Kjeld Møllgård

Monodelphis domestica (grey short-tailed opossum): an accessible model for studies of early neocortical development.

SummaryThe development of the neocortex of the marsupial Monodelphis domestica has been studied from birth until adulthood. Monodelphis is born after a gestational period of 14 days, a time when the neocortex is still at a two-layered “embryonic’ stage of development, that is equivalent to a 13–14 day rat embryo or 6 week human embryo. The cortical plate does not begin to appear until 3 to 5 days postnatal. Thus the whole of neocortical development is a postnatal phenomenon in this species, as has been previously described in other marsupials. The general pattern of development of the characteristic layers of the immature neocortex and the subsequent development of a six-layered adult neocortex is similar to that found in eutherian species. However there are some differences. The depth of the immature cortical plate when compared to the thickness of the neocortical wall is less than in eutherians and the subplate zone is much deeper in Monodelphis; this transient subplate zone consists of widely spaced rows of cells that are aligned parallel to the cortical surface. Unlike eutherians there appears to be no secondary proliferative zone in the subventricular zone of the dorso-lateral neocortical wall.Maturation of the neocortex is apparent by 45 days postnatal and by 60 days (around the time of weaning) the characteristic six-layered adult neocortex is clearly present. The neuronal marker PGP 9.5 was used to define neuronal populations in the adult brain. The density of neurons in Monodelphis appears to be considerably less than in eutherians such as the rat. The suitability of postnatal Monodelphis for studies of neocortical development is discussed.

The development of the human blood-brain and blood-CSF barriers

The development of the human blood‐brain and blood‐CSF barriers

Complex tight junctions of epithelial and of endothelial cells in early foetal brain

SummaryThe morphology of epithelial and of endothelial intercellular junctions in human foetal (9–15 weeks gestation) and sheep foetal (50, 60 and 125 days gestation, term 147 days) brain has been studied using the freeze-fracture technique and thin section transmission electronmicroscopy. Freeze-fracture replicas of the choroid plexus of both early human and sheep foetuses showed that the choroidal ependymal cells are linked at the ventricular surface by tight junctions. Freeze-fracture replicas of foetal cortical endothelial cell junctions showed that they are still more complex than those of choroidal epithelial cells, in all specimens so far examined. In some 60 day sheep foetuses the dye Alcian blue, which binds to plasma albumin and which is electrondense when treated with osmium tetroxide, was injected intravenously a few minutes prior to fixation. The dye penetrated from blood into brain extracellular space and c.s.f. but apparentlynot by anintercellular route. The dye was found in a tubular system (endoplasmic reticulum) in both choroidal epithelial and cortical endothelial cells. The possibility that protein penetrates into the foetal brain and c.s.f. by atranscellular route is discussed. The possible significance of these findings in relation to previous ideas and studies of the development of blood-brain barrier mechanisms is also considered.

Synthesis and localization of plasma proteins in the developing human brain: Integrity of the fetal blood-brain barrier to endogenous proteins of hepatic origin☆

The distribution and possible origins of plasma proteins in the human embryonic and fetal brain at different stages of development have been investigated by a combination of isolation and translation of mRNAs and immunocytochemistry using specific antisera. As many as 23 plasma-like proteins have been identified using immunocytochemical methods at the light microscopical level. The presence of mRNAs for 13 of the immunocytochemically positive plasma proteins was demonstrated by in vitro and in ovo translation followed by crossed immunoelectrophoresis and autoradiography; this indicates in situ synthesis of these proteins (e.g., alpha-fetoprotein, alpha 1-antitrypsin, GC-globulin, alpha 2-macroglobulin, pseudocholinesterase, and transferrin) in some brain regions. The regional distribution of some proteins and the absence of some mRNAs suggest that the presence of certain plasma proteins in developing brain may be accounted for by uptake from csf or via nerve processes extending beyond the blood-brain barrier. In several cases, specific proteins appear to be associated with defined cell types, e.g., alpha-fetoprotein, GC-globulin, and ceruloplasmin with neurons, alpha 2-macroglobulin with endothelial cells, and ferritin with glial cells. Some proteins were associated with two or three cell types, e.g., alpha 1-antitrypsin with neurons and glia, and transferrin and alpha 2HS-glycoprotein with neurons, glia, and endothelial cells. Comparison of the expression of mRNAs from fetal brain and liver injected into Xenopus oocytes showed that a few proteins (transferrin and ceruloplasmin) were secreted when liver mRNA was injected, but not when brain mRNA was injected. This suggests that there may be an important difference in the structure and/or processing of these proteins in the brain which may reflect a function different from that associated with them when they originate from the liver. Staining was generally intracellular rather than extracellular; plasma proteins were not associated with the areas immediately around blood vessels although there was a strong immunoprecipitation for each protein within the lumen of cerebral blood vessels. These immunocytochemical findings together with the identification of mRNAs for a large number of plasma proteins in immature brain are discussed in relation to animal experimental work which suggests that the blood-brain barrier to protein is present even at very early stages of brain development.

