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.

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.

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.

Four-dimensional analysis of nucleogenesis of the pontine nucleus in the hindbrain.

Nuclei in the central nervous system are 3D aggregates of neurons that have common physiological properties, functionalities, and connectivities. To form specific nuclei, neurons migrate from their birthplace towards the presumptive nuclear region where they change their dynamics to aggregate and rearrange into a distinct 3D structure, a process that we term nucleogenesis. Nuclei, together with the laminar structure, form the basic cytoarchitectonic unit for information processing. However, in contrast to much-studied laminar structures, the neuronal dynamics that contribute to the aggregation process to form nuclei are poorly understood. Here, we analyze nucleogenesis by observing the mouse precerebellar pontine nucleus (PN), and provide the first 4D view of nucleogenesis by tracking neuronal behaviors along the three spatial axes over time. Early- and late-born PN neurons were labeled by in utero electroporation and their behaviors on cultured brain slices were recorded by time-lapse imaging. We find that when PN neurons migrate medially into the nuclear region, many of them switch to migrate radially and laterally, to populate the dorsal and lateral PN regions, respectively. The tendency to switch to radial migration is much less in later-born neurons, whereas that to switch to lateral migration is comparable between the two groups. In contrast to the radial and mediolateral axes, very few PN neurons switch to migrate rostrocaudally. These results could thus provide a framework for understanding the mechanisms that regulate this complex yet important process.

Ezh2 orchestrates topographic migration and connectivity of mouse precerebellar neurons.

We investigated the role of histone methyltransferase Ezh2 in tangential migration of mouse precerebellar pontine nuclei, the main relay between neocortex and cerebellum. By counteracting the sonic hedgehog pathway, Ezh2 represses Netrin1 in dorsal hindbrain, which allows normal pontine neuron migration. In Ezh2 mutants, ectopic Netrin1 derepression results in abnormal migration and supernumerary nuclei integrating in brain circuitry. Moreover, intrinsic topographic organization of pontine nuclei according to rostrocaudal progenitor origin is maintained throughout migration and correlates with patterned cortical input. Ezh2 maintains spatially restricted Hox expression, which, in turn, regulates differential expression of the repulsive receptor Unc5b in migrating neurons; together, they generate subsets with distinct responsiveness to environmental Netrin1. Thus, Ezh2-dependent epigenetic regulation of intrinsic and extrinsic transcriptional programs controls topographic neuronal guidance and connectivity in the cortico-ponto-cerebellar pathway.

The interfascicular trigeminal nucleus: a precerebellar nucleus in the mouse defined by retrograde neuronal tracing and genetic fate mapping.

We have found a previously unreported precerebellar nucleus located among the emerging fibers of the motor root of the trigeminal nerve in the mouse, which we have called the interfascicular trigeminal nucleus (IF5). This nucleus had previously been named the tensor tympani part of the motor trigeminal nucleus (5TT) in rodent brain atlases, because it was thought to be a subset of small motor neurons of the motor trigeminal nucleus innervating the tensor tympani muscle. However, following injection of retrograde tracer in the cerebellum, the labeled neurons in IF5 were found to be choline acetyltransferase (ChAT) negative, indicating that they are not motor neurons. The cells of IF5 are strongly labeled in mice from Wnt1Cre and Atoh1 CreER lineage fate mapping, in common with the major precerebellar nuclei that arise from the rhombic lip and that issue mossy fibers. Analysis of sections from mouse Hoxa3, Hoxb1, and Egr2 Cre labeled lineages shows that the neurons of IF5 arise from rhombomeres caudal to rhombomere 4, most likely from rhombomeres 6-8. We conclude that IF5 is a significant precerebellar nucleus in the mouse that shares developmental gene expression characteristics with mossy fiber precerebellar nuclei that arise from the caudal rhombic lip.

The fetal vermis, pons and brainstem: normal longitudinal development as shown by dedicated neurosonography.

