Mutually Repulsive EphA7-EfnA5 Organize Region-to-Region Corticopontine Projection by Inhibiting Collateral Extension.

Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered that EphA7 and EfnA5 are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets.SIGNIFICANCE STATEMENT Our findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization-both within each brain region and between connected brain regions-may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.

Reference Ranges for Transvaginal Examined Fossa Posterior Structures in Fetuses from 45 to 84 mm Crown-Rump Length.

OBJECTIVE: The study aimed to describe reference values for structures of the posterior fossa in fetuses with a crown-rump length (CRL) between 45 and 84 mm. MATERIALS AND METHODS: This was a prospective, cross-sectional study including 216 normal appearing fetuses. In transvaginal acquired 3-dimensional volume blocks, the longest diameter of the vermis (VE), posterior membranous area (PMA), medulla-oblongata-pons angle (MOPA), diameters of the medulla oblongata (MO) and pons (PO), and the area of Blake's pouch (BP) were measured. Polynomial or linear regression analysis were performed to calculate the mean, 5th and 95th centile according to CRL. In 20 fetuses, intra- and interobserver repeatability were calculated. RESULTS: There is a curvilinear correlation between CRL and PO (PO [mean] = 1.3893 + 0.004356 × CRL + 0.000002610 × CRL3; SD = 1.6818 - 0.03765 × CRL + 0.000003831 × CRL3; R2 = 0.489); CRL and MO (MO [mean] = 1.5959-0.001905 × CRL + 0.000003362*CRL3; SD = -0.1417 + 0.005404 × CRL + 0.0000004988 × CRL3; R2 = 0.525); CRL and VE (VE [mean] = -0.3640 + 0.04302 × CRL+ 0.000001486 × CRL3; SD = 0.5854 - 0.004812 × CRL + 0.0000005896 × CRL3; R2 = 0.643); CRL and PMA (PMA [mean] = 0.6901 + 0.04307 × CRL - 0.0000008459 × CRL3; SD = -0.4232 + 0.02026 × CRL - 0.000001320 × CRL3; R2 = 0.272); CRL and BP (mm2; BP [mean] -12.2067 + 0.3334 × CRL - 0.00001262 × CRL3; SD = -1.6431 + 0.06380 × CRL+ 0.0000003257 × CRL3; R2 = 0.289). The relation between CRL and MOPA (°) is best described by a linear regression (MOPA [mean] = 79.6332 + 0.6122 × CRL; SD = 4.8453 + 0.07333 × CRL; R2 = 0.318). CONCLUSION: We provide reference values for anatomical structures of the posterior fossa of fetuses between 45 and 84 mm CRL. The established reference values might ease the diagnosis of fetal malformations in early pregnancy.

Sonoembryology of the fetal posterior fossa at 11 + 3 to 13 + 6 gestational weeks on three-dimensional transvaginal ultrasound.

OBJECTIVE: To describe the sonographic appearance and temporal changes of the structures of the posterior cranial fossa in fetuses at a crown-rump length (CRL) between 45 and 84 mm in transvaginal acquired three-dimensional volume blocks. METHODS: This was a prospective, cross-sectional, observational study including 80 fetuses, whose mothers attended Kepler University Hospital Linz or the Ambulatorium für Fetalmedizin Feldkirch for first-trimester sonography. Three-dimensional volume blocks were acquired in a standardized way and after processing the sonographic characteristics of the brainstem, cerebellar vermis, choroid plexus, anterior membranous area (AMA) and Blake's metapore were described. Measurements of the length of the cerebellar vermis, the length of the AMA and the medulla-oblongata-pons angle (MOPA) were performed. In 20 fetuses the intra- and interobserver repeatability was calculated. RESULTS: The sonomorphologic characteristics of posterior fossa structures as cerebellar vermis, AMA, Blake's metapore, choroid plexus, pons and medulla oblongata were described. There is a significant correlation between CRL and vermis length, CRL and MOPA and CRL and AMA. CONCLUSIONS: Transvaginal three-dimensional sonography allows a detailed depiction of the structures of the posterior fossa and their temporal course in early pregnancy. © 2016 John Wiley & Sons, Ltd.

Assessment of fetal midbrain and hindbrain in mid-sagittal cranial plane by three-dimensional multiplanar sonography. Part 1: comparison of new and established nomograms.

