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The expression of four cadherins (cadherin-6B, cadherin-7, R-cadherin, and N-cadherin) was mapped in the diencephalon of chicken embryos at 11 days and 15 days of incubation and was compared with Nissl stains and radial glial topology. Results showed that each cadherin is expressed in a restricted manner by a different set of embryonic divisions, brain nuclei, and their subregions. An analysis of the segmental organization based on the prosomeric model indicated that, in the mature diencephalon, each prosomere persists and forms a coherent domain of gray matter extending across the entire transverse dimension of the neural tube, from the ventricular surface to the pial surface. Moreover, the results suggest the presence of a novel set of secondary subdivisions for the dorsal thalamus (dorsal, intermediate, and ventral tiers and anteroventral subregion). They also confirm the presence of secondary subdivisions in the pretectum (commissural, juxtacommissural, and precommissural). At most of the borders between the prosomeres and their secondary subdivisions, changes in radial glial fiber density were observed. The diencephalic brain nuclei that derive from each of the subdivisions were determined. In addition, a number of previously less well-characterized gray matter regions of the diencephalon were defined in more detail based on the mapping of cadherin expression. The results demonstrate in detail how the divisions of the early embryonic diencephalon persist and transform into mature gray matter architecture during brain morphogenesis, and they support the hypothesis that cadherins play a role in this process by providing a framework of potentially adhesive specificities. J. Comp. Neurol. 421:481–514, 2000. © 2000 Wiley-Liss, Inc.

MicroRNAs (miRNAs) are single-stranded RNA molecules of approximately 22 nucleotides. They regulate protein expression by binding to and suppressing translation of messenger RNAs. Less than 20 years ago, they were unheard of, but the number of identified miRNAs is growing. Antonio Giraldez This burgeoning field captured Antonio Giraldez's attention while he was a PhD student studying Drosophila wing morphogenesis (1–3), yet he didn't start his own experiments on the subject until after he had started his postdoc. Better late than never, as Giraldez's studies produced important data showing that zebrafish zygotes unable to make their own miRNAs cannot undergo brain morphogenesis, and that one particular miRNA can rescue the defect (4, 5). Now running his own laboratory at Yale, Giraldez has stuck with zebrafish and investigates how miRNAs shape gene expression to orchestrate embryonic development (6).

A large number of genes have been isolated in the last few years that seem to be involved in the processes of induction, specification and regionalization of the brain. The generation of mouse mutant models for such genes has provided a powerful tool of analysis into their in-vivo properties. Among these genes, Otx1 and Otx2 play a crucial role in several processes of brain morphogenesis. Otx2 is required in the early specification of the neuroectoderm destined to become the fore-midbrain, Otx1 is required mainly for correct corticogenesis, and both genes co-operate in patterning the developing brain through a gene-dosage dependent mechanism. Furthermore, an extensive functional equivalence occurs between the mammalian Otx genes and their Drosophila homolog, orthodenticle. This appears particularly fascinating when considering the huge differences between the brains of mice and flies. This review deals with the major aspects related to the involvement of Otx1 and Otx2 in the development of the mammalian brain and, in particular, of the cerebral cortex.

Producción Científica, The expansive force generated by the positive pressure of the neural tube fluid confined inside the brain vesicles has been shown to be a key factor during the earliest stages of brain morphogP.nesis and development of chick embryos. In a previous study,we demonstrated the existence of an intracavity extracellular matrix rich in condroitin sulfate in this species, which could be in­ volved in the regulation of the expa nsive process. In this report, scanning electron microscopy and immunohisto­ chemistry show that,after neurulation,a similar extracel­ lular matrix rich in chondroitin sulfate is present inside the brain vesicles of rat embryos during early enlarge­ ment of the brain anlage. In vitro treatment of rat embryos- Abbreviations used in this paper bryos with B-0-xyloside (a chemical compound which

Pontocerebellar hypoplasias are congenital disorders of brain morphogenesis which include such diverse etiologies as carbohydrate-deficient glycoprotein syndrome type 1, cerebromuscular dystrophies (Walker-Warburg syndrome, Fukuyama syndrome, muscle-eye-brain disease) and at least two types of autosomal recessive neurodegenerations known as pontocerebellar hypoplasia type I and II. Pontocerebellar hypoplasia type 1 is a lethal phenotype and clinical features include congenital contractures, respiratory insufficiency, central and peripheral motor dysfunction and spinal anterior horn degeneration. Type 2 is characterized by progressive microcephaly, extrapyramidal dyskinesia and normal spinal cord findings. In this paper, we describe a girl, born at 33 weeks of gestation, presenting with respiratory insufficiency and multiple contractures. MRI scan of the brain demonstrated pontocerebellar hypoplasia and cortical and diffuse periventricular white matter abnormalities. Postmortem examination showed pontocerebellar hypoplasia with extensive gliosis of the periventricular white matter and of the basal ganglia with normal spinal cord findings. Histology of skeletal muscle was normal. Biochemical analysis demonstrated multiple deficiencies of respiratory chain enzymes in skin fibroblasts. This case demonstrates a lethal phenotype of pontocerebellar hypoplasia without spinal cord abnormalities associated with a respiratory-chain disorder. The diagnostic workup in a patient whose brain image shows pontocerebellar hypoplasia should include a search for respiratory-chain impairment.

Understanding the genetic mechanisms that control brain patterning in vertebrates represents a major challenge for developmental neurobiology. The cloning of genes likely to be involved in the organization of the brain and an analysis of their roles have revealed insights into the molecular pathways leading to neural induction, tissue specification, and regionalization of the brain. Among these genes, both Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, contribute to several steps in brain morphogenesis. Recent findings have demonstrated that Otx2 plays a major role in gastrulation and in the early specification of the anterior neural plate while Otx1 is mainly involved in corticogenesis, and Otx1 and Otx2 genes cooperate in such a way that a minimal level of OTX proteins are required for proper regionalization and subsequent patterning of the developing brain. Finally, experiments have shown functional equivalence between Drosophila otd and vertebrate Otx genes, suggesting a surprising conservation of function required in brain development throughout evolution.

