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. 2010 Aug 25;169(2):920-31.
doi: 10.1016/j.neuroscience.2010.05.026. Epub 2010 May 20.

The expression of twisted gastrulation in postnatal mouse brain and functional implications

M Sun  1 , C Forsman, C Sergi, R Gopalakrishnan, M B O'Connor, A Petryk
Affiliations

The expression of twisted gastrulation in postnatal mouse brain and functional implications

M Sun et al. Neuroscience. .

Abstract

Twisted gastrulation (TWSG1), an extracellular regulator of bone morphogenetic protein (BMP) signaling, is critical for embryonic brain development. Mice deficient in TWSG1 have abnormal forebrain development manifesting as holoprosencephaly. The expression and potential roles of TWSG1 in postnatal brain development are less well understood. We show that Twsg1 is expressed in the adult mouse brain in the choroid plexus (CP), hippocampus, and other regions, with the strongest expression observed in CP. TWSG1 was also detected in a human fetal brain at mid-gestation, with highest levels in the epithelium of CP. Bmp1, Bmp2, Bmp4-Bmp7 as well as BmprIA and BmprII, but not BmprIB, were expressed in CP. BMP antagonists Chordin (Chrd) and Noggin were not detected in CP, however Chrd-like 1 and brain-specific Chrd-like (Brorin) were expressed. Electrophysiological study of synaptic plasticity revealed normal paired-pulse facilitation and long-term potentiation in the CA1 region of hippocampus in Twsg1(-/-) mice. Among the homozygous mutants that survive beyond the first 2 weeks, the prevalence of hydrocephalus was 4.3%, compared to 1.5% in a wild type colony (P=0.0133) between 3 and 10 weeks of life. We detected a high level of BMP signaling in CP in wild type adult mice that was 17-fold higher than in the hippocampus (P=0.005). In contrast, transforming growth factor beta (TGFbeta) signaling was predominant in the hippocampus. Both BMP signaling and the expression of BMP downstream targets Msx1 and Msx2 were reduced in CP in Twsg1(-/-) mice. In summary, we show that Twsg1 is expressed in the adult mouse and human fetal CP. We also show that BMP is a branch of TGFbeta superfamily that is dominant in CP. This presents an interesting avenue for future research in light of the novel roles of CP in neural progenitor differentiation and neuronal repair, especially since TWSG1 appears to be the main regulator of BMP present in CP.