OBJECTIVE: To construct nomograms for fetal midbrain (MB) and hindbrain (HB) dimensions, assessed in the mid-sagittal cranial plane by three-dimensional multiplanar sonographic reconstruction (3D-MPR). METHODS: This was a prospective cross-sectional study of 334 healthy fetuses in low-risk singleton pregnancies between 16 and 35 gestational weeks. All sonographic volumes were obtained by sagittal acquisition. The following MB and HB parameters were evaluated in the mid-sagittal cranial plane using 3D-MPR: MB parameters tectal length (TL) and anteroposterior midbrain diameter (APMD), and HB parameters anteroposterior pons diameter (APPD), superoinferior vermian diameter (SIVD), anteroposterior vermian diameter (APVD) and anteroposterior diameter of the fourth ventricle (APDFV). The measurements were presented as growth charts according to gestational age. RESULTS: MB and HB biometry were best assessed between 19 and 29 weeks. During this period, adequate visualization was achieved for successful measurement of TL in 90.9% of cases, APMD in 86.6%, APPD in 73.7%, SIVD in 74.2%, APVD in 71% and APDFV in 71%. There was a linear growth pattern, with Pearson correlation coefficients of 0.79 for TL, 0.88 for APMD, 0.91 for APPD, 0.95 for SIVD, 0.88 for APVD and 0.88 for APDFV (P < 0.0001 for each). The mean intra- and interobserver variations for the MB measurements and vermian diameters ranged between 4.3% and 9%. APPD and APDFV showed highest mean variations: 9.0% and 19.4% (intraobserver) and 11.6% and 17.7% (interobserver), respectively. CONCLUSION: We present new nomograms for assessment of the fetal MB and HB using 3D-MPR in the mid-sagittal cranial plane. To our knowledge, these are the first proposed nomograms for fetal MB dimensions.

Synaptogenesis in the foetal and neonatal cerebellar system. 2. Pontine nuclei and cerebellar cortex.

Precise temporal and spatial sequences of synaptogenesis were demonstrated in 172 human foetuses and neonates post-mortem in transverse paraffin sections of pons and cerebellar vermis and hemispheres, using synaptophysin immunoreactivity of this protein of synaptic vesicular walls. The pontine nuclei exhibit a transitory patchy pattern not predicted from the uniform histology and reminiscent of the corpus striatum; synaptic vesicle reactivity appears at 20 weeks and is uniform by 34 weeks. In the cerebellar cortex, the vermis matures sooner than the cerebellar hemispheres and the paravermal portions earlier than the lateral folia. The earliest synapses occur around the somata of Purkinje neurons and later in the internal granular layer, but synaptic glomeruli are not well formed until after 26 weeks. The normal patterns here shown, together with earlier data of the Guillain-Mollaret triangle, provide controls for the interpretation of synaptic delay or precociousness and other pathological patterns in malformations, genetic/metabolic conditions and prenatal acquired insults affecting the human foetus.

Role of Neph2 in pontine nuclei formation in the developing hindbrain.

Nuclei are anatomical units of the central nervous system (CNS). Their formation sets the structural basis for the functional organization of the brain, a process known as nucleogenesis. In the present study, we investigated the role of the transmembrane immunoglobulin superfamily molecule Neph2 in the nucleogenesis of the pontine nucleus (PN). Neph2 expression is turned on in migrating PN neurons only after they enter the presumptive nuclear region. Neph2 knockdown disrupted the nuclear organization of PN presumably by changing the migratory behavior of PN neurons inside the nuclear region. Moreover, overexpression of the cytoplasmic region of Neph2, which can sequester intracellular signaling of endogenous Neph2, resulted in similar phenotypes. Overall, these results suggest Neph2 is involved in the nucleogenesis of the PN through the control of neuronal migration inside the nucleus.

A unique expression pattern of Tbx10 in the hindbrain as revealed by Tbx10(LacZ) allele.

To study the expression/function of Tbx10, a T-box gene, Tbx10(LacZ/+) mice were established by replacing the T-box coding region with a LacZ gene. X-gal staining showed that LacZ(+) cells were localized to two-cell populations in rhombomere 4 and rhombomere 6. No significant differences in the locations of LacZ(+) cells were found between Tbx10(LacZ/+) and Tbx10(LacZ/LacZ) mice, and the Tbx10(LacZ/LacZ) mice were viable and fertile. We found that the LacZ(+) cells are present in both embryonic and adult mice. Histological studies suggest that the rhombomere 4-derived LacZ(+) cells are a subpopulation of the ventral interneurons in the pons.

Ultrasonography of the fetal brainstem: a biometric and anatomical, multioperator, cross-sectional study of 913 fetuses of 21-36 weeks of gestation.

