Anat Embryo1 (1991) 183:313-320
Anatomy and Embryology 9 Springer-Verlag1991
Developmental profile of a fetuin-like glycoprotein in neocortex, cerebrospinal fluid and plasma of post-natal tammar wallaby (Macropus eugenil) S.E. Jones 1, D.L. Christie 2, K.M. Dziegielewska 1, L.A. Hinds 3, and N.R. Saunders 1 1 Clinical Neurological Sciences and The Wessex Area Neurosciences Group, University of Southampton, Southampton General Hospital, Tremona Road, Southampton, Hants, SO9 4XY, UK 2 Department of Biochemistry, University of Auckland, Private Bag, Auckland, New Zealand a CSIRO Division of Wildlife & Ecology, PO Box 84, Lyneham, ACT 2602, Australia Accepted November 16, 1990
Summary. A fetuin-like glycoprotein (FLG) has been shown to be present in early cortical plate cells in the developing brain of the tammar wallaby (Macropus eugenii). The developmental sequence of the occurrence of glycoprotein-positive fibres and cells in the dorsolateral telencephalic wall from newborn to day 40 is described. The level of F L G in CSF (cerebrospinal fluid) and plasma of the tammar wallaby has also been measured during pouch life. The presence of FLG in early postnatal fibre systems and in some cells in the primordial plexiform layer, as well as in early cortical plate cells of the tammar is similar to that of fetuin in fetal brain in sheep, pig and cow, and ~2HS glycoprotein in human fetal brain. The sequence of appearance of FLGpositive cells during neocortical development in the tammar is strikingly similar to that of a transient population of early cortical plate cells previously described in fetal cat and sheep cortex. During postnatal development, levels of F L G in tammar plasma and CSF follow a pattern different from that of other species. The developmental expression of all three related glycoproteins in their respective species is discussed. Key words: Brain development - Fetuin
Marsupial- Neocortex
Introduction Several plasma proteins have been identified within cells in the immature brain (see e.g. Mollggtrd et al. 1988). However the function of these proteins in nervous system development is not yet understood. In addition, several studies have shown that there are high concentrations of proteins, that probably originate from plasma, in fetal cerebrospinal fluid (CSF) early in gestation (see Offprint requests to ." N.R. Saunders
Dziegielewska and Saunders 1988 for references). Plasma or serum, usually from bovine fetuses, is commonly used in tissue culture; studies with well-defined culture media have shown the importance of certain plasma proteins (e.g. transferrin) for specific features of cell and tissue development in vitro (e.g. Bottenstein et al. 1980); it may be that these glycoproteins have similar properties in vivo. One fetal protein, fetuin, is a frequent constituent of culture media (e.g. Rizzino and Shermann 1979); it has also been shown to be present in early cortical plate cells in the developing neocortex (MollgSrd et al. 1984; Reynolds et al. 1987). Structurally similar proteins have been identified in plasma, CSF and developing brain of human fetuses (~2HS glycoprotein, Dziegielewska et al. 1987) and in the postnatal tammar wallaby (Jones et al. 1988); e2HS glycoprotein and bovine fetuin have about 65% sequence identity and belong to the superfamily of cystatins (Dziegielewska et al. 1990). The developmental profiles in plasma and CSF of fetuin in sheep and pig fetuses and ~2HS glycoprotein (~2HS) in human fetuses do not follow a similar pattern. Thus in the animals of the order Artiodactyla (sheep and cattle) the level of fetuin in both plasma and CSF is very high early in development; it remains elevated in plasma (e.g. 400 rag/100 ml in fetal sheep) until birth and then falls by an order of magnitude in the adult (Dziegielewska et al. 1980). In contrast, in human fetal and adult plasma the level of ~2HS remains fairly constant at rather more modest levels (50-70 rag/100 ml); nevertheless the appearance and disappearance of these proteins in the developing neocortex follow a similar pattern (Dziegielewska et al. 1987). Recently we have identified a related protein in plasma of the tammar wallaby (Jones et al. 1988). Here we present the data describing the developmental profile of the wallaby equivalent of fetuin (fetuin-like glycoprotein, FLG) in neocortex, CSF and plasma. Fetuin or a related glycoprotein is expressed in the immature cortical plate in
314
Eutheria, very early in gestation (e.g. a r o u n d E35 in sheep fetuses which are b o r n at E150). The results o f this study show that the fetuin-related glycoprotein, F L G , in the t a m m a r brain is also expressed in early cortical plate cells, but as a postnatal p h e n o m e n o n , because o f the very early stage o f brain development at which the y o u n g o f this species are born.
at or close to the interventricular foramen. A description o f the dorsolateral neocortex in the t a m m a r has been published previously (Reynolds et al. 1985). The stages o f cortical plate development used in the description below, are f r o m Reynolds et al. (1985).
Distribution of fetuin-like glycoprotein in the neocortex Materials and methods Animals. The animals used in this study came from an established breeding colony maintained at the CSIRO Division of Wildlife and Ecology Research in Canberra, Australia. Details of breeding and dating of the animals have been described previously (Reynolds et al. 1985).
CSF and plasma samples. CSF and plasma samples from dated animals (ages and numbers are indicated in Table 2) were collected as described previously (Dziegielewska et ai. 1986). All samples were kept at - 2 0 ~ C until used. Fixation and tissuepreparation. Brain tissue was prepared for histological examination as previously described (Reynolds et al. 1985). Briefly, brains were fixed in Bouin's fixative for 6-10 h, followed by a brief wash in tap water; they were dehydrated through graded alcohols, cleared in chloroform and embedded in paraffin wax MP 56~ C. Serial sections, 6 ~tm thick, were cut through the forebrain in the coronal and sometimes sagittal planes. Pairs of sections from every 30 were selected for histological examination; one was stained with toluidine blue or haematoxylin and eosin, the other was subjected to immunocytochemistry to demonstrate FLG. Two to six brains were examined at each age studied (rib, 3d, 5d, 6d, 9d, 15d, 16d, 17d, 20d, 30-210d).
Immunohistochemical staining. Sections were dewaxed, rehydrated and incubated in blocker solution (0.35% bovine serum albumin, 0.05% Tween in phosphate buffered saline, PBS) for 20 rain. Primary antiserum, mouse anti-FLG or anti-FLG peptide (Jones et al. 1988), diluted 1:100 in blocker solution, was applied for 16 h at 4~ C, the sections being kept in humidified boxes. After washing in three changes of PBS, the blocker solution was applied and sections incubated for 10 rain. Sections were then incubated for 30 min with secondary antibody (rabbit anti-mouse IgG, DAKOPATTS, Denmark, diluted 1 : 50 in blocker solution). After three washes in PBS, sections were incubated for 30 rain in PAP complex (Mouse peroxidase-antiperoxidase, DAKOPATTS, Denmark) diluted 1 : 50 in blocker solution. Three washes in PBS were followed by 10 min wash in 0.05 M Tris, pH 7.6, and the colour reaction was developed with 0.025% DAB in 0.05 M Tris buffer, pH 7.6 with l0 ~tl/100 ml of 30% H2Oz. After extensive washing in tap water the sections were dehydrated in graded alcohols, cleared in xylene and mounted with DPX mountant. Positive and negative control sections were included in each run as described previously (Reynolds et al. 1987).
Preparation of protein standard, antiserum and quantitative determination offetuin-Iike glycoprotein. Fetuin-like glycoprotein was separated to purity (Jones et al. 1988) and used as standard. Antibodies were prepared as described previously (Jones et al. 1988). The radial immunodiffusion method of Mancini (Mancini et al. 1965) was used for quantitative determination of the concentration of FLG in plasma and CSF of developing pouch young.
