Die Rolle von B-RAF in der Embryonalentwicklung des Maus-Vorderhirns

Die Familie der RAF-Kinasen umfasst drei Mitglieder, A-RAF, B-RAF und C-RAF. Nur für die B-RAF-Isoform wurde eine wichtige Funktion für die Entwicklung des Zentralen Nervensystems (ZNS) gefunden. Das Fehlen von B-RAF führt bei neu generierten embryonalen Neuronen zum Zelltod, weil sie in vitro nicht auf überlebensfaktoren reagieren können. Bei einer zweiten Zelllinie, die durch die Abwesenheit von B-RAF beeinträchtigt ist, handelt es sich um endotheliale Zellen. Ihr Zelltod führt zu inneren Blutungen und zu Letalität von B-RAF-/--Mäusen zwischen Tag 10.5 (E10.5) und 12.5 (E12.5) der Embryonalentwicklung. Dies verhinderte bisher weitere Untersuchungen der neuralen B-RAF-Funktion bei späteren Stadien. Im Gegensatz zu B-RAF-/--Mäusen überleben B-RAFKIN/KIN-Mäuse die Mitte der Embryonalentwicklung, da ihre Endothelzellen vor Apoptose geschätzt sind. Diese Tiere besitzen kein B-RAF, stattdessen wird im B-RAF-Locus ein chimäres Protein exprimiert, das den N-Terminus von B-RAF sowie alle Domänen von A-RAF umfasst. Der Schutz vor abnormaler neuraler Apoptose im Vorderhirn macht diese Tiere zu einem potentiellen Modell zur Untersuchung der Proliferations- und Differenzierungsfunktion von B-RAF, die die Kinase neben der Überlebensfunktion in der ZNS-Entwicklung ausübt. Die detaillierte Untersuchung der B-RAFKIN/KIN-Tiere konzentrierte sich auf die Entwicklung der Hirnrinde. Augenscheinlich waren kortikale Defekte im B-RAFKIN/KIN Vorderhirn: Der Verlust von B-RAF führte zu einer starken Reduzierung von Brn-2 exprimierenden pyramidalen Projektions-Neuronen begleitet von einer Störung der Dendritenbildung mit weniger und dünneren Dendriten in diesen oberen Schichten. Weitere Untersuchungen mit BrdU-Markierungsexperimenten zeigten in der ventrikulären Schicht reduzierte Zellproliferation für E14.5-E16.5 der Mutantenembryonen und ein Migrationsdefizit der spätgebideten kortikalen Neuronen. Während der Proliferationsdefekt der Hirnrinden-Vorläuferzellen mit einer reduzierten ERK-Aktivierung einherging, bleibt der Mechanismus der gestörten neuralen Migration zu erklären. Unsere Hypothese ist, dass die subzelluläre Lokalisation von Phospho-ERK in den wandernden Hirnrinden-Neuronen der B-RAFKIN/KIN-Mäuse verändert sein könnte. Zur Bestäigung der in vivo-Funktion von B-RAF und weiteren Studien zu ihrer unbekannten Rolle in der embryonalen Neurogenese sowie anderen Morphogenesen wäre die konditionale B-RAF Inaktivierung erforderlich. Durch die Deletion des genetischen Materials bzw. die Inaktivierung der Genfunktion in ausgew�hlten Zellen zu einem bestimmten Zeitpunkt ließen sich die Embryo-Letalität sowie unerwünschte pleiotrope Nebeneffekte vermeiden und akkumulierende, kompensierende Entwicklungsveränderungen von Beginn an ausschließen. Um die Cre Rekombinase-Methode einsetzen zu können, wurden floxed B-RAF embryonale Stammzell (ES)-Zelllinien generiert. Außerdem wurde ein auf dem Tetrazyklin Operator basierendes Schaltallel in den B-RAF Genort von embryonalen Stammzellen integriert, so dass die B-RAF Expression konditional und reversibel durch die Zugabe von Doxyzyklin angeschaltet werden konnte. Bisher wurden hochgradige chimäre Mäuse nach Blastozysten-Injektion geboren. Die Keimbahnübertragung dieser chimären Mäuse wird momentan untersucht. Wenn