OBJECTIVES: To establish the ultrasonographic fetal growth charts of the pons and the vermis/pons ratio on a multioperator basis in low-risk pregnancies and provide a detailed description of the anatomical and ultrasonographic criteria of normal brainstem growth. METHODS: A prospective, multicenter, multioperator, ultrasonographic study was conducted on 913 fetuses aged 21-36 weeks. The anteroposterior diameter of the pons and the greatest vermal height were measured to establish a growth chart, using a mid-sagittal plane with a posterior transfontanellar approach. The LMS semiparametric statistical method was used to construct the growth charts. Three morphological structures were also examined: the pons arch and its echostructure, the bulbo-protuberential sulcus and the primary vermal fissure. RESULTS: The anteroposterior diameter of the pons and the greatest vermal height were measured in 96.7% of cases. The anteroposterior diameter of the pons and vermis increased linearly with gestational age. The vermis/pons ratio was stable during pregnancy. CONCLUSION: We have drawn the growth charts for the pons and vermis during pregnancy and described the normal ultrasound morphology of the brainstem. Knowledge of these morphological and biometric data could facilitate early screening for pontocerebellar hypoplasia.

Change in chemoattractant responsiveness of developing axons at an intermediate target.

Developing axons reach their final targets as a result of a series of axonal projections to successive intermediate targets. Long-range chemoattraction by intermediate targets plays a key role in this process. Growing axons, however, do not stall at the intermediate targets, where the chemoattractant concentration is expected to be maximal. Commissural axons in the metencephalon, initially attracted by a chemoattractant released from the floor plate, were shown to lose responsiveness to the chemoattractant when they crossed the floor plate in vitro. Such changes in axon responsiveness to chemoattractants may enable developing axons to continue to navigate toward their final destinations.

Diffuse Intrinsic Pontine Gliomas Exhibit Cell Biological and Molecular Signatures of Fetal Hindbrain-Derived Neural Progenitor Cells.

Diffuse intrinsic pontine glioma (DIPG) is the main cause of brain tumor-related death among children. Until now, there is still a lack of effective therapy with prolonged overall survival for this disease. A typical strategy for preclinical cancer research is to find out the molecular differences between tumor tissue and para-tumor normal tissue, in order to identify potential therapeutic targets. Unfortunately, it is impossible to obtain normal tissue for DIPG because of the vital functions of the pons. Here we report the human fetal hindbrain-derived neural progenitor cells (pontine progenitor cells, PPCs) as normal control cells for DIPG. The PPCs not only harbored similar cell biological and molecular signatures as DIPG glioma stem cells, but also had the potential to be immortalized by the DIPG-specific mutation H3K27M in vitro. These findings provide researchers with a candidate normal control and a potential medicine carrier for preclinical research on DIPG.

Induction of a mesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en.

The homeobox gene en, homologous to the gene en-grailed of Drosophila, is expressed in the metencephalic-mesencephalic segment of the vertebrate neural tube. Using quail-chick chimeras, an antibody against en proteins, and cytoarchitectonic techniques, we demonstrate that metencephalon transplanted to prosencephalon, at E2, maintains a high level of en proteins and its presumptive cerebellar fate. The ectopic metencephalon induces in the contiguous host prosencephalon the expression of en and, subsequently, a mesencephalic phenotype. These related genetic and phenotypic expressions indicate that the transcriptional regulatory en gene is involved in cerebellar and mesencephalic cyto-differentiation. The expression of en can also be induced in chick prosencephalon by a mammalian metencephalic graft, indicating that the factors regulating the transcription of en are phylogenetically well conserved.

The slit receptor Rig-1/Robo3 controls midline crossing by hindbrain precerebellar neurons and axons.

During development, precerebellar neurons migrate dorsoventrally from the rhombic lip to the floor plate. Some of these neurons cross the midline while others stop. We have identified a role for the slit receptor Rig-1/Robo3 in directing this process. During their tangential migration, neurons of all major hindbrain precerebellar nuclei express high levels of Rig-1 mRNA. Rig-1 expression is rapidly downregulated as their leading process crosses the floor plate. Interestingly, most precerebellar nuclei do not develop normally in Rig-1-deficient mice, as they fail to cross the midline. In addition, inferior olivary neurons, which normally send axons into the contralateral cerebellum, project ipsilaterally in Rig-1 mutant mice. Similarly, neurons of the lateral reticular nucleus and basilar pons are unable to migrate across the floor plate and instead remain ipsilateral. These results demonstrate that Rig-1 controls the ability of both precerebellar neuron cell bodies and their axons to cross the midline.

Axon guidance in the vertebrate central nervous system.

