with closed eyes, the emer- gence of the DA system in the retina is mainly postna- tal, because of its immaturity at birth. DA is first detected biochemically at PN day 7 and reaches a concentration sufficient for its cellular localization by fluorescence histochemistry a few days later s. The ex- pression of tyrosine hydroxylase (TH, the biosynthetic enzyme of DA) immunoreactivity, a marker of DA cells in the retina, is detected earlier, about the day of birth n'~4'28. At this stage, the DA cells have clearly emerged from the neuroblastic layer to occupy the amacrine cell layer, their [3H]thymidine birthdate indi- cating that they were born at E18-E19 during the period of amacrine cell formation (E14-E20, full term

being E21) 3. In rats, the DA system of the retina reaches its full maturity a week after eye opening (PN day 14-15).

In contrast, in guinea-pigs which are born with their eyes open, the DA system of the retina is mature at birth 2°. The DA cells can already be visualized by fluorescence histochemistry during fetal life (E47, full term being E68) and it is highly probable that TH labeling would visualize DA cells earlier in embryonic development.

In the human, which resembles more closely the guinea-pig with respect to retinal development, TH-im- munoreactive (THI) cells have been observed in flat mounted small pieces of retina at 24 post.ovulatory weeks. By this date they exhibit an immature appear- ance, with short thick dendrites terminating in growth cones Is. However, since younger retinas were not ob- served, the date of their initial appearance was un- known.

Human embryos were obtained in conditions suit- able for a rapid fixation in toto which enabled us to observe the early appearance of TH immunoreactivity (THIR) in the neural retina.

Nine human embryos, 5-7 post-ovulatory weeks old

Correspondence: J. Nguyen-Legros, Laboratoire de Neurocytologie Oculaire, 15 rue de I'Ecole de M~decine, 75270 Paris Cedex 06, France. Fax: (33) 40 46 87 77.

Page 2: Early appearance of tyrosine hydroxylase immunoreactivity in the retina of human embryos
Page 3: Early appearance of tyrosine hydroxylase immunoreactivity in the retina of human embryos

TABLE I

Evolution of TH labeling in retinae of human embryos aged 5 to 7 weeks

285

Embryo no. Anatomical e.'ents Age (weeks) Pattern of labeling

20 [Pigment appears in RPE 5 0 ,l Lens vesicle formed

21 ~Retinal fissure unclosed 5 0 23 l, No layer of Chievitz in the retina 5 0

19 RPE uncompletely pigmented 5.5 Lens vesicle with cavity Chievitz layer appears in the central retina

5 f RPE completely pigmented 6 Retinal fissure closed

8 Chievitz layer extends until mid eccentricity 6

9 ( Lens cavity reduced to a thin slit or closed 6.5 (Figs. 1-5) / Chiewitz layer extends until 3 /4 eccentricity 6.5 16 Nerve fiber layer (NFL)

15 Lens cavity closed 6.-7 (Figs. 6-7) Cornea and eyelid folds

diffuse labeling in the ventral retina at about 1/4 eccentricity

I Migrating retinoblasts at mid eccentricity Ganglion-like cells in central retina, with fibers and growth cones

Migrating retinoblasts in far periphery Ganglion-like cells with fibers in the NFL and optic nerve

Fibers of ganglion-like cells in the optic nerve until the optic chiasm

(Streeter stages 15-21t9), were obtained from legal abortions with no postmortem delay. Whole embryos were fixed in 4% buffered paraformaldehyde for 8 h, rinsed and cryoprotected in buffered sucrose and seri- ally cryostat sectioned. The TH antiserum (provided by A. Vigny), which has been extensively used to demon- strate DA cells in the vertebrate retina t6, was used 1/6000. Dopamine-/3-hydroxylase (DBH) antiserum (provided by J.-P. Henry) was also used 1/6000 in alternate sections. The immunoreactivity was demon- strated with the streptavidin-biotin-peroxidase method (Amersham) as described in a previous paper 2s, using DAB as a chromogen. Some sections were treated by classical histological staining methods.

