UCSF UC San Francisco Previously Published Works Title Postnatal growth of the human pons: a morphometric and immunohistochemical analysis. Permalink https://escholarship.org/uc/item/9579m17k Journal The Journal of comparative neurology, 523(3) ISSN 0021-9967 Authors Tate, Matthew C Lindquist, Robert A Nguyen, Thuhien et al. Publication Date 2015-02-01 DOI 10.1002/cne.23690 Peer reviewed eScholarship.org Powered by the California Digital Library University of California RESEARCH ARTICLE Postnatal Growth of the Human Pons: A Morphometric and Immunohistochemical Analysis Matthew C. Tate,1,2† Robert A. Lindquist,1,3,4† Thuhien Nguyen,1,2 Nader Sanai,5 A. James Barkovich,6 Eric J. Huang,3,7 David H. Rowitch,1,2,3,8 and Arturo Alvarez-Buylla1,2,3* 1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California – San Francisco, San Francisco, CA 94143 2Department of Neurological Surgery, University of California – San Francisco, San Francisco, CA 94143 3Neuroscience Graduate Program, University of California – San Francisco, San Francisco, CA 94143 4Medical Scientist Training Program, School of Medicine, University of California – San Francisco, San Francisco, CA 94143 5Department of Neurological Surgery, Barrow Neurological Institute, Phoenix, AZ 85013 6Department of Radiology and Biomedical Imaging, Section of Pediatric Neuroradiology, University of California – San Francisco, San Francisco, CA 94143 7Department of Pathology, University of California – San Francisco, San Francisco, CA 94143 8Howard Hughes Medical Institute and Department of Pediatrics, University of California – San Francisco, San Francisco, CA 94143 ABSTRACT majority of proliferative cells in the postnatal pons Despite its critical importance to global brain function, the expressed the transcription factor Olig2, suggesting an oli- postnatal development of the human pons remains poorly godendrocyte lineage. The proportion of proliferating cells understood. In the present study, we first performed mag- that were Olig21 was similar through the first 7 months netic resonance imaging (MRI)-based morphometric analy- of life and between basis and tegmentum. The number of ses of the postnatal human pons (0–18 years; n 5 6– Ki671 cells declined dramatically from birth to 7 months 14/timepoint). Pons volume increased 6-fold from birth to and further decreased by 3 years, with a small number of 5 years, followed by continued slower growth throughout Ki671 cells observed throughout childhood. In addition, childhood. The observed growth was primarily due to two populations of vimentin/nestin-expressing cells were expansion of the basis pontis. T2-based MRI analysis sug- identified: a dorsal group near the ventricular surface, gests that this growth is linked to increased myelination, which persists throughout childhood, and a parenchymal and histological analysis of myelin basic protein in human population that diminishes by 7 months and was not evi- postmortem specimens confirmed a dramatic increase in dent later in childhood. Together, our data reveal remark- myelination during infancy. Analysis of cellular proliferation able postnatal growth in the ventral pons, particularly revealed many Ki671 cells during the first 7 months of during infancy when cells are most proliferative and myeli- life, particularly during the first month, where proliferation nation increases. J. Comp. Neurol. 523:449–462, 2015. was increased in the basis relative to tegmentum. The VC 2014 Wiley Periodicals, Inc. INDEXING TERMS: brainstem; pediatric; development; basis; pontine glioma; nif-0000-00217; SciRes_000114; AB_304558; AB_2109815; AB_442102; AB_396287; AB_92396; AB_91107; AB_291466; AB_2336877; AB_2336878; AB_261856; AB_2304493 The first two authors contributed equally to this work. Center Way RMB-1038, Box 0525, San Francisco, CA 94143-0525. E-mail: [email protected] Grant sponsor: National Institutes of Health; Grant number: NS28478; Grant sponsor: John G. Bowes Research Fund (to A.A.-B.); Grant num- Received June 23, 2014; Revised September 30, 2014; ber: NRSA 1F32NS067889-01A1 (to M.T.); Grant sponsor: Pediatric Accepted October 6, 2014. Brain Tumor Foundation (to A.A.-B., D.H.R.), University of California; DOI 10.1002/cne.23690 Grant number: MRPI #142675 (to E.J.H.); Grant sponsor: D.H.R. is an Published online October 10, 2014 in Wiley Online Library HHMI Investigator. (wileyonlinelibrary.com) *CORRESPONDENCE TO: Arturo Alvarez-Buylla, PhD, University of California, San Francisco, Department of Neurological Surgery, 35 Medical VC 2014 Wiley Periodicals, Inc. The Journal of Comparative Neurology | Research in Systems Neuroscience 523:449–462 (2015) 449 M.C. Tate et al. During embryogenesis, the pons and cerebellum are formed from the metencephalon, which is derived from the rostral hindbrain (rhombencephalon) (Barkovich et al., 2009). In humans, pontine nuclei are derived from a stream of cells migrating from the rhombic lip termed the corpus pontobulbare between 8 and 20 weeks gestation (Essick, 1912). While a number of studies describe