The development of connections in the central nervous system depends on the ability of the tips of growing axons to find their appropriate, often distant, target field. Factors that regulate axon outgrowth may be distinct from those that influence direction finding. Tissue culture methods have helped to distinguish between possible in vivo mechanisms and, in some cases, have identified candidate molecules.

Development of neurons expressing estrogen receptor alpha transiently in facial nucleus of prenatal and postnatal rat brains.

The transient expression of estrogen receptor alpha (ERalpha) in the facial nucleus of rats during development was already reported. However, how and whether the receptor functions physiologically in the nucleus of developing rats are as yet unclear. In this study, we applied a retrograde tracer into one of the possible target muscles of the motoneurons in the nucleus, that is, the transverse auricular muscle (Mta), and examined whether ERalpha-immunopositive neurons take up the tracer. Because it is probable that neurogenesis, apoptosis, and maturation may be associated with the transient expression of ERalpha, we attempted to analyze the neurons expressing the receptor in the nucleus. We found that ERalpha-immunopositive neurons in the medial facial subnucleus innervate mostly the Mta. Quantitative analyses showed that the number of motoneurons projecting to the Mta remained the same throughout the ages examined, whereas that of ERalpha-immunopositive neurons decreased between postnatal days 6 and 11. Apoptosis and neurogenesis in the nucleus were not affected by the expression of ERalpha during development. ERalpha expression coincided with the maturation of neurons in the nucleus. Thus, it is possible that ERalpha expression in the facial nucleus during development plays important roles in the development of motoneurons and/or external pinna muscles.

Development of the pons in human fetuses.

Morphometric and histological studies of the pons were performed by light microscopy in 28 cases of externally normal human fetuses ranging from 90 to 246 mm in crown-rump length (CRL) and from 13 to 28 weeks of gestation. The brainstems of fetuses were embedded in celloidin or paraffin, and transverse sections were prepared. The pons was divided into two regions at the most ventral margin of the medial lemniscus at the level of the motor trigeminal nucleus. The relationships between the total dorsoventral length, ventral length, and dorsal length of the pons versus CRL and gestational ages were calculated, and empiric formulas were fitted. It was found that the ventral portion increased in size more rapidly than the dorsal portion. The proportion of the ventral portion in the total dorsoventral length was constitutively higher than that of the dorsal portion in the present range of CRL. In the pontine nuclei, from 235 mm in the CRL, some large cells with rich cytoplasm, pale nuclei, and a distinct nucleolus appeared on the dorsal side of the pyramidal tract. According to Weigert stained preparations, the first myelinated fibers in each motor root of the trigeminal, abducent, and facial nerves were recognized at 130-140 mm in CRL and the medial lemniscus at 230-235 mm.

Tumorigenesis in transgenic mice in which the SV40 T antigen is driven by the brain-specific FGF1 promoter.

Gene expression can be manipulated by the introduction of a hybrid gene formed by linking a highly tissue-specific regulatory element to a gene whose expression might be expected to alter cellular function. Previously, we have shown that the human FGF1 gene contains four distinct tissue-specific promoters. In an effort to perturb the programming of proliferation and differentiation in a subset of neural cells, we have produced transgenic mice bearing the brain-specific promoter of the human FGF1 gene joined to the SV40 immediate early gene, which encodes the large T antigen. The resulting mice, and offspring from four individual lines, developed brain tumors that originated in the pontine gray, just rostral to the fourth ventricle. Tumors were moderately vascularized, as demonstrated by staining with both hematoxylin and eosin and antibodies to three different endothelial cell markers, but vessels were histologically normal. Scattered tumor foci were present as early as postnatal day 26; and affected animals died between 5 - 8 months of age. In mature animals, tumors lacked terminal differentiation markers for astrocytes (glial fibrillary acidic protein) or neurons (synaptophysin and neuron-specific enolase). However, they expressed high levels of proliferating cell nuclear antigen and vimentin, markers for proliferating cells. This immunophenotype is consistent with the tumor being at an early stage of differentiation. Therefore, these mice may provide a valuable tool for the study of tumorigenesis, replenishment and differentiation of neural stem cells.

Generation of a novel functional neuronal circuit in Hoxa1 mutant mice.