OBJECTIVE: To describe the normal appearance and the growth of the fetal vermis, pons and midline brainstem by ultrasound from 18 weeks of gestation to term in order to produce developmental nomograms. METHODS: Serial ultrasound examinations of the fetal brain were performed in 21 fetuses between 18 and 39 weeks of gestation every two weeks. A total of 173 examinations were done, 8.2 ± 5.2 examinations per fetus. A mid-sagittal plain of the brain was obtained either by transvaginal or transabdominal sonography. Antero-posterior, cranio-caudal diameters, and surface area of the pons and the vermis were measured. The surface area of the brain stem was also measured. Nomograms were produced according to Royston and Wright. RESULTS: The pons, vermis and brain stem grow in a linear fashion throughout pregnancy. The growth pattern correlates well with gestational age, biparietal diameter, head circumference and the cerebellar transverse diameter. CONCLUSIONS: We have provided nomograms for assessment of the fetal brainstem. The present information supplies tools for the accurate identification of fetal mid-hindbrain anomalies providing a solid basis for a multidisciplinary approach, management and counseling of these conditions.

Morphological and morphometric study on sphenoid and basioccipital ossification in normal human fetuses.

Congenital anomalies of the brain frequently correspond to cranial base anomalies, and a detailed description of morphology and individual variations in the developing cranial base is of clinical importance for diagnosing anomalies. Development of the human cranial base has been studied using dissection, computed tomography, and magnetic resonance imaging, each of which has advantages and disadvantages. We here examined development of the normal human fetal cranial base using bone staining, which allows for direct observation of the ossification centers and precise three-dimensional measurements. We observed alizarin red S-stained sphenoids and basiocciputs of 22 normal formalin-fixed human fetuses with crown-rump lengths (CRL) of 115-175 mm. We defined landmarks and measured sphenoids and basiocciputs using a fine caliper. Growth patterns of these ossifying bones were obtained, and we found similarities and differences among the growth patterns. We also observed individual variations in the ossification patterns, in particular, single- or double-ossification center patterns for the basisphenoid. The orbitosphenoid and basisphenoid widths and ratios of the widths to the total cranial base width were significantly different between the two pattern groups, whereas the other measurements and their ratios to the total cranial base did not differ between the groups. We measured the cerebrum and pons in different sets of 22 human fetuses with CRLs of 105-186 mm and found close relationships with the development of corresponding parts of the cranial base. The results contribute to the quantitative and qualitative information about the growth patterns and variations during human fetal cranial base development.

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.

Formation and maturation of the calyx of Held.

Sound localization requires precise and specialized neural circuitry. A prominent and well-studied specialization is found in the mammalian auditory brainstem. Globular bushy cells of the ventral cochlear nucleus (VCN) project contralaterally to neurons of the medial nucleus of the trapezoid body (MNTB), where their large axons terminate on cell bodies of MNTB principal neurons, forming the calyces of Held. The VCN-MNTB pathway is necessary for the accurate computation of interaural intensity and time differences; MNTB neurons provide inhibitory input to the lateral superior olive, which compares levels of excitation from the ipsilateral ear to levels of tonotopically matched inhibition from the contralateral ear, and to the medial superior olive, where precise inhibition from MNTB neurons tunes the delays of binaural excitation. Here we review the morphological and physiological aspects of the development of the VCN-MNTB pathway and its calyceal termination, along with potential mechanisms that give rise to its precision. During embryonic development, VCN axons grow towards the midline, cross the midline into the region of the presumptive MNTB and then form collateral branches that will terminate in calyces of Held. In rodents, immature calyces of Held appear in MNTB during the first few days of postnatal life. These calyces mature morphologically and physiologically over the next three postnatal weeks, enabling fast, high fidelity transmission in the VCN-MNTB pathway.

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.

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.

Real time analysis of pontine neurons during initial stages of nucleogenesis.

During development, neurons migrate from their origin to their final destinations where they form neuronal architectures such as layers and nuclei. While the mechanisms for the formation of laminated structures have been studied extensively, little is known about nucleogenesis. Previously, we analyzed nucleogenesis in neurons from four types of mossy-fiber projecting precerebellar nuclei neurons by gene transfer, and obtained evidence suggesting that the change from tangential to radial migration occurs at the region that will develop into the nucleus (Kawauchi, D., Taniguchi, H., Watanabe, H., Saito, T., Murakami, F., 2006. Direct visualization of nucleogenesis by precerebellar neurons: involvement of ventricle-directed, radial fibre-associated migration. Development 133, 1113-1123). Here we analyzed the dynamics of these neurons using mice embryos. We electroporated genes of fluorescent proteins to the lower rhombic lip at embryonic day (E) 12.5 and carried out time-lapse analyses at E14.5, when pontine nuclei begin to be formed. We found that many labeled neurons showed transition from tangential to radial migration in the region that will develop into the nucleus. This transition occurred in two ways. One was initiated by a leading process extending radially while the second was caused by a newly developed radial process from the cell soma. Curiously, we observed that many neurons stopped tangentially migration, paused, and then began radial migration. These findings indicate that a signal to stop and cause the change in tangential to radial migration is critical for nucleogenesis by pontine neurons.

