HCMV Infection Reduces Nidogen-1 Expression, Contributing to Impaired Neural Rosette Development in Brain Organoids

Abstract

Human cytomegalovirus (HCMV) is a leading cause of birth defects in humans. These birth defects include microcephaly, sensorineural hearing loss, vision loss, and cognitive impairment. The process by which the developing fetus incurs these neurological defects is poorly understood. To elucidate some of these mechanisms, we have utilized HCMV-infected induced pluripotent stem cells (iPSCs) to generate in vitro brain organoids, modeling the first trimester of fetal brain development. Early during culturing, brain organoids generate neural rosettes. These structures are believed to model neural tube formation. Rosette formation was analyzed in HCMV-infected and mock-infected brain organoids at 17, 24, and 31 days postinfection. Histological analysis revealed fewer neural rosettes in HCMV-infected compared to mock-infected organoids. HCMV-infected organoid rosettes incurred multiple structural deficits, including increased lumen area, decreased ventricular zone depth, and decreased cell count. Immunofluorescent (IF) analysis found that nidogen-1 (NID1) protein expression in the basement membrane surrounding neural rosettes was greatly reduced by virus infection. IF analysis also identified a similar downregulation of laminin in basement membranes of HCMV-infected organoid rosettes. Knockdown of NID1 alone in brain organoids impaired their development, leading to the production of rosettes with increased lumen area, decreased structural integrity, and reduced laminin localization in the basement membrane, paralleling observations in HCMV-infected organoids. Our data strongly suggest that HCMV-induced downregulation of NID1 impairs neural rosette formation and integrity, likely contributing to many of HCMV's most severe birth defects. IMPORTANCE HCMV infection in pregnant women continues to be the leading cause of virus-induced neurologic birth defects. The mechanism through which congenital HCMV (cCMV) infection induces pathological changes to the developing fetal central nervous system (CNS) remains unclear. Our lab previously reproduced identified clinical defects in HCMV-infected infants using a three dimensional (3D) brain organoid model. In this new study, we have striven to discover very early HCMV-induced changes in developing brain organoids. We investigated the development of neural tube-like structures, neural rosettes. HCMV-infected rosettes displayed multiple structural abnormalities and cell loss. HCMV-infected rosettes displayed reduced expression of the key basement membrane protein, NID1. We previously found NID1 to be specifically targeted in HCMV-infected fibroblasts and endothelial cells. Brain organoids generated from NID1 knockdown iPSCs recapitulated the structural defects observed in HCMV-infected rosettes. Findings in this study revealed HCMV infection induced early and dramatic structural changes in 3D brain organoids. We believe our results suggest a major role for infection-induced NID1 downregulation in HCMV-induced CNS birth defects.

Keywords: basement membrane proteins; cerebral organoid; human cytomegalovirus; neural development; neural rosette.

FIG 1

A schematic representation of human neural tube formation. NPC, neural progenitor cells.

FIG 2

HCMV infection impaired rosette formation. A. Schematic diagram describing neural rosette formation in developing brain organoids. Representative mock-infected/control images of 10D embryoid body and 31D brain organoids are shown. Scale bar = 200 μm. B–D. 17, 24 and 31D H&E images of mock- compared to HCMV-infected rosettes, respectively. Bottom panels are 2-fold (17 and 24D) or 3-fold (31D) digital magnifications of the yellow boxed areas in the top panels. Panels B, C and D are representative of 8, 10, and 10 images of mock-infected rosettes and 7, 9, and 10 images of HCMV-infected rosettes, respectively. Scale bar = 20 μm. E. Maximum number of neural rosettes identified on three brain organoid tissue slides with ~24 total sections/organoid (mock- versus HCMV-infected) at 17, 24 and 31D. For each slide, the maximum number of rosettes per section was determined. Then, the largest of these three numbers was plotted on the graph. Number of organoids = 5. Statistical analysis for all graphs used unpaired, two-tailed Student's t test. NS = P value > 0.05. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Error bars are representative of one standard deviation. BM, basement membrane; L, lumen; VZ, ventricular zone. Black arrow: basement membrane layer. Black dashed circle: nonradially organized cells. Yellow dashed lines: radially organized cells. Blue asterisks: holes in the VZ.

FIG 3

HCMV infection induced structural changes to rosettes. A. Schematic diagram of neural rosette structural analysis, indicating areas that were analyzed. See Materials and Methods for details regarding analysis parameters. B. Representative image of mock- versus HCMV-infected neural rosette area at 31D. Scale bar = 20 μm. C. Graph of the area of mock- versus HCMV-infected rosettes at 17, 24, and 31D. D. Representative image of mock- versus HCMV-infected neural rosette lumens at 31D. E. Graph showing the lumen area of mock- versus HCMV-infected rosettes at 17, 24, and 31D. F. Representative image of VZ depth analysis of mock- versus HCMV-infected neural rosettes at 31D. G. Graph showing the VZ depth of mock- and HCMV-infected rosettes at 17, 24, and 31D. The VZ depth was measured by taking an average of five measurements from the edge of the lumen to the edge of the ventricular zone. H. Representative image of VZ cell count analysis of mock- versus HCMV-infected neural rosettes at 31D. I. Graph showing the VZ cell count of mock- versus HCMV-infected rosettes at 17, 24, and 31D. Number of organoids = 5. Yellow line: outline of structural element measured.