Early organization of the vertebrate brainstem is characterized by cellular segmentation into compartments, the rhombomeres, which follow a metameric pattern of neuronal development. Expression of the homeobox genes of the Hox family precedes rhombomere formation, and analysis of mouse Hox mutations revealed that they play an important role in the establishment of rhombomere-specific neuronal patterns. However, segmentation is a transient feature, and a dramatic reconfiguration of neurons and synapses takes place during fetal and postnatal stages. Thus, it is not clear whether the early rhombomeric pattern of Hox expression has any influence on the establishment of the neuronal circuitry of the mature brainstem. The Hoxa1 gene is the earliest Hox gene expressed in the developing hindbrain. Moreover, it is rapidly downregulated. Previous analysis of mouse Hoxa1(-/-) mutants has focused on early alterations of hindbrain segmentation and patterning. Here, we show that ectopic neuronal groups in the hindbrain of Hoxa1(-/-) mice establish a supernumerary neuronal circuit that escapes apoptosis and becomes functional postnatally. This system develops from mutant rhombomere 3 (r3)-r4 levels, includes an ectopic group of progenitors with r2 identity, and integrates the rhythm-generating network controlling respiration at birth. This is the first demonstration that changes in Hox expression patterns allow the selection of novel neuronal circuits regulating vital adaptive behaviors. The implications for the evolution of brainstem neural networks are discussed.

Glial cell line-derived neurotrophic factor (GDNF) is required for differentiation of pontine noradrenergic neurons and patterning of central respiratory output.

Genetic mutations affecting signaling by glial cell line-derived neurotrophic factor (GDNF) perturb development of breathing in mice and are associated with congenital central hypoventilation syndrome in humans. However, the role of GDNF in development of brainstem neurons that control breathing is largely unknown. The present study demonstrates that genetic loss of GDNF decreases the number of tyrosine hydroxylase (TH) neurons in the pontine A5 noradrenergic cell group, a major source of inhibitory input to the medullary respiratory pattern generator. This phenotype is associated with a significant increase in the frequency of central respiratory output recorded from the fetal medulla-spinal cord in vitro. In dissociate cultures of the A5 region from rat embryos, GDNF increases TH cell number and neurite growth without affecting total neuronal survival or proliferation of TH neurons. These effects of GDNF are inhibited by function blocking antibodies against endogenous brain-derived neurotrophic factor (BDNF), indicating that GDNF requires BDNF as a cofactor to stimulate differentiation of A5 neurons. Our findings demonstrate that GDNF is required for development of pontine noradrenergic neurons in vivo and indicate that defects in the A5 cell group may contribute to the effects of genetic disruption of GDNF signaling on respiratory control.

Involvement of Wnt-1 in the formation of the mes/metencephalic boundary.

Wnt-1, a putative signaling molecule, is required before the 7 somite stage (E8.5) for the development of midbrain structures in the mouse. We show here that Wnt-1 is also needed for the formation of a boundary between the mesencephalic and metencephalic domains of the neural tube. In embryos homozygous for the Wnt-1sw allele, mesencephalic and metencephalic markers fail to segregate and the establishment of a straight limit of Otx-2 and Wnt-1 expression at the mid-hindbrain junction is impaired. In addition, as observed previously in heterotopic mes/metencephalic transplantation experiments in avian embryos, Wnt-1 expression is induced at the border of ectopic mes- and metencephalic islands observed in Wnt-1sw/sw mutants, suggesting that, in situ, interactions between mes- and metencephalic cells reinforce Wnt-1 expression at the boundary.

Cooperation between Otx1 and Otx2 genes in developmental patterning of rostral brain.

Otx1 and Otx2 genes are mouse cognates of a Drosophila head gap gene orthodenticle. The homozygous mutants have previously indicated that Otx2 is essential to development of structures anterior to rhombomere 3, probably reflecting its expression around the early primitive streak stage. Otx2 mutation also exhibits craniofacial defects by haplo-insufficiency. Affected structures correspond to the most anterior and most posterior parts of the Otx2 expression where Otx1 is not, or is only weakly, expressed at the time of brain regionalization. No apparent defects are found in early brain development by the Otx1 mutation, suggesting that the Otx1 and Otx2 functions overlap in the regions where both are expressed. To demonstrate this, the Otx1/Otx2 double heterozygous phenotype was examined in this study. Analyses with molecular markers at 9.5 days post coitus suggested the failure in development of mesencephalon and caudal diencephalon with the expansion of anterior metencephalon. Genes expressed in isthmus exhibited a characteristic lateral stripe normally, although rostrally shifted, except that Fgf8 expression was expanded dorsally. The defects were apparent at the 6-somite stage, but not at the 3-somite stage. Broad Fgf8 expression at the 3-somite stage took place normally, but it did not concentrate into a spot corresponding to future isthmus. The double heterozygous phenotype implicates a previously unsuspected mechanism for development of the mes/metencephalic territory; at the 3- to 6-somite stage Otx1 cooperates with Otx2 to establish the mes/diencephalic domain, allowing for the correct development of isthmus/ rhombomere 1.