SDF1/CXCR4 signalling regulates two distinct processes of precerebellar neuronal migration and its depletion leads to abnormal pontine nuclei formation.

The development of mossy-fibre projecting precerebellar neurons (PCN) presents a classical example of tangential neuronal migration. PCN migrate tangentially along marginal streams beneath the pial surface from the lower rhombic lip to specific locations in the hindbrain, where they form precerebellar nuclei. Among them, the pontine neurons follow a stereotypic anteroventral-directed pathway to form the pontine nuclei in the pons. The guidance mechanisms that determine the marginal migration of PCN and the anterior migration of pontine neurons are poorly understood. Here, we report that a chemokine SDF1 (also known as CXCL12) derived from the meningeal tissue regulates the migratory pathways of PCN. PCN are chemoattracted by the meningeal tissue, an effect that is mimicked by an SDF1 source. Analysis of knockout mice for the Sdf1 receptor Cxcr4 shows that both the marginal migration of PCN and the anterior migration of pontine neurons are disrupted. We provide further evidence that SDF1/CXCR4 signalling regulates these two processes cell-autonomously. As a result of disrupted neuronal migration, pontine nuclei formation was highly abnormal, with the presence of multiple ectopic pontine clusters posteriorly. The ectopic pontine clusters led to ectopic collateral branch formation from the corticospinal tract. Our results together demonstrate crucial roles for SDF1/CXCR4 in multiple aspects of PCN migration and highlight the deleterious consequence of derailed migration on proper nuclei formation. Furthermore, we provide the first in vivo evidence that pontine neurons themselves induce collateral branching from the corticospinal axons.

The fetal cerebellum. Pitfalls in diagnosis and management.

Prenatal diagnosis of congenital and acquired cerebellar disorders is possible by the use of ultrasound (US) and magnetic resonance imaging (MRI). Although numerous studies have been conducted in this field, diagnostic uncertainties are still common in daily clinical practice. This review outlines five possible pitfalls in the diagnosis of fetal cerebellar disorders: confusion between different entities describing vermian pathologies (Dandy-Walker variant, vermian hypoplasia and vermian agenesis); premature diagnosis of abnormal vermian formation; difficulties in the ultrasonographic differentiation between the cerebellar hemispheres and the vermis; late development of cerebellar hypoplasia/atrophy and differential diagnosis of unilateral cerebellar findings.

Absence of the transcription factor Nfib delays the formation of the basilar pontine and other mossy fiber nuclei.

Transcription factors of the Nuclear Factor I (Nfi) family are important for the development of specific neuronal and glial populations in the nervous system. One such population, the neurons of the basilar pontine nuclei, expresses high levels of Nfi proteins, and the pontine nuclei are greatly reduced in mice lacking a functional Nfib gene. Pontine neurons, along with other precerebellar neurons that populate the hindbrain, arise from precursors in the lower rhombic lip and migrate anteroventrally to reach their final location. Using immunohistochemistry, we find that NFI-B expression is specific for mossy fiber populations of the precerebellar system. Analysis of the Nfib(-/-) hindbrain indicates that the development of the basilar pontine nuclei is delayed, with pontine neurons migrating 1-2 days later than in control animals, and that significantly fewer pontine neurons are produced. While the mossy fiber nuclei of the caudal medulla do form, they also exhibit a developmental delay. Nfia and Nfix null mice exhibit no apparent pontine phenotype, implying specificity in the action of NFI family members. Collectively, these data demonstrate that Nfib plays an important role in the generation of precerebellar mossy fiber neurons, and may do so at least in part by regulating neurogenesis.

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.