In the last decade, a number of genes related to the induction, specification and regionalization of the brain were isolated and their functional properties currently are being dissected. Among these, Otx1 and Otx2 play a pivotal role in several processes of brain morphogenesis. Findings from several groups now confirm the importance of Otx2 in the early specification of neuroectoderm destined to become fore-midbrain, the existence of an Otx gene dosage-dependent mechanism in patterning the developing brain, and the involvement of Otx1 in corticogenesis. Some of these properties appear particularly fascinating when considered in evolutionary terms and highlight the central role of Otx genes in the establishment of the genetic program defining the complexity of a vertebrate brain. This review deals with the major aspects related to the roles played by Otx1 and Otx2 in the development and evolution of the mammalian brain.

Bacterial collagenase was injected into the ventricular cavity of the optic tectum of chick and quail embryos. Histological examination up to 6 days after enzyme injection revealed that the collagenase disrupted the pial basal lamina, which was evident by the fragmented distribution of basal lamina proteins at the pial surface of the midbrain and the brainstem. Although the disrupted basal lamina was not reestablished at later stages of development, the pial basal lamina of the newly developing neuroepithelium in the caudal part of the tectum was continuous and intact. Western blot analysis showed that the collagenase digested collagens but spared noncollagenous proteins. The disruption of the pial basal lamina caused the neuroepithelial cells to retract their pial end feet and caused tectal axons to exit the brain tissue into the adjacent mesenchyme. The vertical migration of neuroblasts to the pial layers of the tectum was inhibited, leading to a disruption of the tectal histogenesis. In the developing optic pathways, retinal axons were misguided at the optic chiasma and terminated in the head mesenchyme instead of the tectum. None of the abnormalities in histogenesis and axonal pathways were observed when the basal lamina was disrupted at a later stage of embryonic development. The present experiments demonstrate that the pial basal lamina has an important function during brain morphogenesis in restricting axons to the brain, providing an anchoring of the neuroepithelial cells to the pial surface, and allowing the formation of a defined cytoarchitecture of the brain.

The tumor-suppressor protein p53 has been implicated in cell cycle arrest and apoptotic cell death in dividing cells (Yonish-Rouach et al. [1991] Nature 352:342-347. To elucidate possible functions of p53 in the regulation of cell division and cell death during development of the rat central nervous system, we compared the spatial and temporal expressions of p53 mRNA and protein with those of its transcriptional targets Bax, p21Waf1, and cyclin G1 and with the cyclin-dependent kinase inhibitors p27Kip1, p57Kip2, and p16Ink4a. From embryonic day 14 until the second postnatal week, p53 mRNA and protein were found predominantly in proliferating zones, including the cells of the emerging external granular layer of the cerebellum, and the ventricular and the subventricular zones of the forebrain. At all postnatal ages, there was a high expression of p53 mRNA and protein in cells of the rostral migratory stream, a well-defined pathway along which precursor cells of olfactory interneurons migrate from the ventricular and subventricular zones toward the olfactory bulb. In addition to its expression in proliferating cell populations, p53 was expressed in postmitotic cells of the cerebral cortex and hippocampus at embryonic and early postnatal stages. p53, p27Kip1, p16Ink4a, and bax alpha mRNA colocalized in the ventricular and subventricular zones at embryonic and early postnatal stages, but the distribution of p53 differed from that of its transcriptional targets cyclin G1 and p21Waf1 and from that of the cyclin-dependent kinase inhibitor p57Kip2, which were predominantly expressed in fully differentiated neurons. Double-labeling studies showed that p53 mRNA and protein were absent in cells undergoing developmental cell death, as identified by the presence of single- or double-stranded nuclear DNA breaks. Protein levels of p53 decreased during development in all brain areas studied. These results indicate a role for p53 in the control of cell division and early differentiation rather than in the control of cell death during rat brain development. The nonoverlapping temporal and spatial expression patterns of p53 and its transcriptional targets Bax, cyclin G1 and p21Waf1 suggest that each of these gene products fulfill independent roles in brain morphogenesis.

Understanding the genetic mechanisms that control pat- terning of the vertebrate brain represents a major challenge for developmental neurobiology. Previous data suggest that Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, might both contribute to brain morphogenesis. To gain insight into this possibility, the level of OTX proteins was modified by altering in vivo the Otx gene dosage. Here we report that Otx genes may cooperate in brain morphogenesis and that a minimal level of OTX proteins, corresponding either to one copy each of Otx1 and Otx2, or to only two copies of Otx2, is required for proper regionalization and subsequent pat- terning of the developing brain. Thus, as revealed by anatomical and molecular analyses, only Otx1−/−; Otx2+/− embryos lacked mesencephalon, pretectal area, dorsal thalamus and showed an heavy reduction of the Ammon’s horn, while the metencephalon was dramatically enlarged occupying the mesencencephalic area. In 8.5 days post coitum (d.p.c.) Otx1−/−; Otx2+/− embryos, the expression patterns of mesencephalic-metencephalic (mes-met) markers such as En-1 and Wnt-1 confirmed the early presence of the area fated to give rise to mesencephalon and metencephalon while Fgf-8 transcripts were improperly localized in a broader domain. Thus, in Otx1−/−; Otx2+/− embryos, Fgf-8 misexpression is likely to be the consequence of a reduced level of specification between mes-met primitive neuroepithelia that triggers the following repatterning involving the transformation of mesencephalon into metencephalon, the establishment of an isthmic-like structure in the caudal diencephalon and, by 12.5 d.p.c., the telencephalic expression of Wnt-1 and En-2. Taken together these findings support the existence of a molecular mechanism depending on a precise threshold of OTX proteins that is required to specify early regional diversity between adjacent mes-met territories and, in turn, to allow the correct positioning of the isthmic organizer.