Results Adjacent sections were stained with haematoxylin and eosin, and for F L G . The brain region examined was
Newborn to 3 days postnatal (precortical plate stage). On the d a y o f birth, as previously described by Reynolds et al. (1985), the dorsolateral wall o f the forebrain vesicle consists only o f a deep pseudostratified layer o f ventricular zone cells and an outer primordial plexiform layer. The latter contains a very few widely scattered cells o f variable size and shape and w i t h o u t obvious processes. The primordial plexiform layer also contains a fine reticu l u m o f incoming fibres, the origin o f which is unclear. This reticulum is stained with F L G antibodies (Fig. 1). In contrast, few o f the cell bodies in the primordial plexif o r m layer stain for F L G on the day o f birth. Staining for F L G is clearly a p p a r e n t in precipitated C S F within the brain ventricles on the d a y o f birth and subsequently (Table 1). A few cells in the ventricular zone that are immediately adjacent to the ventricular (csf) surface are strongly stained for F L G (Fig. 1, nb). This appearance is consistent with the protein having been taken up by the cells f r o m C S F (see Discussion). By 3 days postnatal, the brain vesicles are slightly enlarged (brain g r o w t h in this species is relatively slow; see Renfree et al. 1982; Reynolds et al. 1985). M o r e cells are present within the primordial plexiform layer but they are still widely dispersed. A few are faintly stained for F L G which is m o r e p r o m i n e n t by 5 days (Fig. 1). Some o f the sparse primordial plexiform layer cells are larger than in the newborn. Staining for F L G in the ventricular zone varies f r o m the presence o f reaction p r o d u c t in occasional cells or g r o u p s o f cells, as in the >
Fig. 1. Distribution of fetuin-like glycoprotein (FLG) in the developing dorsolateral neocortex of the tammar between birth (nb) and 15 days postnatal. Sections are stained for FLG using the PAP method; the sections are not counter-stained. The pial surface is uppermost. All Plates are at the same magnification (bar 50 gin). The original description of developmental of the dorsolateral neocortex of the tammar is in Reynolds et al. (1985). ppl, primordial plexiform layer; vz, ventricular zone; m, marginal zone; cp, cortical plate; sp, subplate; iz, intermediate zone; sz, subventricular zone. newborn (nb): FLG is present in ventricular zone cells, close to the ventricular surface and in fibres of the primordial plexiform layer. 5 days: This illustrates the stronger and more widespread vz cell staining and early appearance of FLG positive cells in ppl. 15 days: This illustrates three different regions of the dorso-lateral neocortex at this age (med, medial; lat, lateral; mid is from the middle region of the dorsal neocortex and corresponds to the regions illustrated for nb and 5 days). Because of the increasing thickness of the dorsal neocortex towards its lateral margin, the mid and lateral sections shown do not extend to the ventricular surface. There is strong immunostaining for FLG in subplate cells and in marginal zone cells. Less mature cortical plate cells are positive for FLG in the more lateral parts of the dorso-lateral neocortex
315
316 Table 1. Distribution of fetuin-like glycoprotein (FLG) in postnatal tammar dorsolateral neocortex and choroid plexus Structure
Ventricular zone Subventricular zone Intermediate zone Subplate zone Cortical plate Primordial plexiform layer Marginal zone Choroid Plexus Plasma CSF
Age
Cells Cells Cells Fibres Cells Fibres Cells Cells Fibres Cells Fibres Cells
nb
3d
5d
9d
+ 0 0 0 0 0 0 + 0 0 +++ ++++ ++++
++ 0 0 0 0 0 0 + ++ 0 0 +++ ++++ ++++
+++ (+) 0 0 -0 +++ 0 + ++ ++ + + splits into marginal (+) zone and subplate zone ++ ++ (+) + +++ +++ ++++ ++++ ++++ ++++
15d (+) + (+) (+ ) ++++ (+) ++
+++ + +++ ++++ ++++
20d --
+++ +++ +++
nb, newborn; other ages indicated are days postnatal + indicates positive staining (range (+) weak staining to + + + + very strong staining); - indicates staining absent; 0 indicates region not yet developed. The cortical plate is first apparent at 5-6 days postnatal, particularly in the more lateral part of the dorsolateral neocortex. The migration of cells into the primordial plexiform layer (ppl) at 5-6 days postnatal appears to divide the ppl into marginal zone and subplate zone (see also Fig. 1)
9-15 days (early cortical plate stage). By 9 days the
There a p p e a r to be several distinct populations of cells present that stain for F L G . These are illustrated in Fig. 1, 15d: 1. Very occasional cells in the marginal zone 2. A few strongly stained, mature-looking cells in the outer layer of the cortical plate, just deep to the scattered cells of the marginal zone proper 3. Lightly stained immature cells in the cortical plate itself, m a n y o f which at this age are positive 4. More mature strongly stained rounded cells with distinct processes in the subplate region (Fig. 1 and Table 1). A striking feature is that the line of F L G positive cells in the primordial plexiform layer and earliest cortical plate at around 5 days (Fig. 1) appears to have split into two populations by 15 days: one in the subplate and one in the upper border o f the cortical plate proper. Between 6 and 15 days new, less mature cortical plate cells (either unstained or only moderately stained for F L G ) seem to have been inserted between the inner and outer lines of cells positive for F L G , particularly in the more lateral part of the cortical plate (Fig. 1).
cortical plate is well formed, consisting of a layer one to two cells deep at the less mature medial part of the neocortex; the cortical plate increases in depth from medial to lateral and is about five to six cells deep at the lateral margin. M a n y of the cells stain for F L G (Table 1). By 15 days, the cortical plate has developed further, containing up to 15 cells in depth at the lateral margin. Only a proportion of the cells in the cortical plate and subplate is positive for F L G ; cells in the subplate are particularly strongly stained (Fig. 1). A few of the sparse cells of the marginal zone are still positive for F L G (Table 1).
16-20 days (compact cortical plate stage). Between 16 and 20 days the cortical plate increases in depth as does the subplate region. The fainter staining for F L G in the immature cells of the cortical plate largely disappears during this period. Distinct staining of the more mature cells in the subplate deep to the cortical plate persists, especially in more anterior regions of the dorsolateral neocortex (Fig. 2, Table 1). On examining less mature areas in the more frontal parts of the neocortex, positive cells are seen in the inner part o f the cortical plate at day 17 (Fig. 2).
newborn, to extensive F L G staining o f m a n y cells (Fig. 1, 5d).
5 days (earliest cortical plate stage). By 5 days a few large rounded cells appear within the primordial plexiform layer (Fig. 1, 5 d), just deep to the widely scattered cells that are the only cells in this layer at 0-3 days postnatal. Some of these larger cells are positive for F L G , particularly towards the lateral margin o f the neocortex. These rounded, rather mature-looking cells, some with processes extending towards the pial surface, form an irregular and discontinuous line of cells between the ventricular zone and the outer part of the primordial plexiform layer. The FLG-positive cells, together with other unstained cells, form the earliest cortical plate. The appearance of these rather mature-looking cells in the primordial plexiform layer subdivides it into an outer marginal zone (eventually layer 1 of the developed cortex) and what will soon become the subplate zone (Marin-Padilla 1978, 1988; Luskin and Shatz 1985).
