The long noncoding RNA Neat1 is required for mammary gland development and lactation

Abstract

The lncRNA Neat1 is an essential architectural component of paraspeckle nuclear bodies. Although cell-based studies identified Neat1-paraspeckles as key regulators of gene expression through retention of hyperdited mRNAs and/or transcription factors, it is unclear under which specific physiological conditions paraspeckles are formed in vivo and whether they have any biological relevance. Herein, we show that paraspeckles are assembled in luminal epithelial cells during mammary gland development. Importantly, genetic ablation of Neat1 results in aberrant mammary gland morphogenesis and lactation defects. We provide evidence that the lactation defect is caused by a decreased ability of Neat1-mutant cells to sustain high rates of proliferation during lobular-alveolar development. This study is the first to assign an important biological function to the lncRNA Neat1 and to link it to the presence of paraspeckles nuclear bodies in vivo.

Keywords: NEAT1; long noncoding RNA; mammary gland development; paraspeckles.

FIGURE 1.

Loss of Neat1 leads to lobuloalveolar development and lactation defects. (A) Average weight of offspring from Neat1 heterozygous (+/− mother) and Neat1 KO (−/− mother) female before (3.5 wk) and after weaning (6 wk) and representative images. Statistical significance determined by two-sided t-test. (**) P = 0.0044 at 3.5 wk and (**) P = 0.0002 at 6 wk. (B) Whole-mount hematoxylin staining of wild-type (WT) and Neat1 KO (Neat1^−/−) lactating inguinal mammary glands at 2 d post-parturition. (C) H&E staining on sections obtained from the inguinal mammary glands contralateral to the ones described in B. Comparative quantification of (D) luminal alveolar compartment (K8/18 immunoreactivity); (*) P = 0.028 and (E) cell number (DAPI stain) in the lactating mammary gland of wild-type versus Neat1 KO females; (***) P = 0.0003. Statistical significance determined by two-sided t-test. (F) Immunohistochemical staining of cytokeratin 8/18 on consecutive sections of samples in C. (G) Immunohistochemical staining of β-casein on consecutive sections of samples in C. (H) Comparative quantification of Ki67-positive cells relative to total cell number in mammary glands of pregnant females at 8.5 (*) P = 0.04 and 12.5 (*) P = 0.01 d post-coitum. (I) Immunohistochemical staining of Ki67 on sections obtained from inguinal mammary gland 8.5 d post-coitum of wild-type and Neat1^−/− females.

FIGURE 2.

Ductal and branching morphogenesis defects in Neat1 KO females. (A) Whole-mount hematoxylin staining of wild-type (WT) and Neat1 KO (Neat1^−/−) prepubertal inguinal mammary gland. (LN) lymph node. (B) Whole-mount hematoxylin staining of wild-type (WT) and Neat1 KO (Neat1^−/−) adult virgin inguinal mammary gland with indication of counting method for secondary branch points shown in C. (LN) Lymph node. (C) Average number of secondary branch points over a distance of 3 mm as indicated in B and average interbranch distance in micrometer. Statistical significance determined by two-sided t-test; (**) P = 0.0031 (upper panel), (**) P = 0.0017 (lower panel). (D) H&E staining on sections obtained from the inguinal mammary gland contralateral to the ones described in B. (E) Immunohistochemical staining of cytokeratin 8/18 on consecutive sections of samples in D. (F) Immunohistochemical staining of α-actin/SMA on consecutive sections of samples in D.

FIGURE 3.

Neat1-paraspeckles are detected in mammary luminal epithelial cells. (A) RNA fluorescence in situ hybridization (FISH) of Neat1 (left column), with nuclear DAPI counterstain (middle column). Wild-type adult virgin duct is represented in the top row, Neat1 KO duct below. The right column shows overlay of DAPI signal (blue) and Neat1 (red). Triangles indicate Neat1-paraspeckles in left column with Neat1 signal. (B) RNA FISH of Neat1 (red) with DAPI counterstain (blue) throughout post-natal mammary gland development: in terminal end bud of prepubertal 27-d-old animal, in duct of 8- to 9-wk-old adult virgin animal, during alveologenesis at 8.5 d post-coitum, and during lactation at 2 d post-parturition (from left to right). Triangles indicate Neat1-paraspeckles. (C) RNA FISH of Neat1 (red) and immunofluorescent (IF) staining of cell type specific markers (green), with nuclear DAPI counterstain (blue). IF in left panel: cytokeratin 8/18, middle panel: cytokeratin 5, right panel: α-actin/SMA. Triangles indicate Neat1-paraspeckles.