Introduction Connexin43 (Cx43) is the main protein of brain astrocytic gap-junctional network, which plays an important role in the ion and glutamate homeostasis, intercellular signaling, cell adhesion and migration during brain morphogenesis. Earlier we obtained monoclonal antibodies to the E2 extracellular loop of Cx43 (MAbE2Cx43) which could block the functions of astrocytic gap-junctions in vitro and in vivo. Aims The aim of the current study was to elucidate the influence of MAbE2Cx43 on the cognitive function in animals treated during neurogenesis. Methods The antibodies were injected intravenously to wild pregnant female rats in a dose of 5 mg/kg on the 10thday of gestation. The saline solution was used as a control medicine. Behavioral experiments were conducted on 1-month old offsprings in the following order: the Y-maze, novel object recognition test (NORT) and passive avoidance (PA) task. Results The Y-maze test revealed a significant decrease in the spontaneous alternation behavior of experimental rats treated with MAbE2Cx43 in the perinatal period compared to control rats. In the NORT rats treated with MAbE2Cx43 have shown significant impairments of the recognition memory after a 24-hour delay. The PA test revealed no significant difference in avoidance latency in the retention trial on the day 2-nd, but it appeared on the day 7-th. Conclusions These findings confirm memory deficiency after one-time administration of MAbE2Cx43 in the perinatal period. We conclude that the injection of anti-Cx43 antibodies in the perinatal period could alter some Cx43-dependent processes during neurogenesis resulting in cognitive deficiency in adulthood.

E-cadherin is transiently expressed in local regions of the embryonic mouse brain, which include several patchy areas on the mesencephalon and diencephalon and their roof plate and part of cerebellar rudiments. In the present study, we compared this E-cadherin expression with that of Wnt-1, which occurs in specific zones in the embryonic brain, and found certain spatiotemporal relations between them: Wnt-1 expression tended to run parallel or overlap with peripheries of the E-cadherin-positive areas. For example, in the dorsal midline, Wnt-1 was expressed at the middle of the roof plate, while E-cadherin was absent in the middle zone but detected in two arrays of marginal roof plate cells. Furthermore, αN-catenin, a cadherin-associated protein, was found to occur at the roof plate of the mesencephalon and diencephalon, coinciding with Wnt-1 expression. The expression of these molecules was then studied in two alleles of the Wnt-1 mutation, Wnt-1sw and Wnt-1neo. In mice homozygous for these mutant genes, E-cadherin expression in the roof plate was up-regulated; the middle E-cadherin-negative zone disappeared. Moreover, E-cadherin expression in the roof plate began earlier in the mutant mice than in wild-type mice. On the contrary, αN-catenin expression in the dorsal midline was suppressed in these mutants. These changes in cadherin and catenin expression occurred at the level of mRNA expression. These results suggest that the Wnt-1 signal is, either directly or indirectly, involved in the regulation of expression of E-cadherin and αN-catenin in restricted regions of the embryonic brain. This mechanism may contribute to the patterning of the expression of these adhesion-related proteins in the embryonic brain.

Experiments were done to investigate the cause of the cranial (mesencephalic) flexure of the chick brain during stages 10 to 14. Measurements of the length and thickness of the roof and floor of the mesencephalon gave values similar to the values obtained previously by others. The labeling index was determined in the roof and floor of the prosencephalon, mesencephalon, and rhombencephalon as a preliminary measure of cell division. The labeling index was about the same in all regions, and was high enough to suggest that most of the cells were dividing. The labeling indices did not suggest that differential growth was caused by differential rates of cell division in the roof and floor of the mesencephalon. It was found through time lapse photography that the foregut and heart remained stationary along the rostrocaudal axis, whereas the prosencephalon moved rostrally and the mesencephalon underwent flexure. Measurements suggested that the neural tube cranial to the otic primordium grew in volume exponentially at a rate consistent with the labeling index. The rostral tip of the neural tube was observed to be linked to the rostral tip of the foregut by the ectoderm that formed Rathke's pouch at the neural tube and the pharyngeal membrane (prospective stomodeum) at the foregut. As the neural tube grew in length, the link between the neural tube and the foregut did not. We suggest that because of this link, the growing neural tube had to bend around the foregut, forming the cranial flexure, and the ectoderm folded where it attached to the prosencephalon, forming Rathke's pouch.

The great discovery of microRNAs (miRNAs) has revolutionized current cell biology and medical science. miRNAs are small conserved non-coding RNA molecules that post-transcriptionally regulate gene expression by targeting the 3' untranslated region of specific messenger RNAs for degradation or translational repression. New members of the miRNA family are being discovered on a daily basis and emerging evidence has demonstrated that miRNAs play a major role in a wide range of developmental process including cell proliferation, cell cycle, cell differentiation, metabolism, apoptosis, developmental timing, neuronal cell fate, neuronal gene expression, brain morphogenesis, muscle differentiation and stem cell division. Moreover, a large number of studies have reported links between alterations of miRNA homeostasis and pathological conditions such as cancer, psychiatric and neurological diseases, cardiovascular disease, and autoimmune disease. Interestingly, in addition, miRNA deficiencies or excesses have been correlated with a number of clinically important diseases ranging from cancer to myocardial infarction. miRNAs can repress the gene translation of hundreds of their targets and are therefore well-positioned to target a multitude of cellular mechanisms. As a consequence of extensive participation in normal functions, it is quite logical to ask the question if abnormalities in miRNAs should have importance in human diseases. Great discoveries and rapid progress in the past few years on miRNAs provide the hope that miRNAs will in the near future have a great potential in the diagnosis and treatment of many diseases. Currently, an explosive literature has focussed on the role of miRNA in human cancer and cardiovascular disease. In this review, I briefly summarize the explosive current studies about involvement of miRNA in various human cancers and cardiovascular disease.