Fig. 2a-d. Comparison of F L G ' s t a i n i n g in less mature (anterior, a) through more mature (b, e, d) regions of dorsal neocortex at 17 days postnatal. The strongest stained cells in a to e are in the subplate region. Stained cells are no longer present in the more
mature d region of the neocortex which is equivalent to the regions illustrated in Fig. 1. Pial surface uppermost, rn, marginal zone; qo, cortical plate; sp, subplate. All plates are at the same magnification (bar 50/am)
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Table 2. Concentration of FLG in plasma and CSF of pouch young tammar, from day of birth (nb) to 210 days Age (days)
n
Plasma (rag/100 ml)
Total protein (%)
Cerebrospinal fluid (rag/100 ml)
Total protein (%) (mg/100ml)
CSF/plasma x 100
nb 10 15 20 30 45 75 150 210
3 3 3 3 6 4 4 3 3
77.0_+1.7 78.0_+11.5 78.0_+4.1 76.0_+3.3 77.0-+13.2 87.5-+2.5 62.0-+7.0 133.3+6.0 207.6-+4.3
6.7 8.1 6.2 4.5 4.4 3.5 1.8 2.9 3.7
35.3 +5.3 30.5 +2.5 23.3 +2.4 23.7 _+2.3 24.0 _+1.7 16.8 -+ 1.1 0.93-+0.1 0.95-+0.1 1.06_+0.2
14.8 9.7 5.8 8.0 7.5 6.7 0.4 0.8 1.8
47.0 38.8 _+2.2 32.7 _+1.3 29.8 -t-2.7 28.0 _+4.3 18.3 -+0.9 1.4 -+0.2 0.68_+0.1 0.48-+0.04
(mean 10.6, range 10-12) (mean 19, range 17-20) (mean 28, range 27-28) (mean 45, range 37-47) (mean 74, range 67-90) (mean 153, range 146156) (mean 208, range 200-215)
Mean_+SEM, n=number of animals at each stage, % total protein = FLG mean concentration expressed as percentage of mean total protein from Dziegielewska et al. 1986; CSF/plasma • 100 = CSF to plasma ratio of concentrations of FLG
After 20 days postnatal. No staining for F L G could be detected in the dorsal wall of the neocortex in animals older than 20 days, except for some rather variable staining in the ventricular zone immediately adjacent to its interface with CSF. This is apparent up to about 40 days. Pronounced F L G staining is apparent in the medial wall of the developing forebrain vesicle. Some of these cells are associated with the anlage of the hippocampus and remain positive for F L G for a longer period than cells in the dorsal neocortex. Positive cells in the developing hippocampus are observed up until about 40 days postnatal (not illustrated). Choroid plexus stroma, CSF and plasma were positive for F L G at all ages examined (Table 1). The presence of this immunoreactivity in brain sections where F L G could no longer be detected in the neocortex is an important internal control indicating that this lack of staining was due to disappearance of the glycoprotein from cells in the neocortex.
Fetuin-like glycoprotein in plasma and CSF The concentration of F L G in plasma and in CSF from day 0 (newborn) to late pouch life (joeys have the pouch permanently at about 250 days postnatal) is shown in Table 2. In plasma the concentration increased from nearly 80 rag/100 ml (77.0_+ 1.7 rag/100 nal) in the newborn to over 200 rag/100 ml at 210 days. In contrast, the concentration in CSF was highest in the neonate (0-10 days) at 30-35 mg/100ml and remained elevated at over 20 rag/100 ml until after day 30. It fell rapidly after day 45 to reach mature levels by day 75 (1.06_+0.20 rag/ 100 ml). The concentration ratio of CSF to plasma for F L G is also shown in Table 2. The ratio was the highest at the newborn stage. It then fell progressively from around 40% to 30% between day 10 and 30 and very rapidly after day 45. In the first two months of life the fall in the ratio is due to the fall in CSF concentration as plasma levels remained constant until day 75. In the mature pouch young, the CSF/plasma ratio of F L G was around 0.5 %.
The contribution of F L G to total protein is also shown in Table 2. The values for total protein were taken from previously published results (Dziegielewska et al. 1986) for the same samples. In plasma the F L G makes a relatively small contribution throughout the period of development, ranging from 8% in the early stages to 2~4% later on. In the CSF the contribution of the glycoprotein to total protein is larger in younger animals, and proportionally greater than in plasma for the first 1-2 months of postnatal life. In the later stages of development, the contribution of F L G in CSF is less than half of that in plasma (Table 2).
Discussion This study of a specific and developmentally regulated plasma protein expressed in the brain confirms earlier reports that there is a structurally related polypeptide present in a defined population of cells forming the early cortical plate in such diverse animals as humans (Dziegielewska etal. 1987), ArtiodactyIa (Reynolds etal. 1987) and marsupials (Jones et al. 1988). The amino acid sequences of human ~2HS glycoprotein (Yoshioka et al. 1986; Lee et al. 1987) and bovine fetuin have been published (Christie et al. 1987; Dziegielewska et al. 1990) as well as the N-terminal sequences of fetuin-like glycoprotein in the tammar wallaby (Jones et al. 1988). The primary amino acid sequence homology is high (50-70% depending on the part of the molecule compared), suggesting that these proteins belong to the same family (Jones et al. 1988). The origin of these proteins in the developing brain is still not clear. There is some evidence that cells in immediate contact with the CSF surface (ventricular zone cells) are able to take up some plasma proteins at some stages of development (see Cavanagh and Warren 1985). The presence of a substantial concentration of F L G in CSF during the period studied (see Table 2) suggests an obvious source of this protein for uptake into ventricular zone cells.. The source of this and other proteins in CSF in the developing brain is probably largely from the plasma (see Dziegielewska and Saunders 1988). Once the cells move out from the ventricular zone to form other parts
319 of the neocortex, they probably lose their contact with CSF. There is now extensive evidence for the presence of specific plasma protein m R N A in the developing brain (MollgSrd et al. 1988) and we have recently shown by in situ hybridisation that the cortical plate in 40d fetal sheep contains m R N A for fetuin (unpublished observations). The immunocytochemical appearance illustrated in Fig. 1 is consistent with the sequence of uptake of FLG from CSF, migration of FLG-containing cells, and onset of synthesis of FLG in subplate/cortical plate neurons. Similar information for human brain is difficult to obtain, and we do not at present have suitable cDNA probes for hybridization studies in the tammar. It does appear though, that the positively immunostained cells in the cortical plate are likely to be able to synthesize fetuin-like proteins at the time that the first cortical plate cells take up their position within the primordial plexiform layer. The proportion of glycoprotein-positive cells in the early neocortex varies in different species : in some eutherian mammals (sheep, cow) the majority of early cortical plate cells are immunopositive, whereas, as shown in this study, in marsupial species like the tammar and also in Monodelphis domestica (unpublished observation) the positive cells are more sparsely distributed. Also in eutherian mammals (Reynolds and Mollgfird 1985; Reynolds et al. 1987) the staining persists over a much longer period of development. This may be a reflection of the differences in cortical plate development in marsupial species (Reynolds et al. 1985; Reynolds 1987; Reynolds and Saunders 1988) as compared with eutherians. In spite of these differences, the general pattern of fetuin-like glycoprotein staining in the developing neocortex is similar in different species. The rather fleeting apperance of these glycoproteins in cortical plate cells would suggest some developmentally regulated role in general formation and modulation of the cortex. The distribution of strongly-staining glycoprotein-positive cells in two zones flanking the immature cortical plate (Fig. 1, 15d) is similar to the distribution of [3H]-thymidine labelling described in the early cortical plate of the cat fetus (Luskin and Shatz 1985; Shatz et al. 1988). A similar pattern for both [3H]-thymidine labelling and fetuin in the same sections has been found in fetal sheep brain at the equivalent stage of early cortical plate formation; these cells are thought to be a transient population (Reynolds et al. 1990). Similar quantitative autoradiographic studies have not been carried out in the tammar. It may be that the much earlier disappearance of the fetuin-like glycoprotein from early cortical plate cells in the tammar pouch young compared with the sheep fetus reflects much earlier disappearance of these cells in the tammar. This in turn may be associated with the generally more mature appearance of the subplate and developing layer 6 in the marsupial compared with eutherians (see Reynolds et al. 1985 and Reynolds 1987 for discussion). Shatz and her colleagues have demonstrated a number of peptides in the early generated population of cortical plate neurons that largely disappear during development; these peptides include somatostatin, neu-