Studies of the development of brain barrier systems to lipid insoluble molecules in fetal sheep.

1. The development of the blood‐brain and blood‐c.s.f barriers to lipid insoluble substances of different molecular radii has been studied in fetal sheep, early (60 days) and late (125 days) in gestation, using labelled erythritol (C14), sucrose (3H or 14C), inulin (3H or 14C) and albumin (125I), or albumin and IgG detected by immunoassay. 2. Morphological studies of fetal brain and choroid plexus at the same gestational stages were carried out using thin section electron microscopy and the freeze fracture techniques. 3. Penetration of markers into c.s.f. was substantially greater at 60 days than at 125 days, but at both ages the steady‐state level achieved appeared to be related to molecular size. 4. A simple model describing penetration from blood into c.s.f. at 60 days is proposed. It involves the assumption that c.s.f. and brain extracellular fluid are effectively separate compartments; morphological and permeability data which supports this assumption is presented. The data for c.s.f. at 60 days are consistent with the suggestion that the markers penetrate into c.s.f. by diffusion and are not restricted by small pores in the interface between blood and c.s.f. 5. The reduction in penetration which occurred by 125 days for all markers except erythritol appears to be accounted for by an increase in the sink effect and a decrease in the effective surface area for exchange between blood and c.s.f. 6. Intercellular tight junctions of both cerebral endothelial cells and choroid plexus epithelial cells were well formed at 60 days gestation. There was no change in junctional characteristics previously thought to correlate with transepithelial permeability (tight junction depth and strand number) between the two ages studied, although there were marked changes in permeability. 7. Evidence is advanced in support of the hypothesis that in the fetus much of the penetration of lipid insoluble non‐polar substances across the blood‐c.s.f. barrier and perhaps across the blood‐brain barrier occurs via a transcellular route consisting of a system of tubulo‐cisternal endoplasmic reticulum. Penetration via the choroid plexus appears to be the dominant route for penetration from blood into c.s.f. in the 60 day fetus.

Immunohistochemical evidence for an intracellular localization of plasma proteins in human foetal choroid plexus and brain

The intracellular distribution of plasma proteins in human foetal choroid plexus and brain was investigated by immunohistochemistry. Small groups of cells or single scattered cells in the epithelial layer of the choroid plexus exhibited positive staining for alpha-fetoprotein, albumin and transferrin, whereas prealbumin was found in the majority of the epithelial cells. Numerous nerve cells in the cerebral wall and in various brain stem nuclei were positively stained for alpha-fetoprotein, albumin and prealbumin. All appropriate controls were negative. The presence of plasma proteins within choroid plexus epithelial cells suggest that these proteins are transported from blood to CSF by a transcellular route across the choroid plexus epithelium. The intracellular distribution of plasma proteins in developing neurons may indicate that these proteins play some important role in neuronal differentiation or development.