Individuals with partial HSA21 trisomies and mice with partial MMU16 trisomies containing an extra copy of the DYRK1A gene present various alterations in brain morphogenesis. They present also learning impairments modeling those encountered in Down syndrome. Previous MRI and histological analyses of a transgenic mice generated using a human YAC construct that contains five genes including DYRK1A reveal that DYRK1A is involved, during development, in the control of brain volume and cell density of specific brain regions. Gene dosage correction induces a rescue of the brain volume alterations. DYRK1A is also involved in the control of synaptic plasticity and memory consolidation. Increased gene dosage results in brain morphogenesis defects, low BDNF levels and mnemonic deficits in these mice. Epigallocatechin gallate (EGCG) - a member of a natural polyphenols family, found in great amount in green tea leaves - is a specific and safe DYRK1A inhibitor. We maintained control and transgenic mice overexpressing DYRK1A on two different polyphenol-based diets, from gestation to adulthood. The major features of the transgenic phenotype were rescued in these mice.

Microtubule-associated proteins (MAPs) are a diverse family of cytoskeletal proteins that copurify with tubulin in vitro. Recently a number of novel approaches have been used to learn more about the functions of MAPs during brain development, including: localization of MAPs and their mRNA in the developing brain, comparisons of MAPs between species to learn potential fundamental characteristics, biochemical analysis of changes in MAPs in process-bearing cell lines, and sequence analysis of MAP cDNAs and cDNA transfection studies. Taken together, these data allow us to assign roles to MAPs which are abundant in the developing brain, and to develop models for future studies. Four MAPs are particularly abundant in the developing brain: MAP1B, the high and low-molecular weight forms of MAP2, and juvenile tau. MAP1B is the only MAP to be found consistently in extending processes in both the developing and adult brain, making it a likely regulator of neurite outgrowth. High-molecular weight MAP2 and tau crosslink microtubules in dendrites and axons, respectively. Low-molecular weight MAP2 may be able to regulate MAP2-mediated crosslinking to make processes more labile during development and in adult brain regions where synaptogenesis is active. Tau-mediated crosslinking may be regulated by temporal regulation of the expression of tau forms with different binding affinities to tubulin. High-molecular weight MAP2 is sequestered into dendrites by the selective transport of its mRNA. This allows rapid and local regulation of MAP2 synthesis.

αN-catenin is a cadherin-binding protein, widely expressed in the nervous system; and it plays a crucial role in cadherin-mediated cell-cell adhesion. Here we report the effects of αN-catenin gene deficiency on brain morphogenesis. In addition to the previously reported phenotypes, we found that some of the axon tracts did not normally develop, in particular, axons of the anterior commissure failed to cross the midline, migrating, rather, to ectopic places. In restricted nuclei, a population of neurons was missing or their laminar arrangement was distorted. The ventricular structures were also deformed. These results indicate that αN-catenin has diverse roles in the organization of the central nervous system, but only in limited portions of the brain. Developmental Dynamics 235:2559–2566, 2006. © 2006 Wiley-Liss, Inc.

Homeobox-containing genes encode transcription factors implicated in the development and differentiation of multiple cell systems, including hematopoiesis. Several lines of evidence, obtained in our laboratory, indicate that Otx1, a homeobox gene of the paired class strictly required for brain morphogenesis, plays an important role also in blood cell production. It is differentially expressed in hematopoietic progenitors, particularly within the erythroid lineage and loss of Otx1 function results in a cell-autonomous erythroid impairment. Furthermore, Otx1 contributes to the control of erythropoiesis through a direct action on Scl/Tal1, a major hematopoietic regulator. In this study we have investigated whether Otx1 is also implicated in the regulation of myelo-monocytic differentiation. Analysis of Otx1 expression indicated that the gene is transcriptionally active in myelo-monocytic precursors. Moreover, using clonogenic assays we observed that Otx1−/− mice display abnormal frequencies of bone marrow myeloid subpopulations, although the total number of myeloid precursors is normal, as compared to wild type animals. Inactivation of Otx1 leads to a significant decrease of myelo-monocytic (CFU-GM) and monocytic (CFU-M) progenitors, and a concomitant increase of granulocytic precursors (CFU-G). In addition, morphological analysis of Otx1−/− bone marrow cells confirmed a high percentage of cells of the granulocytic lineage. Furthermore, at the molecular level, we detected in null mutant cells the upregulation of the gene encoding the G-CSF receptor and the modulation of other key myeloid regulators. Finally, to understand whether the action of Otx1 in myeloid cells is, like in erythoid cells, mediated by Scl we tested whether expression of a Scl transgene in Otx1−/− mice can re-establish normal ratios between the myeloid subpopulations. Results showed that, in contrast to the findings within the erythroid compartment, constitutive Scl expression does not fully rescue the alterations within the myelo-monocytic lineage. Taken together our data indicate that the brain morphogenetic gene Otx1 is an important regulator of the blood system, as it plays a relevant role in erythropoiesis, and contributes to control also myelo-monocytic differentiation, likely at the level of the commitment events of bipotential progenitor cells.

Strabismus is a frequently recognized manifestation of Williams syndrome [Greenberg and Lewis, 1988: Ophthalmology 95:1608-1612; Kapp et al., 1995: Am J Ophthalmol 119:355-360]. We recently evaluated the ophthalmologic function of 12 patients with Williams syndrome (WS), with an emphasis on binocularity. Four of 12 patients (33%) had measurable strabismus. Of the 8 remaining patients, examination of binocular function was possible in 6, all of whom demonstrated reduced stereoacuity. We speculate that subnormal binocular vision and the poor visuospatial performance observed in patients with WS may be related to abnormal brain morphogenesis in the region of the occipitoparietal cortex.