beide konditionale Mauslinien bereit sind, k�nnte die Entwicklung ihres Zentralnervensystems untersucht werden, um die Rolle von B-RAF in der Entwicklung des Nervensystems herauszufinden.The RAF family of protein kinases consists of three members, A-RAF, B-RAF and C-RAF. Unlike the other isotypes, B-RAF has been found to have an important function for normal development of the central nervous system (CNS), because newly generated embryonic neurons lacking B-RAF cannot respond to survival factors and undergo cell death in vitro. A second cell lineage affected by the absence of B-RAF are endothelial cells and their death leads to internal bleedings and lethality of B-RAF-/- mice between embryonic day 10.5 (E10.5) and E12.5 precluding an opportunity to further analyze neural B-RAF function at a later stage. In contrast to B-RAF-/- mice, B-RAFKIN/KIN mice, which are B-RAF deficient but express a chimeric protein consisting of the unique N terminus of B-RAF and all the domains of A-RAF in the B-RAF gene locus, survive after midgestation because their endothelial cells are protected from apoptosis. More importantly, overall prevention of abnormal neural apoptosis in the forebrain allows us to study proliferation- or differentiation-oriented function of B-RAF other than its survival effects in CNS development. The detailed investigation of B-RAFKIN/KIN animals was concentrated on cortical development. There were apparent cortical defects in B-RAFKIN/KIN forebrain: Loss of B-RAF led to severe reduction of Brn-2 expressing pyramidal projection neurons accompanied by a disruption of dendrite formation in the upper layers. In further analysis, BrdU labelling experiments showed that from E14.5 to E16.5 cell proliferation in the ventricular zone of the mutant mice was reduced and that the late-born cortical neurons failed to migrate properly. While the proliferation defect of cortical progenitors was associated with reduced ERK activation, the mechanism causing impaired neuronal migration remains to be determined. Our hypothesis is that the subcellular localization of phospho-ERK may be altered in migrating cortical neurons in B-RAFKIN/KIN mice. To confirm in vivo function of B-RAF and further study unknown roles in embryonic neurogenesis as well as other morphogenesis, conditional B-RAF knockouts would be the ideal models, which can efficiently avoid embryonic lethality, prevent unwanted pleiotropic side effects and exclude accumulative compensatory developmental changes from the earliest developmental stage on, through the deletion of genetic material/gene function in selected cells at a specific time. The use of site-specific recombinases such as Cre and the successful development of the reversible tetracycline-based switch have provided powerful venues for creating conditional loss-of-function mouse models. Generation of tetracycline-regulated B-RAF and floxed B-RAF mouse embryonic stem (ES) cell lines was performed. Up to now, high-grade chimeric mice were obtained after blastocyst injection of the modified ES cell clones. The germline transmission from these chimeric mice is currently under investigation. When either of conditional mouse lines is ready, detailed examination in their CNS development would be done to reveal how B-RAF plays a real role for normal development of the nervous system

