M.I. Alonso

Disruption of proteoglycans in neural tube fluid by β-D-xyloside alters brain enlargement in chick embryos

Following neurulation, the anterior end of the neural tube undergoes a dramatic increase in size due mainly to the enlarging of the brain cavity. This cavity is filled with so‐called neural tube fluid (NTF), whose positive pressure has been shown to play a key role in brain morphogenesis. This fluid contains a watersoluble matrix, rich in chondroitin sulfate (CS), which has been proposed as an osmotic regulator of NTF pressure genesis. The purpose of the present study is to observe the influence of CS on NTF osmolality and its relation to NTF hydrostatic pressure and brain expansion.

Embryonic cerebrospinal fluid collaborates with the isthmic organizer to regulate mesencephalic gene expression

Early in development, the behavior of neuroepithelial cells is controlled by several factors acting in a developmentally regulated manner. Recently it has been shown that diffusible factors contained within embryonic cerebrospinal fluid (CSF) promote neuroepithelial cell survival, proliferation, and neurogenesis in mesencephalic explants lacking any known organizing center. In this paper, we show that mesencephalic and mesencephalic + isthmic organizer explants cultured only with basal medium do not express the typically expressed mesencephalic or isthmic organizer genes analyzed (otx2 and fgf8, respectively) and that mesencephalic explants cultured with embryonic CSF‐supplemented medium do effect such expression, although they exhibit an altered pattern of gene expression, including ectopic shh expression domains. Other trophic sources that are able to maintain normal neuroepithelial cell behavior, i.e., fibroblast growth factor‐2, fail to activate this ectopic shh expression. Conversely, the expression pattern of the analyzed genes in mesencephalic + isthmic organizer explants cultured with embryonic cerebrospinal fluid‐supplemented medium mimics the pattern for control embryos developed in ovo. We demonstrate that embryonic CSF collaborates with the isthmic organizer in regulation of the expression pattern of some characteristic neuroectodermal genes during early stages of central nervous system (CNS) development, and we suggest that this collaboration is not restricted to the maintenance of neuroepithelial cell survival. Data reported in this paper corroborate the hypothesis that factors contained within embryonic CSF contribute to the patterning of the CNS during early embryonic development.

Basal lamina heparan sulphate proteoglycan is involved in otic placode invagination in chick embryos.

Formation of the otocyst from the otic placode appears to differ from invagination of other cup-shaped organ primordia. It is known that the cellular cytoskeleton plays a limited role in otic placode invagination, whilst the extracellular matrix underlying the otic primordium intervenes in the folding process. In this study we have analysed the role of the basal lamina heparan sulphate proteoglycan in otic primordium invagination. At 10 H.H. stage, heparan sulphate proteoglycan immunomarking begins to appear on the otic placode basal lamina, increasing noticeably at 13 H.H. stage, coinciding with maximum folding of the otic epithelium, and is still present at later stages. Enzyme degradation of heparan sulphate proteoglycan in the otic primordium basal lamina, by means of microinjection with heparinase III prior to folding, significantly disrupts invagination of the otic placode, which remains practically flat, with a significant reduction in the depth of the otic pit and an increase in the diameter of the otic opening. The immunocytochemistry analysis revealed a notable depletion of basal lamina heparan sulphate proteoglycan in the otic primordia microinjected with heparinase, with no statistically significant differences observed in the volume or rate of cell proliferation in the otic epithelium relative to the control, which suggests that heparan sulphate proteoglycan disruption does not interfere with the epithelial growth. In addition, a study of apoptosis distribution by the TUNEL method confirmed that treatment with heparinase does not cause interference with cell survival in the otic epithelium. Our findings support the theory that otic primordium invagination may be regulated, at least in part, by the basal lamina components, which might contribute towards anchoring the otic epithelium to adjacent structures.

Cerebrospinal Fluid Control of Neurogenesis Induced by Retinoic Acid During Early Brain Development

Embryonic‐cerebrospinal fluid (E‐CSF) plays crucial roles in early brain development including the control of neurogenesis. Although FGF2 and lipoproteins present in the E‐CSF have previously been shown to be involved in neurogenesis, the main factor triggering this process remains unknown. E‐CSF contains all‐trans‐retinol and retinol‐binding protein involved in the synthesis of retinoic acid (RA), a neurogenesis inducer. In early chick embryo brain, only the mesencephalic‐rombencephalic isthmus (IsO) is able to synthesize RA. Here we show that in chick embryo brain development: (1) E‐CSF helps to control RA synthesis in the IsO by means of the RBP and all‐trans‐retinol it contains; (2) E‐CSF has retinoic acid activity, which suggests it may act as a diffusion pathway for RA; and (3) the influence of E‐CSF on embryonic brain neurogenesis is to a large extent due to its involvement in RA synthesis. These data help to understand neurogenesis from neural progenitor cells. Developmental Dynamics 240:1650–1659, 2011.