the development of the fetal pons (Nozaki et al., 1992; Fischbein et al., 1996; Hatta et al., 2007), less is known about postnatal pontine growth in humans, particularly at the cellular level. An improved understanding of the dynamics of growth and develop- ment of the human pons, particularly postnatal stem/ progenitor populations, may provide insight into the development of this critical brain region and into abnor- malities of the pons such as developmental malforma- tions and pediatric gliomas (Barkovich et al., 2009). In this study, we quantified spatial and temporal growth profiles of the human pons throughout childhood using magnetic resonance imaging (MRI)-based morphometry. In addition, we examined cell proliferation and expres- sion of progenitor markers as a function of age and location within the pons. MATERIALS AND METHODS Figure 1. Brainstem morphometric measurements. The upper panel Brainstem morphometry (A) is an example mid-sagittal T1-weighted MRI showing the basis pontis (yellow), pontine tegmentum (red), and medulla (blue). All brain MRI studies were approved by the University Dashed lines illustrate the axial sections extending from proximal of California, San Francisco (UCSF) Committee on pons to distal medulla that were used to estimate volumes. For Human Research. MRI scans obtained between 2005 each cross-section, the relevant brainstem subregions (tegmentum, and 2010, for which there were no intracranial abnor- basis, and medulla) were traced to determine the volume. The malities (as noted by the official report of the neurora- lower panels illustrate the tracing of axial images through mid-pons (B, left) and mid-medulla (C, right) levels. D 5 dorsal, V 5 ventral, diologist who interpreted the scan as part of routine R 5 rostral, C 5 caudal. clinical care), were included in the study (n 5 123). For volumetric assessments (total pons, medulla, pontine tegmentum, basis pontis), T1-weighted sequences were The human pons, part of the brainstem, is an impor- evaluated. In addition, average T2 values for both basis tant relay for sensory and motor information from the and tegmentum were measured from mid-pons axial forebrain to the cerebellum. In addition, the pons plays images, and the ratios of T2 intensities were used to essential roles in respiration, sleep, swallowing, eye estimate myelination in these two regions (modification movement, hearing, facial movement/sensation, pos- of a method described by Abe et al., 2004). For each ture, and maintenance of consciousness (Saladin, MRI, consecutive axial images extending from rostral 2012). Anatomically, the pons can be divided into three pons (at midbrain-pons junction, just caudal to cerebral regions: tectum, tegmentum, and basis. The tectum is peduncles) to distal medulla (cervicomedullary junction) the most dorsal component and forms the roof of the were analyzed. The areas (in mm2) of regions of interest 4th ventricle. The tegmentum is also located dorsally (pontine tegmentum, pontine basis, medulla) were man- within the pons and contains important autonomic ually traced for each axial image and the volume (in structures, as well as nuclei of cranial nerves V–VIII, mm3) estimated using measurement tools from Philips which control various facial movements and sensation. iSite Enterprise (v. 3.6) radiology software (Fig. 1). Age The ventral portion of the pons is occupied by the basis groups evaluated were age 0 (within 3 days of birth, pontis. Pontine nuclei within the basis receive inputs normal gestational age), 3 months, 6 months, 9 from descending corticopontine fibers and project to months, 1 year, 3 years, 5 years, 7 years, 9 years, 11 the cerebellum via the middle cerebellar peduncle. years, 13 years, and 18 years (n 5 123). Average 450 The Journal of Comparative Neurology | Research in Systems Neuroscience Human postnatal pons development TABLE 1. List of Human Specimens Specimen Postnatal Gestational APGAR Postmortem Year of Number Age Length (wks) (1,5 min) Gender Diagnosis Interval (hrs) Autopsy Source 1 1 day 38 1/7 1,0 F Pulmonary failure 72 2011 1 2 1 mo 35 6/7 8,8 M Cardiac anomaly 36 2008 1 3 2 mo 37 5,6 M Hepatic failure 24 2011 1 4 7 mo 31 4/7 — M Cardiac anomaly 24 2011 1 5 1.5 yr — — F Sepsis 29 2007 1 6 3 yr — — M Asphyxia 16 2009 2 7 7 yr — — F Lymphoma 2 2007 1 8 8 yr — — M Trauma 16 2009 2 9 13 yr — — M Trauma 15 2009 2 10 13.5 yr — — M Trauma 19 2010 2 1 UCSF Dept. Pathology. 2 NICHD Brain and Tissue Bank. volumetric growth rates for basis, tegmentum, and Immunohistochemistry medulla (mm3/mo) were calculated between age After rinses in TNT wash buffer (13 phosphate- groups (0–3 months, 3–6 months, 6 months to 1 year, buffered saline, 0.05% Triton X-100), microwave or water 1–3 years, 3–5 years, 5–7 years, 7–18 years) by calcu- bath antigen retrieval was performed for all sections in lating the difference in volumetric averages of the two 0.01M citrate buffer (pH 6.0) at 95Cfor10minutes,fol- age groups (in mm3) and dividing by the time between lowed by a 20-minute cooling period.