Cortical Migration Defects in Mice Expressing A-RAF from the B-RAF Locus

We have previously shown that mice lacking the protein kinase B-RAF have defects in both neural and endothelial cell lineages and die around embryonic day 12 (E12). To delineate the function of B-RAF in the brain, B-RAF(KIN/KIN) mice lacking B-RAF and expressing A-RAF under the control of the B-RAF locus were created. B-RAF(KIN/KIN) embryos displayed no vascular defects, no endothelial and neuronal apoptosis, or gross developmental abnormalities, and a significant proportion of these animals survived for up to 8 weeks. Cell proliferation in the neocortex was reduced from E14.5 onwards. Newborn cortical neurons were impaired in their migration toward the cortical plate, causing a depletion of Brn-2-expressing pyramidal neurons in layers II, III, and V of the postnatal cortex. Our data reveal that B-RAF is an important mediator of neuronal survival, migration, and dendrite formation and that A-RAF cannot fully compensate for these functions

Ablation of BRaf impairs neuronal differentiation in the postnatal hippocampus and cerebellum.

This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures

Dependence of dentate gyrus growth on BRaf.

<p>(A) Generation of conditional <i>BRaf</i> mice. In the conditional <i>BRaf</i> allele exon 3 encoding part of the Ras-binding domain is flanked by <i>loxP</i> sites (arrowheads). Deletion of the neomycin resistance gene in <i>BRaf ^nfl</i> mice generated the <i>BRaf ^fl</i> allele. Deletion of both exon 3 and the neomycin resistance gene generated the <i>BRraf ^del</i> allele. Positions of primers used in PCR reactions to distinguish the different alleles are shown, for details see text. (B) Analysis of BRaf expression in embryos. Western blot analysis of BRaf expression in E10.5 embryos resulting from <i>BRaf ^wt/del</i> intercrossing reacted with antibodies against BRaf N-terminal or C-terminal epitopes. Note that the C-terminal-specific antibody detects a ∼82 kDa BRaf band in extracts from <i>BRaf ^del/del</i> and <i>BRaf ^wt/del</i> embryos that is smaller than the BRaf doublet bands of ∼92 and ∼89 kDa seen in wild-type embryos. Detection of β-actin served as loading control. (C) Analysis of downstream targets of BRaf signalling. The phosphorylation levels of the kinases ERK1,2, as well as the levels of the early growth response 1 transcription factor Egr1 were significantly reduced in the hippocampus of cKO mice compared to ctrl mice whereas the expression of Erk1,2 was unaltered. The residual level of BRaf in cKO may occur from “escaper” cells. Gapdh served as loading control. (D) Analysis of BRaf expression by immunohistochemistry. Upper panels are representative sagittal sections of P21 hippocampus immunostained for BRaf with an antibody against the BRaf N-terminus. Lower panels are images taken from boxed regions in upper panels; note presence of BRaf stain in cell body of singular granule neurons (arrows) and their dendrite extending into the molecular layer that might have “escaped” Cre recombinase-mediated <i>BRaf</i> deletion in <i>Nestin-Cre/BRaf ^fl,fl</i> mice. Scale bars; upper row, 200 µm; lower row, 25 µm. (E) Representative sagittal sections of P12 and P21 hippocampus stained with Nissl. Scale bars; 800 µm. (F) Volume of hippocampal granule cell layer (gcl) in 12 and 21 day old mice. Data are mean ±s.e.m.; P12, n = 3; P21, n = 7. (G) Exon organization and location of regulatory regions in BRaf isoforms. Boxes indicate exons with their sizes in nucleotides aligned to the regulatory, catalytic and RAS-binding domains (RBD) of BRaf protein. The vertical arrows above exon 3 indicate the positions of the 5′ end and 3′ end, respectively of an intron that has been spliced out in the small cDNA harbouring exon 3* in embryonic RNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058259#pone.0058259.s002" target="_blank">Figure S2</a>). This in-frame splicing retains the reading frame and is predicted to encode the 89 kDa BRaf isoform. The scheme is deduced from cDNA sequencing of wild-type and exon 2–4 spliced BRaf del samples (see text). The molecular masses of BRaf proteins present on the gel (Fig. 1B) are shown.</p

<i>Nestin-Cre</i> mediated deletion of <i>BRaf</i> causes postnatal death and abnormal behaviour.

<p>(A) Kaplan-Meier survival curves of mice with <i>Nestin-Cre</i> driven <i>BRaf</i> deletion. Mice were monitored daily. CKO, n = 13; ctrl mice, n = 10. (B) Abnormal behaviour of P21 cKO mice, indicated by autoaggression was observed in 13 out of 15 cKO mice. (C) Quantification of fraction of animals capable to balance on a small rod. CKO, n = 13; ctrl mice, n = 11.</p

Cell cycle and cell fate analysis in postnatal hippocampus lacking BRaf.