ropeptide Y and cholecystokinin (see Shatz et al. 1988). However they are not expressed until late fetal or early neonatal life, whereas fetuin and equivalent glycoproreins are expressed from the earliest stages of subplate and cortical plate formation. In detailed studies of the development of the tammar telencephalon (Reynolds et al. 1985) experiments with [3H]-thymidine injections indicate that the cells born on day 0 take up their position in the subventricular zone by day 5; by day 40-50 such labelled cells are present only in the subplate zone. In more recent experiments (unpublished) we have shown that injecting the animals with [3H] thymidine at day 2, was already too late to label FLG-positive cells. It is therefore likely that the FLG-positive cells are born sometime between day 0 and day 2. The role of fetuin-like glycoproteins in brain development is not known, nor is it even clear what their function may be in plasma. These proteins are not fetalspecific, as adult plasma contains variable amounts, depending on the species studied. Thus in the sheep and cow, fetuin is a major plasma protein in development, constituting 30% of all plasma protein in sheep fetuses (Dziegielewska et al. 1980) and up to 50% in late gestation cow fetuses (Bergmann et al. 1962). In adult animals the levels are of the order of 20~40 mg per 100 ml. In the human fetus the data are more limited, but it seems that the levels of e2HS glycoprotein remain similar throughout the developmental period, and do not differ much from the adult (50-70 rag/100 ml, Dziegielewska et al. 1987). In this paper we present data on the ontogeny of fetuin-like glycoprotein in the tammar wallaby; in contrast to the above species the levels of FLG in early pouch young are lower and more like e2HS in human than fetuin in sheep and cow (around 70 rag/ 100 ml). From this low concentration, FLG then increases with age to reach over 200 rag/100 ml in the mature joey, which is 3-4 times higher than c~zHS in the adult human, and an order of magnitude more than fetuin in sheep. Thus the developmental change in plasma concentrations of these glycoproteins follows a completely different pattern in all species studied so far. Several biological roles have been ascribed to fetuin and cr glycoprotein (see Dziegielewska et al. 1987 for references and discussion). The wallaby equivalent of these proteins has not been studied. No common property of fetuin and ezHS has been so far found. Nevertheless because of the similarity of their developmental expression in the brain, combined with differences in plasma during development, we would propose that their functions in the brain may be similar in all species, and perhaps confined to only some parts of the molecule, as has been shown for proenkephalin (Noda et al. 1982) and opiomelanocortin (Kita et al. 1979). The more overall function of fetuin-like glycoproteins in other tissue development and in plasma may be different and dependent on more general properties of these molecules. Marsupial species are born at an extremely early (precortical plate) stage of neocortical developmental (Reynolds etal. 1985; Reynolds and Saunders 1988; Saunders et al. 1989). The demonstration in this paper
320 o f p o s t n a t a l e x p r e s s i o n o f a g l y c o p r o t e i n r e l a t e d to fetuin a n d e 2 H S in early c o r t i c a l p l a t e cells in the t a m m a r a n d in Monodelphis domestica ( u n p u b l i s h e d o b s e r v a tions) opens the p o s s i b i l i t y o f m o r e direct studies o f the f u n c t i o n a l significance o f this g l y c o p r o t e i n f o r early neoc o r t i c a l d e v e l o p m e n t t h a n is p o s s i b l e in less accessible e u t h e r i a n fetuses.
Acknowledgements. We should like to thank Action Research for the Crippled Child (SJ) and The Welicome Trust (KMD) for their generous support of this work. LAH was supported by the Australian Academies and Royal Society Exchange Programme. We should particularly like to thank Professor Kjeld Mollg~rd for his painstaking advice and discussion of our experimental material and manuscript and Professor M Szelke and Dr. J Sueras-Diaz for synthesizing FLG peptide sequences. We should also like to thank Mrs Mary Eldridge and Mrs Lynn Ford for typing the manuscript.
References Bergmann FH, Levine L, Spiro RG (1962) Fetuin: immunochemistry and quantitative estimation in serum. Biochim Biophys Acta 58:41-51 Bottenstein JE, Skaper SD, Varon SS, Sato GH (1980) Selective sm'vival of neurons from chick embryo sensory ganglionic dissociates utilizing serum-free supplemented medium. Exp Cell Res 125:183-190 Cavanagh ME, Warren A (1985) The distribution of native albumin and foreign albumin injected into lateral ventricles of prenatal and neonatal rat forebrains. Anat Embryot 172:345-351 Christie DL, Dziegielewska KM, Hill RM, Saunders NR (1987) Fetuin: the bovine homologue of human c~2HS glycoprotein. FEBS Lett 214:45-49 Dziegielewska KM, Saunders NR (1988) The development of the blood-brain barrier: proteins in fetal and neonatal CSF, their nature and origins. In: Meisami E, Timiras PS (eds) Handbook of Human Growth and Developmental Biology, vol 1. CRC Press, Boca Raton, Florida Dziegielewska KM, Evans CAN, Fossan G, Lorscheider FL, Malinowska DH, MollgSrd K, Reynolds ML, Saunders NR, Wilkinson S (1980) Proteins in cerebrospinal fluid and plasma of fetal sheep during development. J Physiol 300:441-455 Dziegielewska KM, Hinds LA, Sarantis MEP, Saunders NR, Tyndale-Biscoe CH (1986) Proteins in cerebrospinal fluid and plasma of the pouch young tammar wallaby (Macropus eugenii) during development. Comp Biochem Physiol 83 B: 561 567 Dziegielewska KM, MollgSrd K, Reynolds ML, Saunders NR (1987) A fetuin-related glycoprotein (~2HS) in human embryonic and fetal development. Cell Tissue Res 248:33-41 Dziegielewska KM, Brown WM, Casey S-J, Christie DL, Foreman RC, Hill RM, Saunders NR (1990) The complete cDNA and amino acid sequence of bovine fetuin. Its homology with c~2HS glycoprotein and relation to other members of the cystatin superfamily. J Biol Chem 265:4354-4357 Jones SE, Dziegielewska KM, Saunders NR, Christie DL, SueirasDiaz J, Szelke M (1988) Early cortical plate specific glycoprotein in a marsupial species belongs to the same family as fetuin and ~zHS glycoprotein. FEBS Lett 236:411-414 Kita T, Inoue A, Nakanishi S, Numa S (1979) Purification and characterization of the messenger RNA coding for bovine corticotropin fl-lipotropin precursor. Eur J Biochem 93:213-220 Lee C-C, Bowman BH, Yang F (1987) Human-eaHS-glycoprotein: the A and B chains with a connecting sequence are encoded