The adult arrangement and the development of stem vessels and capillaries was studied in the rat cerebellum. In principle, stem vessels branch and terminate at three levels: (1) the molecular layer, (2) the Purkinje cell-granular layer, and (3) the cerebellar white matter. All stem vessels are interconnected by the capillary network which is most dense in the Purkinje cell—granular layer. As in the neocortex, the stem vessels of the cerebellum are formed successively during development, so that the later they are formed the more superficial are their terminations. The formation of multiple stem vessels in the depths of fissures and sulci during both pre- and postnatal development may correlate to regional variations in, e.g., mitotic frequency or thickness of the external granular layer. The earliest “endo-parenchymal” branches are formed before the first neurons are present. Capillary growth by sprouting during the postnatal period parallels known regional differences in the timing of the neuronal maturation, e.g., increased synaptic density and oxidative metabolism. The findings in this investigation confirm and extend the results of an earlier morphometric study on capillary development in the cerebellar cortex. Although the angiogenetic factors remain unknown, the hypothesis of a link between the vascularization and the functional maturation of the brain is corroborated by the results. Knowledge of the normal vascular development seems necessary for an understanding of brain morphogenesis and for interpretation of primary pathogenetic mechanisms in various intoxications etc.

Morphogenesis of the brain in a cetacean species has been investigated by means of reconstructions from serial sections of successive prenatal stages of the harbour porpoise (Phocoena phocoena). Four specimens ranging from 10 to 46 mm crown-rump length (CRL) were selected and three-dimensional reconstructions of the developing brains were obtained with the plate model method. External and internal characteristics, established as criteria for staging embryonic development of primates and rodents, revealed that a common ontogenetic plan regarding the chronological sequence of morphogenetic events exists in mammalian orders as different as primates and odontocetes. Comparison of the 10-mm and 11.5-mm CRL harbour porpoise brains with those in other mammalian embryos of a similar ontogenetic stage (stages 16 and 17) showed a high degree of correspondence in morphological features. This ontogenetic age group therefore might still be considered as a generalized mammalian one. However, during succeeding morphogenesis of the Phocoena brain, qualitative and quantitative divergences from other mammalian groups became manifest, such as those found in the 24-mm CRL specimen (corresponding to mammalian stages 20, 21). Early foetuses of the harbour porpoise (46 and 65 mm CRL) already exhibited a variety of typical odontocete brain features, such as absence of olfactory bulb, thick cochlear nerve, and strong progression of brainstem structures. Morphogenesis of the harbour porpoise brain is discussed from a comparative perspective, incorporating the literature on the development of mammalian brains. Part of this study has been published in abstract form (Buhl and Oelschlager: Acta Anat. (Basel) 120:15-16 (Abstract), '84).

The present study deals with aspects of the brain development in the roach, Rutilus rutilus, a common mid-European cyprinid fish. The morphogenesis of selected brain areas from hatching to early juveniles was examined on serial paraffin cross-sections. From early juveniles to large adults, brain growth was quantitatively analyzed by computer-aided planimetry. The hatchlings of roach show a cytologically distinct optic tectum, but a poorly differentiated brainstem, reflecting the predominance of the optic sense during the larval planktivorous period. The differentiation and outgrowth of chemosensory brainstem centers is related to the onset and development of benthivorous feeding in juveniles. The optic tectum decreases in size relative to the total brain volume from juveniles through adults. The corpus cerebelli increases in relative size, whereas chemosensory and acousticolateral centers grow isometrically with the brain as a whole.

There is a functional device in embryonic ectodermal cells that we propose causes them to differentiate into either neuroepithelial or epidermal tissue during the process called primary neural induction. We call this apparatus the “cell state splitter”. Its main components are the apical microfilament ring and the coplanar apical mat of microtubules, which exert forces in opposite radial directions. We analyze the mechanical interaction between these cytoskeletal components and show that they are in anunstable mechanical equilibrium. The role of the cell state splitter is thus to create a mechanical instability corresponding to the embryonic state of “competence” in an otherwise mechanically stable cell. When the equilibrium of the cell state splitter is disturbed so as to produce a slight contraction of the apical end, apical contraction continues and the distinctive columnar neuroepithelial cells are produced. A slight expansion from the equilibrium state, on the other hand, results in flattened epidermal cells. The calculated forces are consistent with the know constitutive and force-generating properties and morphology of microfilaments and microtubules, and with free tubulin concentration. There are no free parameters in the analysis. The first cells to assume the neuroepithelial state lie over the notochord. Propagation of the neuroepithelial state (homoiogenetic induction) then proceeds via stretch-induced constriction of the apical microfilament rings, until ahemisphere is covered, at which point the high rate of change of the meridional stress component necessary for further propagation vanishes. The remaining cells are stretched somewhat by this process and become epidermis. A sharp boundary between the tissues is thus formed (explaining “compartmentalization” and the binary nature of differentiation in general). Normal induction apparently involves setup of the cell state splitters in all of the ectoderm cells, perhaps synchronously timed by global embryo tension. The initial transition of cells from the ectodermal to the neuroepithelial state begins at the notoplate, where cell attachments to the notochord may both cause basal actin deposition and significantly reduce the stress induced in the ectoderm by the global tension, biasing the notoplate cell state splitters toward the neuroepithelial state. Introduction of an organizer or other solid substrate (artificial inducer) elsewhere, to which ectodermal cells can adhere, may likewise have both of these effects. Differentiation to either epidermis or neuroepithelium is thus a machanical eventfollowed by the synthesis of specific proteins. This model of differentiation suggests that the genome responds to, rather than directly causes, differentiation.