<p>(A) Quantification of activated caspase-3-positive cells in the dentate gyrus at P24. Representative sagittal sections of the dentate gyrus of ctrl or cKO mice were stained for activated caspase-3 (brown); tissue was counterstained with Nissl. Data are mean ±s.e.m.; n = 7. Scale bar = 50 µm. (B) Quantification of BrdU-labelled cells in the dentate gyrus at P20 (2 h BrdU pulse) of ctrl or cKO mice. Data are mean ±s.e.m.; n = 4. (C) Quantification of Ki67-labelled cells in the dentate gyrus at P20 of ctrl or cKO mice. Data are mean ±s.e.m.; n = 4. (D) BrdU-positive Ki67-negative cells as a fraction of BrdU-labelled cells in the dentate gyrus at P20 of ctrl or cKO mice. Data are mean ±s.e.m.; n = 4. (E) Representative sagittal sections of the dentate gyrus of ctrl or cKO mice stained with the S-phase marker BrdU (green) and the proliferation marker Ki67 (red). Double positive cells are marked with an arrow; arrowheads depict BrdU-positive, Ki-67-negative cells. Scale bar = 50 µm.</p

<i>Nestin-Cre</i> mediated deletion of <i>braf</i> impairs neuronal differentiation in the granular cell layer of the dentate gyrus.

<p>(A) Quantification of BrdU-labelled cells in the dentate gyrus of ctrl or cKO mice. Cells were labelled in vivo with BrdU at days P10 and P11, followed by sacrification 24 hours after the second BrdU pulse. Representative sagittal sections of the dentate gyrus stained with the proliferation marker BrdU (green). Data are mean ±s.e.m.; n = 4. Scale bar = 50 µm. (B) Quantification of BrdU-labelled cells in the dentate gyrus at P22 of ctrl or cKO mice. Neural progenitor cells were labelled in vivo with BrdU at days P10 and P11, followed by sacrification of mice at P22. Representative sagittal sections of the dentate gyrus stained with proliferation marker BrdU (green). Data are mean ±s.e.m.; n = 4. Scale bar = 50 µm. (C) Quantification of BrdU/NeuN-positive cells in the granular cell layer of the dentate gyrus of ctrl cKO mice. Neural progenitor cells were labelled in vivo with BrdU at days P10 and P11, followed by sacrification of mice at P22. Representative sagittal sections of the dentate gyrus stained with proliferation marker BrdU (green) and neuronal marker NeuN (red) 11–12 days after BrdU labelling. Double positive cells are marked with an arrow. Data are mean ±s.e.m.; n = 4. Scale bar = 50 µm. (D) Quantification of BrdU/GFAP-positive radial glia cells in the granular cell layer of the dentate gyrus of ctrl or cKO mice. Neural progenitor cells were labelled in vivo with BrdU at days P10 and P11, followed by sacrification of mice at P22. Representative sagittal sections of the dentate gyrus stained with proliferation marker BrdU (green) and neural precursor/astrocyte marker GFAP (red) 11–12 days after BrdU labelling. Expanded region is indicated by an arrow; the arrowhead depicts a double-positive cell. Data are mean ±s.e.m.; n = 4. Scale bar = 50 µm.</p

Cerebellar abnormalities caused by <i>Nestin-Cre</i> mediated deletion of <i>BRaf</i>.

<p>(A, A’) Representative sagittal sections of P21 cerebellum stained with haematoxylin and eosin (HE) are shown in the upper panel. (B,B’) HE-stained pictures display the reduced size of lobe X with disorganized glomeruli. (C, C’) Calbindin staining was used to visualize the elongated primary dendrite and the reduced and irregular arborization of Purkinje neurons in the molecular layer. Scale bars; 25 µm or as indicated. (D) Quantification of cerebellar lobule length in LV. Comparable Nissl stained slices were analysed from P21 ctrl and cKO mice. Data are mean ±s.e.m.; n = 3. (E) Glomeruli/granule cell distribution in cerebellar lobe LX. Glomeruli distribution was analysed in a defined area in three different positions of comparable slices of P21 ctrl and cKO mice. Data are mean ±s.e.m.; n = 7.</p