by a single mRNA transcript. Proc Nat1 Acad Sci USA 84: 4403-4407 Luskin MB, Shatz CJ (1985) Studies of the earliest generated cells of the cat's visual cortex : cogeneration of the cells of the marginal zone and subplate. J Neurosci 5:1062-1075 Mancini G, Carbonara AO, Heremans JF (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Int J Immunochem 2:235 254 Marin-Padilla M (1978) Dual origin of mammalian neocortex and evolution of the cortical plate. Anat Embryol 152:109-126 Marin-Padilla M (1988) Early ontogenesis of the human cerebral cortex. In: Peters A, Jones EG (eds) Cerebral Cortex Vol 7, Plenum Press, New York, pp I 34 MollgSrd K, Reynolds ML, Jacobsen M, Dziegielewska KM, Saunders NR (1984) Differential immunocytochemical staining for fetuin and transferrin in the developing cortical plate. J Neurocytol 13 : 497-502 Mollg~rd K, Dziegielewska KM, Saunders NR, Zakut H, Soreq H (1988) Synthesis and localisation of plasma proteins in the developing human brain: integrity of the fetal blood-brain barrier to endogenous proteins of hepatic origin. Dev Biol 128:207-221 Noda M, Furutani Y, Takahashi H, Toyosato M, Hirose T, Inayama S, Nakanishi S, Numa S (1982) Cloning and sequence analysis of cDNA for bovine adrenal preproenkephalin. Nature 295:202-206 Renfree MB, Holt AB, Green SW, Carr JP, Cheek DB (1982) Ontogeny of the brain in a marsupial (Macropus eugenii) throughout pouch life. Brain Behav Evol 20 : 57-71 Reynolds ML (1987) Early development of the neocortex in Eutheria and Metatheria: differentiation and plasma protein distribution. PhD thesis, University of London Reynolds ML, Mollg~.rd K (1985) The distribution of plasma proteins in the neocortex and early allocortex of the developing sheep brain. Anat Embryol 171:41-60 Reynolds ML, Saunders NR (1988) Differentiation of the neocortex. In: Tyndale-Biscoe CH, Janssens PA (eds) The developing marsupial: models for biomedical research. Springer, Berlin Heidelberg, pp 101-116 Reynolds ML, Cavanagh ME, Dziegielewska KM, Hinds LA, Saunders NR, Tyndale-Biscoe CH (1985) Postnatal development of the telencephalon of the tammar wallaby (Macropus eugenii). An accessible model of neocortical differentiation. Anat Embryol 173:81-94 Reynolds ML, Sarantis MEP, Lorscheider FL, Saunders NR (1987) Fetuin as a marker of cortical plate cells in the fetal cow neocortex : a comparison of the distribution of fetuin, c~2HS glycoprotein, c~-fetoprotein and albumin during early development. Anat Embryol 175 : 355-363 Reynolds ML, Habgood MD, Ward RA, Saunders NR (1990) Origin and fate of fetuin-containing neurons in the developing neocortex of the fetal sheep. Submitted to J. Neurosci Rizzino A, Shermann M[ (1979) Development and differentiation of mouse blastocysts in serum-free medium. Exp Cell Res 121:221-233 Saunders NR, Adam E, Reader M, Mollg~rd K (1989) Monodelphis domestica (grey short-tailed opossum): an accessible model for studies of early neocortical development. Anat Embryol 180:227-236 Shatz CJ, Chun JJM, Luskin MB (1988) The role of the subplate in the development of the mammalian telencephalon. In: Peters A, Jones EG (eds) Cerebral cortex Vol 7, Plenum Press, New York, pp 35-58 Yoshioka Y, Gejyo F, Marti T, Rickli EE, Burgi W, Offner GD, Troxler RF, Schmid K (1986) The complete amino acid sequence of the A-chain of human plasma ~2HS-glycoprotein. J Biol Chem 261 : 1665-1676
Anatomy and Embryology 9 Springer-Verlag1991
Developmental profile of a fetuin-like glycoprotein in neocortex, cerebrospinal fluid and plasma of post-natal tammar wallaby (Macropus eugenil) S.E. Jones 1, D.L. Christie 2, K.M. Dziegielewska 1, L.A. Hinds 3, and N.R. Saunders 1 1 Clinical Neurological Sciences and The Wessex Area Neurosciences Group, University of Southampton, Southampton General Hospital, Tremona Road, Southampton, Hants, SO9 4XY, UK 2 Department of Biochemistry, University of Auckland, Private Bag, Auckland, New Zealand a CSIRO Division of Wildlife & Ecology, PO Box 84, Lyneham, ACT 2602, Australia Accepted November 16, 1990
Summary. A fetuin-like glycoprotein (FLG) has been shown to be present in early cortical plate cells in the developing brain of the tammar wallaby (Macropus eugenii). The developmental sequence of the occurrence of glycoprotein-positive fibres and cells in the dorsolateral telencephalic wall from newborn to day 40 is described. The level of F L G in CSF (cerebrospinal fluid) and plasma of the tammar wallaby has also been measured during pouch life. The presence of FLG in early postnatal fibre systems and in some cells in the primordial plexiform layer, as well as in early cortical plate cells of the tammar is similar to that of fetuin in fetal brain in sheep, pig and cow, and ~2HS glycoprotein in human fetal brain. The sequence of appearance of FLGpositive cells during neocortical development in the tammar is strikingly similar to that of a transient population of early cortical plate cells previously described in fetal cat and sheep cortex. During postnatal development, levels of F L G in tammar plasma and CSF follow a pattern different from that of other species. The developmental expression of all three related glycoproteins in their respective species is discussed. Key words: Brain development - Fetuin
Marsupial- Neocortex
Introduction Several plasma proteins have been identified within cells in the immature brain (see e.g. Mollggtrd et al. 1988). However the function of these proteins in nervous system development is not yet understood. In addition, several studies have shown that there are high concentrations of proteins, that probably originate from plasma, in fetal cerebrospinal fluid (CSF) early in gestation (see Offprint requests to ." N.R. Saunders
Dziegielewska and Saunders 1988 for references). Plasma or serum, usually from bovine fetuses, is commonly used in tissue culture; studies with well-defined culture media have shown the importance of certain plasma proteins (e.g. transferrin) for specific features of cell and tissue development in vitro (e.g. Bottenstein et al. 1980); it may be that these glycoproteins have similar properties in vivo. One fetal protein, fetuin, is a frequent constituent of culture media (e.g. Rizzino and Shermann 1979); it has also been shown to be present in early cortical plate cells in the developing neocortex (MollgSrd et al. 1984; Reynolds et al. 1987). Structurally similar proteins have been identified in plasma, CSF and developing brain of human fetuses (~2HS glycoprotein, Dziegielewska et al. 1987) and in the postnatal tammar wallaby (Jones et al. 1988); e2HS glycoprotein and bovine fetuin have about 65% sequence identity and belong to the superfamily of cystatins (Dziegielewska et al. 1990). The developmental profiles in plasma and CSF of fetuin in sheep and pig fetuses and ~2HS glycoprotein (~2HS) in human fetuses do not follow a similar pattern. Thus in the animals of the order Artiodactyla (sheep and cattle) the level of fetuin in both plasma and CSF is very high early in development; it remains elevated in plasma (e.g. 400 rag/100 ml in fetal sheep) until birth and then falls by an order of magnitude in the adult (Dziegielewska et al. 1980). In contrast, in human fetal and adult plasma the level of ~2HS remains fairly constant at rather more modest levels (50-70 rag/100 ml); nevertheless the appearance and disappearance of these proteins in the developing neocortex follow a similar pattern (Dziegielewska et al. 1987). Recently we have identified a related protein in plasma of the tammar wallaby (Jones et al. 1988). Here we present the data describing the developmental profile of the wallaby equivalent of fetuin (fetuin-like glycoprotein, FLG) in neocortex, CSF and plasma. Fetuin or a related glycoprotein is expressed in the immature cortical plate in
314
Eutheria, very early in gestation (e.g. a r o u n d E35 in sheep fetuses which are b o r n at E150). The results o f this study show that the fetuin-related glycoprotein, F L G , in the t a m m a r brain is also expressed in early cortical plate cells, but as a postnatal p h e n o m e n o n , because o f the very early stage o f brain development at which the y o u n g o f this species are born.
at or close to the interventricular foramen. A description o f the dorsolateral neocortex in the t a m m a r has been published previously (Reynolds et al. 1985). The stages o f cortical plate development used in the description below, are f r o m Reynolds et al. (1985).