Migration of newly-generated neuroblasts to their definitive locations to form the laminated architecture of the brain is crucial in brain morphogenesis. In order to understand the molecular mechanism operative in the neuroblast migration, cell surface or pericellular glycoconjugates were isolated from the rat brains at various developmental stages. Hyaluronate (HA), chondroitin sulfate proteoglycans (CS-PGs), heparan sulfate proteoglycans (HS-PGs) and polysialosyl glycoproteins (N-CAMs) have been characterized so far. Our biochemical and immunohistochemical experiments indicate that HA and a CS-PG participate in construction of the hydrated extracellular milieu for the directional migration of neuroblasts. Dissociated fetal brain cells reaggregated and formed a tissue configuration, in aggregating culture for 48 h, similar to that observed in the original brain primordium. Therefore, one can consider that the in vitro process is regulated at least in part by the mechanism operative in brain morphogenesis in vivo. It was suggested that HS-PGs and N-CAMs were involved in the in vitro process of the tissue self-organization as cell membrane components. From these findings and others, we conclude that some developmental changes in the structure and the contents of these cell surface glycoconjugates of the brain are implicated in neuroblast migration in brain morphogenesis.

1. (1) This communication reports the amidophosphoribosyltransferase (PRPP-At; EC 2.4.2.14), hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.7) and adenine phosphoribosyltransferase (APRT; EC 2.4.2.8) activities and the phosphoribosylpyrophosphate (PRPP) content of rat brain at different stages of development. The results are not age-related in the foetal and neonatal animals and the data for whole brain homogenates are similar to the average results for the individual regions of the brain at the same stage of development. 2. (2) The enzyme activities and PRPP content are similar in the different regions of the rat central nervous system. PRPP-At has the lowest activity of the 3 enzymes studied and this decreases gradually from birth until 8 weeks. HPRT is the most active of the three enzymes, its activity increases markedly between birth and the end of the third week of life. The time course of these changes shows only minor differences between the regions of the brain studied. The ratio of HPRT activity to PRPP-At activity increases from age 1 week in all parts of the rat brain. 3. (3) The APRT activities in rat brain are intermediate between those of PRPP-At and HPRT and essentially steady except for a decrease in the cerebellum during the first 3 weeks of life. 4. (4) The PRPP concentrations in rat brain decrease between birth and the end of the 3rd week of life. 5. (5) The systemic tissues examined have PRPP-At, HPRT and APRT activities. The relationship between the activities of the different enzymes appears to be characteristic of the tissue concerned. 6. (6) Correlating the observed time course of the changes in the ratio of hypoxanthine phosphoribosyltransferase activity to amidophosphoribosyltransferase activity in the rat with other workers' data on changes in the rate of DNA accretion in human brain during development indicates that the main increase in this ratio is after the major bursts of neuroblast and neuroglia proliferation. We suggest that the neurological dysfunction in the Lesch-Nyhan syndrome is due to lack of a purine derivative with a physiological or neuropharmacological function, rather than to an effect of the biochemical lesion on brain morphogenesis.

Hatched chicks with chimeric brains containing cells from both the domestic chicken (Gallus gallus domesticus) and the Japanese quail (Coturnix coturnix japonica) have been produced by transplantation of various regions of the neural tube at the 8- to 15- somite stage. The positions of host and donor cells relative to graft boundaries observed throughout embryonic development and after hatching implicated both radial and tangential cell movements in brain morphogenesis. In addition, transplants containing the entire quail mesencephalon and diencephalon resulted in the transfer of certain aspects of species-typical crowing behavior.

The morphological development of the rat cerebral cortex was investigated following hypophysectomy at 4–5 days of age. Although the body and endocrineorgan weights of the hypophysectomized rats were much less than those of their littermate controls at 33–35 days of age, the brain weights of the hypophysectomized rats were equal to those of the controls. Early hypophysectomy had no effect on the depth of the somatosensory or visual cortices, nor on the basal dendritic parameters of pyramidal cells from layers II and III of the somatosensory cortex. Thyroid hormones-either from residual secretion or obtained in maternal milk, or both-probably accounted for the normal morphological development of the rat brain after hypophysectomy. Although growth hormone is known to influence prenatal and postnatal brain development, the presence of growth hormone in the postnatal period is not essential for normal brain growth.

1. Cellular morphogenesis during postembryonic brain development inDanaus plexippus plexippus L. was examined using histological techniques including radioautography. 2. The production of new neurones is continuous throughout larval and pupal stages and shows no fluctuations corresponding to ecdysis. Glial cell production, on the other hand, occurs at the time of molting. 3. New ganglion cells are formed by the division of neuroblasts found in aggregates or isolated among larval ganglion cells. Asymmetrical neuroblast divisions yield one neuroblast and one ganglion-mother cell which then divides at least once to form the new ganglion cells. Such divisions begin earlier inDanaus than in other investigated Lepidoptera. Symmetrical divisions yielding two neuroblasts also occur, but only among aggregated neuroblasts. 4. Radioautographs of brains fixed at progressive intervals after Tritiated Thymidine (H3TdR) injection have permitted description of the basic pattern by which cells of the adult brain cortex are laid out and progressive changes in the relationship of new ganglion cells derived from a single neuroblast. Ganglion-mother cells are deposited between the neuroblast and the neuropile, thus forming a row of cells which move the neuroblast progressively farther from the neuropile. New ganglion cells produced by ganglion-mother cell mitoses, which usually are oriented at 45° angles to the neuropile, expand the cell cluster. Differentiating fibers of these cells are apparent within a few days of their production and seem to enter the neuropile in one bundle. Later with increased neuropile volume and further cell differentiation the cells are no longer clumped and thus are not recognizable as offspring of a single neuroblast. 5. Neuroblasts found scattered among the larval ganglion cells arise from cells near the neuropile. These cells, at first indistinguishable from their neighbors, gradually assume the size and ready stainability of neuroblasts and subsequently divide according to the pattern described above. 6. Scattered neuroblasts degenerate beginning shortly after pupation and have completely disappeared by the end of the fourth day. 7. Except in the developing optic lobe, glial cell numbers increase through the proliferation of already existing glial cells. All glial cells show H3TdR uptake during a 12 hour period surrounding each larval-larval molt and for a somewhat longer period after pupation. However, in the larval stages mitotic figures were seen only among glial I, II, and IV. Glial I cells divide through the entire last larval stage and for two days following pupation. Large irregular mitoses seen among glial III cells at pupation indicate that these cells are probably polyploid. 8. In the newly forming adult optic lobe glial II, III, and IV cells appear to develop from preganglion cells or cells indistinguishable from them. These cells gradually stain more and more darkly, segregate into the normal glial positions, and subsequently divide in accord with other glial cells. 9. At the end of the fifth instar the perineurium (glial I cells), which begins to thicken during the third larval instar, is multilayered and contains many vacuolar cells. Just prior to pupation the neurilemma begins to disintegrate and during the next five days all but the cells closest to the brain disappear. Hemocytes are seen to engulf portions of the disintegrating neurilemma and already degenerating perineurial cells, but do not seem to engulf live cells. The glial I cells remaining adjacent to the brain secrete a new neurilemma. 10. There is no evidence for mass destruction of larval ganglion cells by either autolysis or phagocytosis, and only in the antennal center is there evidence of degeneration of larval cells (NORDLANDER andEDWARDS, in press).

