XB-ART-53810
Dev Biol.
September 1, 2017;
429
(1):
200-212.
Developmental neurogenesis in mouse and Xenopus is impaired in the absence of Nosip.
Hoffmeister M
,
Krieg J
,
Ehrke A
,
Seigfried FA
,
Wischmann L
,
Dietmann P
,
Kühl SJ
,
Oess S
.
Genetic deletion of
Nosip in mice causes holoprosencephaly, however, the function of
Nosip in neurogenesis is currently unknown. We combined two vertebrate model organisms, the mouse and the South African clawed frog, Xenopus laevis, to study the function of
Nosip in neurogenesis. We found, that size and cortical thickness of the developing
brain of
Nosip knockout mice were reduced. Accordingly, the formation of postmitotic neurons was greatly diminished, concomitant with a reduced number of apical and basal neural progenitor cells in vivo. Neurospheres derived from
Nosip knockout embryos exhibited reduced growth and the differentiation capability into neurons in vitro was almost completely abolished. Mass spectrometry analysis of the neurospheres proteome revealed a reduced expression of
Rbp1, a regulator of retinoic acid synthesis, when
Nosip was absent. We identified the homologous
nosip gene to be expressed in differentiated neurons in the developing
brain of Xenopus embryos. Knockdown of
Nosip in Xenopus resulted in a reduction of
brain size that could be rescued by reintroducing human
NOSIP mRNA. Furthermore, the expression of pro-neurogenic transcription factors was reduced and the differentiation of neuronal cells was impaired upon
Nosip knockdown. In Xenopus as well as in mouse we identified reduced proliferation and increased apoptosis as underlying cause of microcephaly upon
Nosip depletion. In Xenopus
Nosip and
Rbp1 are similarly expressed and knockdown of
Nosip resulted in down regulation of
Rbp1. Knockdown of
Rbp1 caused a similar microcephaly phenotype as the depletion of
Nosip and synergy experiments indicated that both proteins act in the same signalling pathway.
Nosip is a novel factor critical for neural stem cell/progenitor self-renewal and neurogenesis during mouse and Xenopus development and functions upstream of
Rbp1 during early neurogenesis.
PubMed ID:
28663132
Article link:
Dev Biol.
Genes referenced:
egr2
emx1
en2
gla
h3f3a
neurog1
nosip
opa1
pax6
rbp1
rras
syne2
tubb2b
Morpholinos referenced:
nosip MO1
rbp1 MO2
Article Images:
[+] show captions
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Fig. 4.
Nosipis expressed in the developing neural tissue ofXenopusand knockdown of Nosip causes microcephaly (A) Expression analysis of Nosip by WMISH at indicated Xenopus laevis developmental stages; white arrowheads point to eyes, black arrowheads to anterior neural tissue (St.13) or brain (St. 23/St. 30), and red arrowhead to pharyngeal arches (B) Transversal vibratome section of the embryo head with arrowheads pointing to differentiated neurons of the brain; the dotted line in A indicates the level of the section shown in B. (C) Immunoblot analysis of Xenopus embryo lysates using a Nosip-specific antiserum; immunoblot with an α-tubulin-specific antibody served as loading control; lysates were generated from Xenopus embryos at stage 20 after bilateral injection of 23 ng Nosip MO or Control MO together with 0.5 ng human NOSIP mRNA in 2-cell stage Xenopus embryos as indicated (D) Bright field images of Xenopus brains at stage 42, anterior to the top after unilateral injection of Nosip MO or Control MO and human NOSIP mRNA as indicated; scale bar 200 µm (E) Statistical evaluation of the data shown in D, N = number of independently evaluated brains.
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Fig. 5.
Nosip deficiency results in neurogenesis and brain differentiation defects in Xenopus (A) pax6-, neurogenin- and tubb2b-specific WMISH of Xenopus embryos at stage 13 after unilateral injection of 23 ng Nosip MO or Control MO as indicated, injected side at the right, arrowhead points to the posterior neural plate (B) Statistical evaluation of the data shown in A (C)tubb2b-specific WMISH of Xenopus embryos at stage 17 treated as described in A, arrowhead points to reduced number and misaligned tubb2b-positive cells (D) Statistical evaluation of the data shown in C (E)emx1-, en2-, and egr2-specific WMISH of Xenopus embryos at stage 23 treated as described in A, arrowheads point to reduced brain marker gene expression (F) Statistical evaluation of the data shown in E (G)Xenopus embryos at stage 23 stained with a pH3-specific antibody after unilateral injection of Nosip MO or Control MO as indicated (H) Statistical evaluation of the data shown in G (I) TUNEL staining of Xenopus embryos of stage 23 after unilateral injection of Nosip MO or Control MO as indicated (J) Statistical evaluation of the data shown in I.
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Fig. 6.
Nosip and Rbp1 act synergistically duringXenopusbrain development (A)rbp1-, opa1- and syne2-specific WMISH of Xenopus embryos at indicated stages; black arrowheads pointing to anterior neural plate at stage 13 and brain at stages 23 and 30, white arrowhead pointing to the eye, red arrowhead to pharyngeal arches (B)rbp1-specific WMISH of Xenopus embryos at stage 23 after unilateral injection of Nosip MO or Control MO as indicated; injected side at the right, arrowhead pointing to reduced rbp1 expression in the brain (C) Statistical evaluation of the data shown in B (D) Bright field images of Xenopus brains at stage 42, anterior to the top, after unilateral injection of Rbp1 MO or Control MO and GFP mRNA as indicated; scale bar 200 µm (E) Statistical evaluation of the data shown in D, N = number of independently evaluated brains (F) Synergy experiment. Bright field images of Xenopus brains at stage 42, after unilateral injection of either 10 ng Nosip MO or 30 ng Rbp1 MO alone or in combination. 0.5 ng GFP mRNA was co-injected as lineage tracer; scale bar 200 µm (G) Statistical evaluation of the data shown in F, N = number of independently evaluated brains.
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Fig. S1.
Synteny analysis; AP2A1 - Adaptor-related protein complex 2, alpha 1; APOC2 - Apolipoprotein C2; EMC10 - ER Membrane Protein Complex Subunit 10; MIR150 - MicroRNA 150; PRR12 - Proline-rich 12; PRRG2 - Proline Rich And Gla Domain 2; RCN3 - Reticulocalbin 3; RRAS - Related RAS Viral (R-Ras) Oncogene Homologue; SCAF1 - Serine/Arginine-Related CTD Associated Factor 1.
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nosip (nitric oxide synthase interacting protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, anterior view, dorsal up.
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nosip (nitric oxide synthase interacting protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior right, dorsal up.
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opa1 (optic atrophy 1 (autosomal dominant)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior right, dorsal up.
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rbp1 (retinol binding protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior right, dorsal up.
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syne2 (spectrin repeat containing, nuclear envelope 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior right, dorsal up.
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