No THIR was observed in the eyes of 5 post-ovula- tory week old embryos, but first became detectable a few days later. No DBH immunoreactivity (DBHIR) was observed at any stage. The eyeball in which the first consistent THIR was detected exhibited morpho- logical characteristics in agreement with the descrip- tion of 6-7 post-ovulatory week human embryo eyem'23'26: the optic fissure was closed, the lens cavity was closed or reduced to a thin slit, and the cornea and eyelids were developing. The hyaloid vessel system was well developed in the primitive vitreous. The retinal pigment epithelium (RPE) was reduced to a single layer of cylindrical cells and the inverted layer (neural retina) was closely apposed to it. The optic stalk was

¢-.

Fig. 1. a: morphological characteristics of the eyeball "Jt the stage of first THIR observation. Nissl staiaing. The lens cavity is completely closed; the cornea and eyelids are developing; the RPE is reduced to a single layer and completely pigmented; the neural retina is divided into two neuroblastic layers by the transient layer of Chievitz, except in the far periphery (arrow) where a single neuroblastic layer is observed. Tire small

detachment is artefactuaL Bar -- 100 ~m. b: mitotic figure observed in the outermost row of ventricular cells in the same retina. Bar = 4 pm.

Fig. 2. a: migrating retinoblasts exhibit THIR in the peripheral retina. L, crystallin lens. Bar = 50/~m. b: in the posterior retina THIR is observed in post-migratory rounded somata and fibers in the nerve fiber layer. Bar = 50/~m.

Fig. 3. An enlarged view of the TH! retinoblasts in the peripheral retina: the nuclei observed at different levels of the single neuroblastic layer exhibit attachment processes to both limiting membranes of the neural retina. Some are still located very near the ventricular cell layer (arrows). On the left, which represents more central positioned retina, post migratory cells which have lost their attachment processes are observed

(arrowhead). Bar = 10 pro.

Fig. 4. Post.migratory TH! cells in the inner neuroblastic layer and TH! fibers in the NFL of the posterior retina. Although the cells exhibit short processes, they cannot be traced to fibers in the NFL. Bar = 10 ,¢m.

Fig. 5. In this oblique section, some THI fibers can clearly be traced to their parent cells (arrows). Bar = 10/~m.

Fig. 6. THI fibers in the NFL can be seen entering the optic nerve head on both sides of the hyaloid artery (arrow). Bar = 10/~m.

Fig. 7. Section of the optic nerve at a short distance from the eyeball. A number of THI fibers are running parallel to the optic nerve fibers toward the optic chiasm. Bar = 10/~m.

Page 4: Early appearance of tyrosine hydroxylase immunoreactivity in the retina of human embryos

286

filled with fibers from the developing ganglion cells of the retina. As would be expected from the center-pe- riphery gradient of differentiation, the posterior retina was separated into two neuroblastic layers by the tran- sient layer of Chievitz. The outer neuroblastic layer was composed of vertically elongated retinoblasts, while the inner neuroblastic layer contained several rows of rounded somata. A nerve fiber layer (NFL) was ob- served vitreally. In the periphery, a single neuroblastic layer contained numerous rows of retinoblasts and ventricular cells (Fig. ia). Mitotic figures occurred in the most scleral cell rows, i.e. in ventricular cells (Fig. lb).

THIR was found in several different structures: (i) in the peripheral retina, elongated neuroblastic cells with attachments to both inner and outer limiting membranes were heavily labeled. Their nuclei were located at different levels within the neuroblastic layer except in ventricular cell layer (Figs. 2a and 3); (ii) in the posterior retina, THIR was observed in a number of the round somata forming the inner neuroblastic layer (Figs. 2b and 4); (iii) labeled fibers were observed in the NFL (Fig. 4). In favourable instances, these fibers could be traced to their parent cells among the labeled round somata (Fig. 5). The optic nerve head contained numerous widely spaced THI fibers which could be followed in optic nerve sections through the head of the embryos (Figs. 6 and 7) until the optic chiasm. The zone of transition between the two pat- terns of labeling paralleled the extension of the layer of Chievitz and was observed at slightly different eccen- tricities among the specimens.

Table I summarizes the evolution of TH labeling during this period.

We report here the first appearance of THIR in the retina of 6 week old human embryos. In the retina, TH is considered as a specific marker of dopaminergic neurons because noradrenalin and adrenalin can not be biochemically detected in significant amount 2. THIR is expressed in two sorts of cells in the human embry- onic retina, retinoblasts and primitive ganglion and/or amacrine cells, each raising specific questions.