The possibility that mechanisms of retinal determination may be similar between vertebrates and Drosophila has been supported by the observations that Pax6/eyeless genes are necessary and sufficient for retinal development. These studies suggest that the function of other gene families, operating during early eye development, might also be conserved. One candidate is the retinal homeobox (Rx) family of transcription factors. Vertebrate Rx is expressed in the prospective eye and forebrain and is required for eye morphogenesis, retinal precursor appearance, and normal forebrain development, indicating that it is an essential regulator of early eye and brain formation. Here, we test the hypothesis that Drosophila Rx (drx) is required for adult and larval eye development. We have isolated a drx null allele and demonstrate that the mutant compound eye and larval visual system is not detectably abnormal. However, we find that drx is required for development of a central brain structure, the ellipsoid body, suggesting that Rx function in the brain may be conserved. Finally, we characterize a novel anterior head phenotype and demonstrate that drx is required for clypeus development. Thus, our data suggest that drx may be required for the regulation of genes involved in brain morphogenesis and clypeus precursor development. We propose that differences in insect and vertebrate eye development may be explained by changes in gene regulation and/or the tissue of origin for eye precursor cells.

Constitutional heterozygous mutations of ATP1A2 and ATP1A3, encoding for two distinct isoforms of the Na+/K+-ATPase (NKA) alpha-subunit, have been associated with familial hemiplegic migraine (ATP1A2), alternating hemiplegia of childhood (ATP1A2/A3), rapid-onset dystonia-parkinsonism, cerebellar ataxia-areflexia-progressive optic atrophy, and relapsing encephalopathy with cerebellar ataxia (all ATP1A3). A few reports have described single individuals with heterozygous mutations of ATP1A2/A3 associated with severe childhood epilepsies. Early lethal hydrops fetalis, arthrogryposis, microcephaly, and polymicrogyria have been associated with homozygous truncating mutations in ATP1A2. We investigated the genetic causes of developmental and epileptic encephalopathies variably associated with malformations of cortical development in a large cohort and identified 22 patients with de novo or inherited heterozygous ATP1A2/A3 mutations. We characterized clinical, neuroimaging and neuropathological findings, performed in silico and in vitro assays of the mutations’ effects on the NKA-pump function, and studied genotype-phenotype correlations. Twenty-two patients harboured 19 distinct heterozygous mutations of ATP1A2 (six patients, five mutations) and ATP1A3 (16 patients, 14 mutations, including a mosaic individual). Polymicrogyria occurred in 10 (45%) patients, showing a mainly bilateral perisylvian pattern. Most patients manifested early, often neonatal, onset seizures with a multifocal or migrating pattern. A distinctive, ‘profound’ phenotype, featuring polymicrogyria or progressive brain atrophy and epilepsy, resulted in early lethality in seven patients (32%). In silico evaluation predicted all mutations to be detrimental. We tested 14 mutations in transfected COS-1 cells and demonstrated impaired NKA-pump activity, consistent with severe loss of function. Genotype-phenotype analysis suggested a link between the most severe phenotypes and lack of COS-1 cell survival, and also revealed a wide continuum of severity distributed across mutations that variably impair NKA-pump activity. We performed neuropathological analysis of the whole brain in two individuals with polymicrogyria respectively related to a heterozygous ATP1A3 mutation and a homozygous ATP1A2 mutation and found close similarities with findings suggesting a mainly neural pathogenesis, compounded by vascular and leptomeningeal abnormalities. Combining our report with other studies, we estimate that ∼5% of mutations in ATP1A2 and 12% in ATP1A3 can be associated with the severe and novel phenotypes that we describe here. Notably, a few of these mutations were associated with more than one phenotype. These findings assign novel, ‘profound’ and early lethal phenotypes of developmental and epileptic encephalopathies and polymicrogyria to the phenotypic spectrum associated with heterozygous ATP1A2/A3 mutations and indicate that severely impaired NKA pump function can disrupt brain morphogenesis.

A relationship has been deduced between aberrant scalp hair directional patterning and problems in early brain development and function. The unusual scalp hair patterns noted by Smith and Gong1,2 were frontal upsweep and aberrant parietal whorl(s). The purpose of this article is to report a third type of unusual scalp hair patterning, unruly hair over the top of the scalp, and its association with problems in brain development. This feature was initially observed by Freund in 1910,3 who noted its association with neuropathic dysfunction in infants. METHODS The definition of unruly hair is that which naturally and persistently tends to stand up over the top of the head from the area of the posterior parietal whorl toward the frontal hairline.