FIG 4

HCMV infection altered the staining pattern of the gap junction protein, ZO1. Representative IF images of ZO1 staining at 31D, in mock- (top row) versus HCMV-infected rosettes (rows 2 to 4). White arrows point out discontinuity in apical membrane staining after infection. Scale bar = 20 μm.

FIG 5

Infection downregulated NID1 expression along basement membrane in HCMV-infected rosettes. A. Representative IF images of mock-infected rosettes at 17, 24, 31D. B. HCMV-infected rosettes at 17, 24, 31D. C. Graph of percent basement membrane perimeter positive for NID1. Each point represents a single scored rosette.

FIG 6

HCMV infection reduced laminin expression in rosette basement membrane. A. Representative IF images of mock rosettes at 17, 24, and 31D. B. HCMV-infected rosettes at 17, 24, and 31D. C. Graph of percent basement membrane perimeter positive for laminin. Lo: HCMV-infected rosette with less laminin. Hi: HCMV infected rosette with more laminin. LAM, laminin B1.

FIG 7

NID1 KD compromised embryoid body development. A. Western blot analysis of NID1 KD and V2.0 iPSCs differentiated for 4D. Lamin B was used as the loading control. The normalized values, calculated as described in Materials and Methods, of V2.0/V2.0 and NID1 KD/V2.0 are displayed below the blots. B, D, F, H, and J. Representative white light images of V2.0 versus NID1 KD embryoid bodies at 2, 4, 6, 8, and 10D, respectively. C, E, G, I, and K. Graphs showing embryoid body area at 2, 4, 6, 8, and 10D as measured using ImageJ software. Yellow arrows: embryoid body interior region. Red arrow: neuroectoderm layer. Scale bar = 200 μm.

FIG 8

NID1 KD compromised brain organoid development. A and B. Representative brightfield images of NID1 KD brain organoids versus V2.0 at 12 and 24D, respectively. Black arrow: budding neuroectoderm layer in V2.0. Red arrow: neural rosettes in V2.0. Arrows point out aberrant cysts in NID1 KD organoids. Scale bar= 200 μm.

FIG 9

NID1 KD compromised rosette formation. A–C. Representative H&E images of V2.0 versus NID1 KD rosettes at 17, 24, and 31D. Bottom panels are magnifications of the yellow boxed area, with level of magnification indicated. D. Neural rosette counts per brain organoid. Rosettes were enumerated as described in legend to Fig. 2E and Materials and Methods. Number of organoids = 6. +, V2.0; −, NID1 KD; L, lumen; VZ, ventricular zone. Black arrow: basement membrane layer. Black dashed circle: nonradially organized cell. Yellow dashed lines: radial organization. Blue asterisks: holes in the VZ. Scale bar = 20 μm.

FIG 10

NID1 KD induced structural changes to rosettes. A. Schematic diagram of neural rosette structural analysis, indicating areas that were analyzed. See Materials and Methods for details regarding analysis parameters. B. Representative image of V2.0 and NID1 KD neural rosette area at 31D. C. Graph of the rosette area, V2.0 and NID1 KD rosette at 17, 24, and 31D. D. Representative image of V2.0 and NID1 KD neural rosette lumen area at 31D. E. Graph of the rosette lumen area, V2.0, and NID1 KD rosette at 17, 24, and 31D. F. Representative image of V2.0 and NID1 KD neural rosette VZ depth at 31D. G. Graph of the rosette VZ depth, V2.0 and NID1 KD rosette at 17, 24, and 31D. VZ depth was measured by taking an average of five measurements from the edge of the lumen to the edge of the ventricular zone. H. Representative image of V2.0 and NID1 KD neural rosette cell count at 31D. I. Graph of the rosette cell count, V2.0 and NID1 KD rosette at 17, 24, and 31D. +, V2.0; −, NID1 KD. Yellow line: outline of structural element measured.

FIG 11

NID1 KD altered the staining pattern of gap junction protein, ZO1. Representative IF image of ZO1 staining at 24D and 31D in V2.0 (top and fourth rows, respectively) versus NID1 KD rosettes (second, third, and last rows, respectively). White arrows point out discontinuity in apical membrane staining after NID1 KD. Scale bar = 20 μm.

FIG 12

NID1 KD reduced laminin expression. A. Representative IF images of NID1-stained rosettes (NID1 KD versus V2.0) at 17D. B and C. Representative images of laminin IF staining of NID1 KD rosette versus V2.0 at 17, 24, and 31D. D. Graph of percent basement membrane perimeter positive for laminin staining. Number of organoids = 6.