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Figures

Figure 1
Figure 1. Expression pattern of Twsg1 in a mouse adult brain
(A) In situ hybridization image from Allen Brain Atlas; sagittal section, 55 days old male. Twsg1 is expressed in the choroid plexus, hippocampus, and cerebellum. (B) Close-up view of the choroid plexus with digital analysis of signal intensity. (C) RT-PCR data showing expression of Twsg1 from different brain regions of WT mice, and absence of the transcript in Twsg1−/− mice. (D) Q-PCR data showing the highest level of Twsg1 expression in the choroid plexus compared to other brain regions; no signal is detected from Twsg1−/− hippocampus (KO). BS, brain stem; CP, choroid plexus; CR, cerebellum; CX, cerebral cortex; HP, hippocampus; OB, olfactory bulb; TH, thalamus;. Beta-actin is the reference gene for the reactions. Scale bars: 824 μm in A and 314 μm in B.
Figure 2
Figure 2. Immunolocalization of TWSG1 in adult mouse brain
(A) TWSG1 is detected at highest level in choroid plexus. (B) Negative control (no primary antibody). (C) Higher magnification of the region in A (box) showing patch-like staining of TWSG1 on cell surface and in the extracellular matrix in the choroid plexus. (D) Negative control. (E) Hippocampus. (F) Higher magnification of the region in E (box); CA3 region of hippocampus shows patch-like TWSG1 staining. (G) Purkinje cell layer (white arrow) shows high TWSG1 expression in cerebellum. Weak immunoreactivity is present in the granular layer (green arrow), but not molecular layer (yellow arrow). (H) Cortical neurons show weak immunoreactivity for TWSG1, particularly in layer II/III. Scale bars: 200 μm in A, B, E, G, H and 20 μm in C, D, F.
Figure 3
Figure 3. Immunolocalization of TWSG1 in human fetal brain at mid-gestation
(A) Cerebral cortex. Arrowheads point to the molecular layer. (B) Cerebral cortex. Intense staining in the perikarya of neurons with rosette pattern formation (Fig. 6B, arrows). (C) Control staining of fetal brain without primary antibody (negative control); 630x magnification. (D) Ventricular epithelium of the 4th ventricle showed moderate immunoreactivity. Arrowheads point to the ependymal epithelium. (E) Negative control; 100x magnification. (F) Choroid plexus. The strongest expression was observed in the choroid plexus. Inset at 630x magnification, all papillary projections of the choroid plexus show an intense signal of the lining epithelium, whereas no signal is seen in the fibrovascular core of the papillary projections. Scale bars: 20 μm in A–D and inset and 200 μm in E, F.
Figure 4
Figure 4. Electrophysiological evaluation of synaptic plasticity in the hippocampus
(A) Normal input-output curve in hippocampal synaptic transmission in Twsg1−/− mice. Relationship between the slope of fEPSP (field excitatory postsynaptic potential) and stimulation strength for Twsg1−/− and WT mice. Data are expressed as mean ± SEM. (B) Normal Paired-pulse facilitation (PPF) in Twsg1−/− hippocampal slices. PPF was measured as the ratio between the slopes of fEPSPs evoked by the second and first pulses and plotted for several inter-pulse intervals (ISI). Field EPSPs were evoked with a stimulus that evoked 30% of the maximal fEPSP. Values represent mean ± SEM. (C) TBS protocol induced a similar level of LTP in Twsg1−/− and WT slices. Mean amplitudes of fEPSPs recorded 0–20 min before induction of LTP were set as a baseline. Above the summary plot are shown an average baseline sweep and a sweep 1 hour after induction.
Figure 5
Figure 5. Severe hydrocephalus in TWSG1-deficient mice
(A) WT male mouse at 1 month of age. (B) A gross view of a WT brain. (C) Twsg1−/− mice have smaller body size but larger brain volume (D) in comparison with WT littermates in C57BL/6 background. Knockout mice show enlarged head with dome-shape skull and partly closed eyes. Scale bars: 2 mm in B, D.
Figure 6
Figure 6. Ventricular system in Twsg1−/− mice and controls
(A) Coronal section through WT brain. (B) Coronal section through a Twsg1−/− brain with hydrocephalus showing enlarged lateral ventricles and third ventricle; the asterisk indicates the lateral ventricle. Also the cortex is atrophic and much thinner than control. (C) WT brain; arrow points to the periaqueductal grey matter. (D) Compressed hippocampi and aqueduct. (E–H) Serial sections through the aqueduct; DAPI staining with pseudocolor. (G) WT aqueduct; the largest opening is at midbrain interaural -1mm level. (H) Closed aqueduct in a mutant. (I, J) Normal fourth ventricle and (K,L) normal choroid plexus tissue in the dorsal part of third ventricle. There was no evidence of papilloma in any of the ventricles. Scale bars: 2 mm in A–D, 100 μm in E–H, 200 μm in I–L.
Figure 7
Figure 7. Expression of BMP ligands, their receptors, and BMP-binding proteins in a mouse brain at 1 month of age by RT-PCR
The brain regions tested included hippocampus (HP), cerebellum (CR), cerebral cortex (CX), and choroid plexus (CP). All Bmps tested, BmprIA, BmprII, Chrd-like 1 and Brorin were expressed in the choroid plexus, but not Chrd, Chrd-like 2, or Nog.
Figure 8
Figure 8. Comparison between the levels of BMP and TGFβ signaling in the choroid plexus and the hippocampus by western blotting
(A) Representative image shows the western blot double-staining with anti-P-Smads and GAPDH antibodies. (B) BMP signaling is more active in the choroid plexus than in the hippocampus in WT mice. Conversely, P-Smad2 signaling is significantly lower in the choroid plexus than in the hippocampus. (C) Representative gel image shows reduced P-Smad1/5/8 signal in Twsg1−/− choroid plexus with GAPDH as a control. (D) The graph shows significantly lower P-Smad1/5/8 level in Twsg1−/−choroid plexus compared to WT.
Figure 9
Figure 9. Downstream targets of BMP signaling pathway are suppressed in Twsg1−/− choroid plexus and hippocampus
Both Msx1 and Msx2, two major BMP regulated genes in neural tissues, are reduced in Twsg1−/− choroid plexus and hippocampus. The samples were obtained from 3 wild type and 3 Twsg1−/− mice.
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