In the peripheral retina THIR is expressed in undif- ferentiated but postmitotic retinoblasts (mitoses being restricted to the ventricular cell layer where no l~bel is observed). It is unknown as to whether this early ex- pression is specific to human retina as there are no reports on the developmental appearance of THIR in the retina of other precocial mammalian species. A possible comparison concerns the addition of newly formed THI cells in the germinal zone of the tadpole retina, which grows continuously during the larval pe- riod, recapitulating the early stages of development 22.

The THI cells observed in the marginal zone possess small weakly THI somata without neurites or attach- ment processes, and are located in the position of amacrine cells. In rats, the first THI cells observed in the retina at birth have the same morphological char- acteristics. At most they sometimes bear a remnant of a trailing process but no longer have any attachments to the limiting membranes, indicating that they have finished their migration ~. Conversely, the early detec- tion .of catecholamine markers in neurons undergoing migration has been reported in rodent and ~uman brain ~. In the retina, it has been suggested that differ- entiation as defined by expression of cell type specific markers occurs rapidly after final mitosis and before significant migration ~. The present observation of THIR in postmitotic retinoblasts would indicate that not only is the cell type programmed just after mitosis, but also the neurotransmitter used by a specific cell type, at least for a subtype of dopaminergic amacrine cells.

The second sub-population of THI cells in embry- onic human retina is more puzzling. In the posterior pole of the eye, THIR is found in postmigratory cells a number of which possess an axon in the NFL. Since these cells derive from the internal neuroblastic layer in which are segregated the future amacrine and gan- glion cells (which are born simultaneously), it is likely that the two cell types are represented in the THI cells. Although some THI axons were observed in the NFL of adult human retina 17, they were very rare as com- pared with the number observed in these embryos. In addition, they could not be clearly traced to THI somata, which are classically thought to be amacrine cells even located in the ganglion cell layer. Thus the presence of a great number of THI ganglion-like cells in early development raises the question of their tran- sient existence. Whether it is the expression of THIR, the presence of an axon or the survival of the cells which is transient is currently unknown, and evidence exists for all three possibilities. Firstly there are exam- ples of transient expression of THIR during develop- ment of the central nervous system z4. Secondly, in the retina, about 80% of the ganglion cells die during development 9. And thirdly, two modes of formation have been described for amacrine cells in rodents 7. Either they derive from retinoblasts which stop their migration upon reaching the amacrine cell layer, or they come from primitive ganglion cells whose axon degenerates as a result of competition for connections in visual relays. This latter mode of formation which leads to 'displaced' amacrine cells could be favored in the primate retina whose proportion of displaced THI amacrine cells (25% of the total THI cell population) is

Page 5: Early appearance of tyrosine hydroxylase immunoreactivity in the retina of human embryos

the highest among mammalian species ~°,t3,tT. The ob-

servation of older specimens would be needed to fol-

low this evolution.

Finally, since there is no data available on concomit-

tent D A synthesis in the retina of the human embryo,

it is possible that this early T H I R constitutes the

expression of ~a catecholamine cell type o ther than the

D A cell population. This hypothesis is interesting be-

cause of the existence of two THI cell populations in

the primate retina, one of which, the 2CA cells, cannot

be clearly referred to as dopaminergic cells at the

present t ime ~°aT. The same suggestion was recently

proposed concerning the early appearance of T H I R in

the rat retina, which also contains a sub-population of

2CA cells 2s. However, the absence of labeling with

D B H antibody in the developing human retina, whereas

it occurs in the brain of the same staged embryos,

eliminates the possibility of this populat ion being nora-

drenergic or adrenergic.

This research project obtained approval from the Ethical Committee of University Clinical Center Belgrade and from the French Com- mit6 Consultatif National d'Ethique pour les Sciences de la Vie et de la Sant6. The authors thank Drs A. Vigny and J-P. Henry for providing TH and DBH antisera, Mrs. C. Aivarez and G. Prenant for technical assistance, and Dr. D. Hicks for correcting the English. This work was partly supported by Grant Cll CT90 0848 from CEE.

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2 Da Prada, M., Dopamine content and synthesis in retina and nucleus accumbens septi: pharmacological and light-induced modifications. In E. Costa and G.L. Gessa (Eds.), Advance in Biochemical Psychopharmaeology, Vol. 16, Raven Press, New York, 1977, pp. 311-319.

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