Neural development proceeds in an ordered fashion in which a variety of genetic and epigenetic factors exert an influence at well defined times. Using a cloned chicken genomic fragment for the nerve growth factor (NGF) receptor, we have detected strong expression in chicken brain at early stages of embryonic development. Expression of the receptor gene was greatly diminished at birth. This pattern of NGF receptor mRNA level was observed in all cranial regions and was further correlated with the appearance and disappearance of cell surface receptors. The transient developmental expression of NGF receptors in chick brain and the requirement for receptors to mediate NGF's effects suggests that NGF may possess a broader range of actions during development of the nervous system.

The embryonic development of total specific activities as well as of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and of butyrylcholinesterase (BChE, EC 3.1.1.8) have been studied in the chick brain. A comparison of the development in different brain parts shows that cholinesterases first develop in diencephalon, then in tectum and telencephalon; cholinesterase development in retina is delayed by about 2-3 days; and the development in rhombencephalon [not studied until embryonic day 6 (E6)] and cerebellum is last. Both enzymes show complex and independent developmental patterns. During the early period (E3-E7) first BChE expresses high specific activities that decline rapidly, but in contrast AChE increases more or less constantly with a short temporal delay. Thereafter the developmental courses approach a late phase (E14-E20), during which AChE reaches very high specific activities and BChE follows at much lower but about parallel levels. By extraction of tissues from brain and retina in high salt plus 1% Triton X-100, we find that both cholinesterases are present in two major molecular forms, AChE sedimenting at 5.9S and 11.6S (corresponding to G2 and G4 globular forms) and BChE at 2.9S and 10.3S (G1 and G4, globular). During development there is a continuous increase of G4 over G2 AChE, the G4 form reaching 80% in brain but only 30% in retina. The proportion of G1 BChE in brain remains almost constant at 55%, but in retina there is a drastic shift from 65% G1 before E5 to 70% G4 form at E7.(ABSTRACT TRUNCATED AT 250 WORDS)

Cephalometric analyses were performed on five caucasian patients with AHPS to further investigate the craniofacial abnormalities observed in this disorder. The study included four females and one male ranging in age from 11 to 33 years. Analysis of twenty-two angular and linear relationships measured on lateral cranial radiographs demonstrated the following significant abnormalities: microcephaly, short anterior and posterior cranial base, mid-face retrusion and decreased vertical face height. The sella turcica, reported as being displaced in previous studies, was normal in configuration and position in all our cases. In addition, despite the fact that four of our five patients appeared clinically prognathic, all had normal mandibular length, width and shape. It was concluded that this relative prognathism was caused by upward and forward rotation of the mandible as a consequence of the shortening and retrusion of the mid-face. We postulate that these abnormalities, responsible for the characteristic craniofacial configuration in patients with the AHPS, result from the shortening of the anterior cranial base which is secondary to abnormal brain morphogenesis and growth.

Studies of normal calvarial morphogenesis in human fetuses were found to support the concepts of Tondury and of Moss. The early growth and form of the brain causes growth stretch on the dura with the development of reflections which extend from the sites of dural attachment at the cranial base and conform to the major recesses in the early brain. The dura is the guiding tissue in the morphogenesis of the overlying calvarium and its major sutures, which develop from 10 to 16 weeks of fetal life. In the quiet zones between the dural reflections ossification occurs, whereas over the reflections of dura no ossification occurs, these being the sites of sutures. Supportive evidence for the role of the dural reflections in determining the sites of sutures was obtained from the evaluation of instances of major brain malformations which must have antedated calvarial morphogenesis. These included holoprosencephaly (13 cases), craniopagus (7 cases) and dicephaly (2 cases). In each case the gross brain malformation gave rise to unusual sites of dural reflection, which were found to coincide with the unusual sites of sutures in the calvarium. Furthermore, a lack of a dural reflection was accompanied by a lack of development of a suture at that site. The relevance of these findings to the evaluation of problems of brain morphogenesis and the diagnosis and management of craniostenosis will be discussed.

Background Cerebellar corticogenesis begins with the assembly of Purkinje cells into the Purkinje plate (PP) by embryonic day 14.5 (E14.5) in mice. Although the dependence of PP formation on the secreted protein Reelin is well known and a prevailing model suggests that Purkinje cells migrate along the 'radial glial' fibers connecting the ventricular and pial surfaces, it is not clear how Purkinje cells behave in response to Reelin to initiate the PP. Furthermore, it is not known what nascent Purkinje cells look like in vivo. When and how Purkinje cells start axonogenesis must also be elucidated. Results We show that Purkinje cells generated on E10.5 in the posterior periventricular region of the lateral cerebellum migrate tangentially, after only transiently migrating radially, towards the anterior, exhibiting an elongated morphology consistent with axonogenesis at E12.5. After their somata reach the outer/dorsal region by E13.5, they change 'posture' by E14.5 through remodeling of non-axon (dendrite-like) processes and a switchback-like mode of somal movement towards a superficial Reelin-rich zone, while their axon-like fibers remain relatively deep, which demarcates the somata-packed portion as a plate. In reeler cerebella, the early born posterior lateral Purkinje cells are initially normal during migration with anteriorly extended axon-like fibers until E13.5, but then fail to form the PP due to lack of the posture-change step. Conclusions Previously unknown behaviors are revealed for a subset of Purkinje cells born early in the posteior lateral cerebellum: tangential migration; early axonogenesis; and Reelin-dependent reorientation initiating PP formation. This study provides a solid basis for further elucidation of Reelin's function and the mechanisms underlying the cerebellar corticogenesis, and will contribute to the understanding of how polarization of individual cells drives overall brain morphogenesis.