Distribution of fetuin-like glycoprotein in the neocortex Materials and methods Animals. The animals used in this study came from an established breeding colony maintained at the CSIRO Division of Wildlife and Ecology Research in Canberra, Australia. Details of breeding and dating of the animals have been described previously (Reynolds et al. 1985).
CSF and plasma samples. CSF and plasma samples from dated animals (ages and numbers are indicated in Table 2) were collected as described previously (Dziegielewska et ai. 1986). All samples were kept at - 2 0 ~ C until used. Fixation and tissuepreparation. Brain tissue was prepared for histological examination as previously described (Reynolds et al. 1985). Briefly, brains were fixed in Bouin's fixative for 6-10 h, followed by a brief wash in tap water; they were dehydrated through graded alcohols, cleared in chloroform and embedded in paraffin wax MP 56~ C. Serial sections, 6 ~tm thick, were cut through the forebrain in the coronal and sometimes sagittal planes. Pairs of sections from every 30 were selected for histological examination; one was stained with toluidine blue or haematoxylin and eosin, the other was subjected to immunocytochemistry to demonstrate FLG. Two to six brains were examined at each age studied (rib, 3d, 5d, 6d, 9d, 15d, 16d, 17d, 20d, 30-210d).
Immunohistochemical staining. Sections were dewaxed, rehydrated and incubated in blocker solution (0.35% bovine serum albumin, 0.05% Tween in phosphate buffered saline, PBS) for 20 rain. Primary antiserum, mouse anti-FLG or anti-FLG peptide (Jones et al. 1988), diluted 1:100 in blocker solution, was applied for 16 h at 4~ C, the sections being kept in humidified boxes. After washing in three changes of PBS, the blocker solution was applied and sections incubated for 10 rain. Sections were then incubated for 30 min with secondary antibody (rabbit anti-mouse IgG, DAKOPATTS, Denmark, diluted 1 : 50 in blocker solution). After three washes in PBS, sections were incubated for 30 rain in PAP complex (Mouse peroxidase-antiperoxidase, DAKOPATTS, Denmark) diluted 1 : 50 in blocker solution. Three washes in PBS were followed by 10 min wash in 0.05 M Tris, pH 7.6, and the colour reaction was developed with 0.025% DAB in 0.05 M Tris buffer, pH 7.6 with l0 ~tl/100 ml of 30% H2Oz. After extensive washing in tap water the sections were dehydrated in graded alcohols, cleared in xylene and mounted with DPX mountant. Positive and negative control sections were included in each run as described previously (Reynolds et al. 1987).
Preparation of protein standard, antiserum and quantitative determination offetuin-Iike glycoprotein. Fetuin-like glycoprotein was separated to purity (Jones et al. 1988) and used as standard. Antibodies were prepared as described previously (Jones et al. 1988). The radial immunodiffusion method of Mancini (Mancini et al. 1965) was used for quantitative determination of the concentration of FLG in plasma and CSF of developing pouch young.
Results Adjacent sections were stained with haematoxylin and eosin, and for F L G . The brain region examined was
Newborn to 3 days postnatal (precortical plate stage). On the d a y o f birth, as previously described by Reynolds et al. (1985), the dorsolateral wall o f the forebrain vesicle consists only o f a deep pseudostratified layer o f ventricular zone cells and an outer primordial plexiform layer. The latter contains a very few widely scattered cells o f variable size and shape and w i t h o u t obvious processes. The primordial plexiform layer also contains a fine reticu l u m o f incoming fibres, the origin o f which is unclear. This reticulum is stained with F L G antibodies (Fig. 1). In contrast, few o f the cell bodies in the primordial plexif o r m layer stain for F L G on the day o f birth. Staining for F L G is clearly a p p a r e n t in precipitated C S F within the brain ventricles on the d a y o f birth and subsequently (Table 1). A few cells in the ventricular zone that are immediately adjacent to the ventricular (csf) surface are strongly stained for F L G (Fig. 1, nb). This appearance is consistent with the protein having been taken up by the cells f r o m C S F (see Discussion). By 3 days postnatal, the brain vesicles are slightly enlarged (brain g r o w t h in this species is relatively slow; see Renfree et al. 1982; Reynolds et al. 1985). M o r e cells are present within the primordial plexiform layer but they are still widely dispersed. A few are faintly stained for F L G which is m o r e p r o m i n e n t by 5 days (Fig. 1). Some o f the sparse primordial plexiform layer cells are larger than in the newborn. Staining for F L G in the ventricular zone varies f r o m the presence o f reaction p r o d u c t in occasional cells or g r o u p s o f cells, as in the >
Fig. 1. Distribution of fetuin-like glycoprotein (FLG) in the developing dorsolateral neocortex of the tammar between birth (nb) and 15 days postnatal. Sections are stained for FLG using the PAP method; the sections are not counter-stained. The pial surface is uppermost. All Plates are at the same magnification (bar 50 gin). The original description of developmental of the dorsolateral neocortex of the tammar is in Reynolds et al. (1985). ppl, primordial plexiform layer; vz, ventricular zone; m, marginal zone; cp, cortical plate; sp, subplate; iz, intermediate zone; sz, subventricular zone. newborn (nb): FLG is present in ventricular zone cells, close to the ventricular surface and in fibres of the primordial plexiform layer. 5 days: This illustrates the stronger and more widespread vz cell staining and early appearance of FLG positive cells in ppl. 15 days: This illustrates three different regions of the dorso-lateral neocortex at this age (med, medial; lat, lateral; mid is from the middle region of the dorsal neocortex and corresponds to the regions illustrated for nb and 5 days). Because of the increasing thickness of the dorsal neocortex towards its lateral margin, the mid and lateral sections shown do not extend to the ventricular surface. There is strong immunostaining for FLG in subplate cells and in marginal zone cells. Less mature cortical plate cells are positive for FLG in the more lateral parts of the dorso-lateral neocortex
315
316 Table 1. Distribution of fetuin-like glycoprotein (FLG) in postnatal tammar dorsolateral neocortex and choroid plexus Structure
Ventricular zone Subventricular zone Intermediate zone Subplate zone Cortical plate Primordial plexiform layer Marginal zone Choroid Plexus Plasma CSF
Age
Cells Cells Cells Fibres Cells Fibres Cells Cells Fibres Cells Fibres Cells
nb
3d
5d
9d
+ 0 0 0 0 0 0 + 0 0 +++ ++++ ++++
++ 0 0 0 0 0 0 + ++ 0 0 +++ ++++ ++++
+++ (+) 0 0 -0 +++ 0 + ++ ++ + + splits into marginal (+) zone and subplate zone ++ ++ (+) + +++ +++ ++++ ++++ ++++ ++++
15d (+) + (+) (+ ) ++++ (+) ++
+++ + +++ ++++ ++++
20d --
+++ +++ +++
nb, newborn; other ages indicated are days postnatal + indicates positive staining (range (+) weak staining to + + + + very strong staining); - indicates staining absent; 0 indicates region not yet developed. The cortical plate is first apparent at 5-6 days postnatal, particularly in the more lateral part of the dorsolateral neocortex. The migration of cells into the primordial plexiform layer (ppl) at 5-6 days postnatal appears to divide the ppl into marginal zone and subplate zone (see also Fig. 1)
9-15 days (early cortical plate stage). By 9 days the
There a p p e a r to be several distinct populations of cells present that stain for F L G . These are illustrated in Fig. 1, 15d: 1. Very occasional cells in the marginal zone 2. A few strongly stained, mature-looking cells in the outer layer of the cortical plate, just deep to the scattered cells of the marginal zone proper 3. Lightly stained immature cells in the cortical plate itself, m a n y o f which at this age are positive 4. More mature strongly stained rounded cells with distinct processes in the subplate region (Fig. 1 and Table 1). A striking feature is that the line of F L G positive cells in the primordial plexiform layer and earliest cortical plate at around 5 days (Fig. 1) appears to have split into two populations by 15 days: one in the subplate and one in the upper border o f the cortical plate proper. Between 6 and 15 days new, less mature cortical plate cells (either unstained or only moderately stained for F L G ) seem to have been inserted between the inner and outer lines of cells positive for F L G , particularly in the more lateral part of the cortical plate (Fig. 1).