Differential immunocytochemical staining for glial fibrillary acidic (GFA) protein, S-100 protein and glutamine synthetase in the rat subcommissural organ, nonspecialized ventricular ependyma and adjacent neuropil

SummaryAntibodies raised against glial fibrillary acidic protein (GFA), S-100 protein (S100) and glutamine synthetase (GS) are currently used as glial markers. The distribution of GFA, S100 and GS in the ependyma of the rat subcommissural organ (SCO), as well as in the adjacent nonspecialized ventricular ependyma and neuropil of the periaqueductal grey matter, was studied by use of the immunocytochemical peroxidase-antiperoxidase technique. In the neuropil, GFA, S100 and GS were found in glial elements, i.e., in fibrous (GFA, S100) and protoplasmic astrocytes (S100, GS). The presence of S100 in the majority of the ventricular ependymal cells and tanycytes, and the presence of GFA in a limited number of ventricular ependymal cells and tanycytes confirm the glial nature of these cells. The absence of S100, GFA and GS from the ependymocytes of the SCO, which are considered to be modified ependymal cells, suggests either a non-astrocytic lineage of these cells or an extreme specialization of the SCO-cells as glycoprotein-synthesizing and secreting elements, a process that may have led to the disappearance of the glial markers.

Proteins in cerebrospinal fluid and plasma of fetal sheep during development

1. The concentration of total protein in c.s.f. and plasma has been measured in fetal sheep of different gestational ages and in the adult. In c.s.f. it was highest (approximately 840 mg/100 ml.) in the youngest fetuses (35 days) and declined steeply by 60 days (260 mg/100 ml.). It decreased less markedly in the last half of gestation to reach about 50 mg/100 ml. at 125 days which is twice the adult value. Protein concentration in plasma was lowest in the youngest fetuses and did not rise much until the second half of gestation during which time it doubled. There was a further rather larger increase between the late fetal (125 days) stage and the adult.

In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24 women). Expressions of androgen receptor (AR) mRNA levels in granulosa cells, and of androstenedione and testosterone in follicular fluid, were correlated to the expression of the FSH receptor (FSHR), LH receptor (LHR), CYP19 and anti-Müllerian Hormone-receptor II (AMHRII) mRNA in the granulosa cells and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the follicular fluid levels of androgen and FSHR expression. This suggests that follicular sensitivity towards FSH stimulation may be augmented by stimulation of androgens via the AR.

Stem cell factor and c-Kit in human primordial germ cells and fetal ovaries.

The distribution of the tyrosine kinase receptor c-Kit and its ligand stem cell factor (SCF) was evaluated by immunohistochemistry in primordial germ cells (PGCs) and human embryonic gonads during weeks 5-8 of prenatal life, and fetal ovaries during weeks 9-36 of prenatal life. Distinct c-Kit and SCF staining was present in primordial germ cells in the wall of the hindgut and in the dorsal mesentery, particularly on level with the 10th thoracic columnar segment. Several PGCs were in close contact with c-Kit-negative but SCF-positive autonomic nerve fibers of the dorsal mesentery. Many fibroblasts and mesothelial cells of the dorsal mesentery were clearly stained for SCF, but not for c-Kit. Prominent c-Kit and SCF staining was present in germ cells of the embryonic gonadal anlage and in oogonia during further ovarian development. However, oocytes were either unstained or faintly stained for SCF. Oocytes not yet enclosed in follicles or present in primordial follicles were either unstained or exhibited faint cytoplasmic c-Kit staining, whereas oocytes of growing preantral follicles again showed distinct cell membrane staining which decreased during further follicular growth. Theca cells did not stain for c-Kit. Some pregranulosa cells and the first formed granulosa cells of primordial follicles were c-Kit stained. Granulosa cells of other follicles were not c-Kit stained. In the inner part of the cortex, SCF immunolabeling was detected in some pregranulosa cells surrounding cords containing germ cells and involved in formation of primordial follicles. Granulosa cells of primordial and growing follicles, including medium-sized antral follicles also revealed SCF staining. In conclusion, this first report on SCF in human PGCs and embryonic and fetal ovaries together with the c-Kit data lend substantial countenance to the notion that c-Kit and SCF play important roles during ascent of primordial germ cells towards the gonadal anlage, and during oogenesis and folliculogenesis in the human fetal ovary. We suggest that both autocrine and paracrine mechanisms are involved in the proposed anti-apoptotic effect of the c-Kit/SCF duet while PGCs are present in the dorsal mesentery. The SCF-positive autonomic nerve fibers of the dorsal mesentery, mesothelial cells and fibroblasts may nurse and perhaps guide PGCs during their ascent.