Prenatal expression of interleukin 1β and interleukin 6 in the rat pituitary gland

It is known that interleukin 1beta (IL-1beta) and interleukin 6 (IL-6) are expressed post-natally in normal and tumoral cells in the anterior pituitary, and that they play a role in both the liberation of different hormones and in the growth, proliferation and tumor formation of the pituitary gland. However, their expression and role during embryonic and fetal development remain unknown. We have performed an immunocytochemistry study of prenatal expression and distribution of IL-1beta and IL-6 in isolated embryonic rat Rathkes pouch prior to birth, more specifically between 13.5 and 19.5 days p.c. Western-blot analysis carried out on 19.5-day p.c. embryos showed positive immunolabelling for IL-1beta and IL-6. These interleukins were initially expressed simultaneously in the rostral and ventral portions of Rathkes pouch in 15.5-day p.c. embryos, and this expression progressed caudodorsally in later developmental stages, extending to most of the hypophysis before birth. The number of cells expressing these interleukins increased throughout this period: 48.22% of anterior pituitary cells expressed IL-6 in 19.5-day embryos, whilst IL-1beta was positive in 39.8% of the cells. Moreover, we have demonstrated that some adenohypophyseal cells co-express both interleukins. Such findings represent the first step towards an understanding of the physiological role of these interleukins in anterior pituitary development.

Embryonic Cerebrospinal Fluid Activates Neurogenesis of Neural Precursors within the Subventricular Zone of the Adult Mouse Brain

Introduction: There is a nondeveloped neurogenic potential in the adult mammalian brain, which could be the basis for neuroregenerative strategies. Many research efforts have been made to understand the control mechanisms which regulate the transition from a neural precursor to a neuron in the adult brain. Embryonic cerebrospinal fluid (CSF) is a complex fluid which has been shown to play a key role in neural precursor behavior during development, working as a powerful neurogenic inductor. We tested if the neurogenic properties of embryonic CSF are able to increase the neurogenic activity of neuronal precursors from the subventricular zone (SVZ) in the brains of adult mice. Results: Our results show that mouse embryonic CSF significantly increases the neurogenic activity in precursor cells from adult brain SVZ. This intense neurogenic effect was specific for embryonic CSF and was not induced by adult CSF. Conclusions: Embryonic CSF is a powerful neurogenesis inductor in homologous neuronal precursors in the adult brain. This property of embryonic CSF could be a useful tool in neuroregeneration strategies.

Embryonic Cerebrospinal Fluid Increases Neurogenic Activity in the Brain Ventricular-Subventricular Zone of Adult Mice

Neurogenesis is a very intensive process during early embryonic brain development, becoming dramatically restricted in the adult brain in terms of extension and intensity. We have previously demonstrated the key role of embryonic cerebrospinal fluid (CSF) in developing brain neurogenic activity. We also showed that cultured adult brain neural stem cells (NSCs) remain competent when responding to the neurogenic influence of embryonic CSF. However, adult CSF loses its neurogenic inductive properties. Here, by means of an organotypic culture of adult mouse brain sections, we show that local administration of embryonic CSF in the subventricular zone (SVZ) niche is able to trigger a neurogenic program in NSCs. This leads to a significant increase in the number of non-differentiated NSCs, and also in the number of new neurons which show normal migration, differentiation and maturation. These new data reveal that embryonic CSF activates adult brain NSCs, supporting the previous idea that it contains key instructive components which could be useful in adult brain neuroregenerative strategies.

Contents Vol. 189, 2009

Stem Cells and Tissue Engineering A. Bader, Leipzig E-Mail: augustinus.bader@bbz.uni-leipzig.de S.F. Badylak, Pittsburgh, Pa. E-Mail: badylaks@msx.upmc.edu A. Müller, Würzburg E-Mail: albrecht.müller@mail.uni-wuerzburg.de A. Ratcliffe, San Diego, Calif. E-Mail: anthonyratcliffe@synthasome.com A.M. Wobus, Gatersleben E-Mail: wobusam@ipk-gatersleben.de Neurosciences M. Frotscher, Freiburg i.Br. E-Mail: frotsch@sun2.ruf.uni-freiburg.de W.L. Neuhuber, Erlangen E-Mail: winfried.neuhuber@rzmail.uni-erlangen.de