cortical plate is well formed, consisting of a layer one to two cells deep at the less mature medial part of the neocortex; the cortical plate increases in depth from medial to lateral and is about five to six cells deep at the lateral margin. M a n y of the cells stain for F L G (Table 1). By 15 days, the cortical plate has developed further, containing up to 15 cells in depth at the lateral margin. Only a proportion of the cells in the cortical plate and subplate is positive for F L G ; cells in the subplate are particularly strongly stained (Fig. 1). A few of the sparse cells of the marginal zone are still positive for F L G (Table 1).
16-20 days (compact cortical plate stage). Between 16 and 20 days the cortical plate increases in depth as does the subplate region. The fainter staining for F L G in the immature cells of the cortical plate largely disappears during this period. Distinct staining of the more mature cells in the subplate deep to the cortical plate persists, especially in more anterior regions of the dorsolateral neocortex (Fig. 2, Table 1). On examining less mature areas in the more frontal parts of the neocortex, positive cells are seen in the inner part o f the cortical plate at day 17 (Fig. 2).
newborn, to extensive F L G staining o f m a n y cells (Fig. 1, 5d).
5 days (earliest cortical plate stage). By 5 days a few large rounded cells appear within the primordial plexiform layer (Fig. 1, 5 d), just deep to the widely scattered cells that are the only cells in this layer at 0-3 days postnatal. Some of these larger cells are positive for F L G , particularly towards the lateral margin o f the neocortex. These rounded, rather mature-looking cells, some with processes extending towards the pial surface, form an irregular and discontinuous line of cells between the ventricular zone and the outer part of the primordial plexiform layer. The FLG-positive cells, together with other unstained cells, form the earliest cortical plate. The appearance of these rather mature-looking cells in the primordial plexiform layer subdivides it into an outer marginal zone (eventually layer 1 of the developed cortex) and what will soon become the subplate zone (Marin-Padilla 1978, 1988; Luskin and Shatz 1985).
Fig. 2a-d. Comparison of F L G ' s t a i n i n g in less mature (anterior, a) through more mature (b, e, d) regions of dorsal neocortex at 17 days postnatal. The strongest stained cells in a to e are in the subplate region. Stained cells are no longer present in the more
mature d region of the neocortex which is equivalent to the regions illustrated in Fig. 1. Pial surface uppermost, rn, marginal zone; qo, cortical plate; sp, subplate. All plates are at the same magnification (bar 50/am)
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Table 2. Concentration of FLG in plasma and CSF of pouch young tammar, from day of birth (nb) to 210 days Age (days)
n
Plasma (rag/100 ml)
Total protein (%)
Cerebrospinal fluid (rag/100 ml)
Total protein (%) (mg/100ml)
CSF/plasma x 100
nb 10 15 20 30 45 75 150 210
3 3 3 3 6 4 4 3 3
77.0_+1.7 78.0_+11.5 78.0_+4.1 76.0_+3.3 77.0-+13.2 87.5-+2.5 62.0-+7.0 133.3+6.0 207.6-+4.3
6.7 8.1 6.2 4.5 4.4 3.5 1.8 2.9 3.7
35.3 +5.3 30.5 +2.5 23.3 +2.4 23.7 _+2.3 24.0 _+1.7 16.8 -+ 1.1 0.93-+0.1 0.95-+0.1 1.06_+0.2
14.8 9.7 5.8 8.0 7.5 6.7 0.4 0.8 1.8
47.0 38.8 _+2.2 32.7 _+1.3 29.8 -t-2.7 28.0 _+4.3 18.3 -+0.9 1.4 -+0.2 0.68_+0.1 0.48-+0.04
(mean 10.6, range 10-12) (mean 19, range 17-20) (mean 28, range 27-28) (mean 45, range 37-47) (mean 74, range 67-90) (mean 153, range 146156) (mean 208, range 200-215)
Mean_+SEM, n=number of animals at each stage, % total protein = FLG mean concentration expressed as percentage of mean total protein from Dziegielewska et al. 1986; CSF/plasma • 100 = CSF to plasma ratio of concentrations of FLG
After 20 days postnatal. No staining for F L G could be detected in the dorsal wall of the neocortex in animals older than 20 days, except for some rather variable staining in the ventricular zone immediately adjacent to its interface with CSF. This is apparent up to about 40 days. Pronounced F L G staining is apparent in the medial wall of the developing forebrain vesicle. Some of these cells are associated with the anlage of the hippocampus and remain positive for F L G for a longer period than cells in the dorsal neocortex. Positive cells in the developing hippocampus are observed up until about 40 days postnatal (not illustrated). Choroid plexus stroma, CSF and plasma were positive for F L G at all ages examined (Table 1). The presence of this immunoreactivity in brain sections where F L G could no longer be detected in the neocortex is an important internal control indicating that this lack of staining was due to disappearance of the glycoprotein from cells in the neocortex.
Fetuin-like glycoprotein in plasma and CSF The concentration of F L G in plasma and in CSF from day 0 (newborn) to late pouch life (joeys have the pouch permanently at about 250 days postnatal) is shown in Table 2. In plasma the concentration increased from nearly 80 rag/100 ml (77.0_+ 1.7 rag/100 nal) in the newborn to over 200 rag/100 ml at 210 days. In contrast, the concentration in CSF was highest in the neonate (0-10 days) at 30-35 mg/100ml and remained elevated at over 20 rag/100 ml until after day 30. It fell rapidly after day 45 to reach mature levels by day 75 (1.06_+0.20 rag/ 100 ml). The concentration ratio of CSF to plasma for F L G is also shown in Table 2. The ratio was the highest at the newborn stage. It then fell progressively from around 40% to 30% between day 10 and 30 and very rapidly after day 45. In the first two months of life the fall in the ratio is due to the fall in CSF concentration as plasma levels remained constant until day 75. In the mature pouch young, the CSF/plasma ratio of F L G was around 0.5 %.
The contribution of F L G to total protein is also shown in Table 2. The values for total protein were taken from previously published results (Dziegielewska et al. 1986) for the same samples. In plasma the F L G makes a relatively small contribution throughout the period of development, ranging from 8% in the early stages to 2~4% later on. In the CSF the contribution of the glycoprotein to total protein is larger in younger animals, and proportionally greater than in plasma for the first 1-2 months of postnatal life. In the later stages of development, the contribution of F L G in CSF is less than half of that in plasma (Table 2).
Discussion This study of a specific and developmentally regulated plasma protein expressed in the brain confirms earlier reports that there is a structurally related polypeptide present in a defined population of cells forming the early cortical plate in such diverse animals as humans (Dziegielewska etal. 1987), ArtiodactyIa (Reynolds etal. 1987) and marsupials (Jones et al. 1988). The amino acid sequences of human ~2HS glycoprotein (Yoshioka et al. 1986; Lee et al. 1987) and bovine fetuin have been published (Christie et al. 1987; Dziegielewska et al. 1990) as well as the N-terminal sequences of fetuin-like glycoprotein in the tammar wallaby (Jones et al. 1988). The primary amino acid sequence homology is high (50-70% depending on the part of the molecule compared), suggesting that these proteins belong to the same family (Jones et al. 1988). The origin of these proteins in the developing brain is still not clear. There is some evidence that cells in immediate contact with the CSF surface (ventricular zone cells) are able to take up some plasma proteins at some stages of development (see Cavanagh and Warren 1985). The presence of a substantial concentration of F L G in CSF during the period studied (see Table 2) suggests an obvious source of this protein for uptake into ventricular zone cells.. The source of this and other proteins in CSF in the developing brain is probably largely from the plasma (see Dziegielewska and Saunders 1988). Once the cells move out from the ventricular zone to form other parts
319 of the neocortex, they probably lose their contact with CSF. There is now extensive evidence for the presence of specific plasma protein m R N A in the developing brain (MollgSrd et al. 1988) and we have recently shown by in situ hybridisation that the cortical plate in 40d fetal sheep contains m R N A for fetuin (unpublished observations). The immunocytochemical appearance illustrated in Fig. 1 is consistent with the sequence of uptake of FLG from CSF, migration of FLG-containing cells, and onset of synthesis of FLG in subplate/cortical plate neurons. Similar information for human brain is difficult to obtain, and we do not at present have suitable cDNA probes for hybridization studies in the tammar. It does appear though, that the positively immunostained cells in the cortical plate are likely to be able to synthesize fetuin-like proteins at the time that the first cortical plate cells take up their position within the primordial plexiform layer. The proportion of glycoprotein-positive cells in the early neocortex varies in different species : in some eutherian mammals (sheep, cow) the majority of early cortical plate cells are immunopositive, whereas, as shown in this study, in marsupial species like the tammar and also in Monodelphis domestica (unpublished observation) the positive cells are more sparsely distributed. Also in eutherian mammals (Reynolds and Mollgfird 1985; Reynolds et al. 1987) the staining persists over a much longer period of development. This may be a reflection of the differences in cortical plate development in marsupial species (Reynolds et al. 1985; Reynolds 1987; Reynolds and Saunders 1988) as compared with eutherians. In spite of these differences, the general pattern of fetuin-like glycoprotein staining in the developing neocortex is similar in different species. The rather fleeting apperance of these glycoproteins in cortical plate cells would suggest some developmentally regulated role in general formation and modulation of the cortex. The distribution of strongly-staining glycoprotein-positive cells in two zones flanking the immature cortical plate (Fig. 1, 15d) is similar to the distribution of [3H]-thymidine labelling described in the early cortical plate of the cat fetus (Luskin and Shatz 1985; Shatz et al. 1988). A similar pattern for both [3H]-thymidine labelling and fetuin in the same sections has been found in fetal sheep brain at the equivalent stage of early cortical plate formation; these cells are thought to be a transient population (Reynolds et al. 1990). Similar quantitative autoradiographic studies have not been carried out in the tammar. It may be that the much earlier disappearance of the fetuin-like glycoprotein from early cortical plate cells in the tammar pouch young compared with the sheep fetus reflects much earlier disappearance of these cells in the tammar. This in turn may be associated with the generally more mature appearance of the subplate and developing layer 6 in the marsupial compared with eutherians (see Reynolds et al. 1985 and Reynolds 1987 for discussion). Shatz and her colleagues have demonstrated a number of peptides in the early generated population of cortical plate neurons that largely disappear during development; these peptides include somatostatin, neu-
ropeptide Y and cholecystokinin (see Shatz et al. 1988). However they are not expressed until late fetal or early neonatal life, whereas fetuin and equivalent glycoproreins are expressed from the earliest stages of subplate and cortical plate formation. In detailed studies of the development of the tammar telencephalon (Reynolds et al. 1985) experiments with [3H]-thymidine injections indicate that the cells born on day 0 take up their position in the subventricular zone by day 5; by day 40-50 such labelled cells are present only in the subplate zone. In more recent experiments (unpublished) we have shown that injecting the animals with [3H] thymidine at day 2, was already too late to label FLG-positive cells. It is therefore likely that the FLG-positive cells are born sometime between day 0 and day 2. The role of fetuin-like glycoproteins in brain development is not known, nor is it even clear what their function may be in plasma. These proteins are not fetalspecific, as adult plasma contains variable amounts, depending on the species studied. Thus in the sheep and cow, fetuin is a major plasma protein in development, constituting 30% of all plasma protein in sheep fetuses (Dziegielewska et al. 1980) and up to 50% in late gestation cow fetuses (Bergmann et al. 1962). In adult animals the levels are of the order of 20~40 mg per 100 ml. In the human fetus the data are more limited, but it seems that the levels of e2HS glycoprotein remain similar throughout the developmental period, and do not differ much from the adult (50-70 rag/100 ml, Dziegielewska et al. 1987). In this paper we present data on the ontogeny of fetuin-like glycoprotein in the tammar wallaby; in contrast to the above species the levels of FLG in early pouch young are lower and more like e2HS in human than fetuin in sheep and cow (around 70 rag/ 100 ml). From this low concentration, FLG then increases with age to reach over 200 rag/100 ml in the mature joey, which is 3-4 times higher than c~zHS in the adult human, and an order of magnitude more than fetuin in sheep. Thus the developmental change in plasma concentrations of these glycoproteins follows a completely different pattern in all species studied so far. Several biological roles have been ascribed to fetuin and cr glycoprotein (see Dziegielewska et al. 1987 for references and discussion). The wallaby equivalent of these proteins has not been studied. No common property of fetuin and ezHS has been so far found. Nevertheless because of the similarity of their developmental expression in the brain, combined with differences in plasma during development, we would propose that their functions in the brain may be similar in all species, and perhaps confined to only some parts of the molecule, as has been shown for proenkephalin (Noda et al. 1982) and opiomelanocortin (Kita et al. 1979). The more overall function of fetuin-like glycoproteins in other tissue development and in plasma may be different and dependent on more general properties of these molecules. Marsupial species are born at an extremely early (precortical plate) stage of neocortical developmental (Reynolds etal. 1985; Reynolds and Saunders 1988; Saunders et al. 1989). The demonstration in this paper
320 o f p o s t n a t a l e x p r e s s i o n o f a g l y c o p r o t e i n r e l a t e d to fetuin a n d e 2 H S in early c o r t i c a l p l a t e cells in the t a m m a r a n d in Monodelphis domestica ( u n p u b l i s h e d o b s e r v a tions) opens the p o s s i b i l i t y o f m o r e direct studies o f the f u n c t i o n a l significance o f this g l y c o p r o t e i n f o r early neoc o r t i c a l d e v e l o p m e n t t h a n is p o s s i b l e in less accessible e u t h e r i a n fetuses.
Acknowledgements. We should like to thank Action Research for the Crippled Child (SJ) and The Welicome Trust (KMD) for their generous support of this work. LAH was supported by the Australian Academies and Royal Society Exchange Programme. We should particularly like to thank Professor Kjeld Mollg~rd for his painstaking advice and discussion of our experimental material and manuscript and Professor M Szelke and Dr. J Sueras-Diaz for synthesizing FLG peptide sequences. We should also like to thank Mrs Mary Eldridge and Mrs Lynn Ford for typing the manuscript.
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