Published On: Tue, Mar 26th, 2019

Sonic hedgehog expression in the postnatal brain [RESEARCH ARTICLE]

Endogenous Shh expression levels in the rat brain during embryonic and postnatal development

Starting with Shh mRNA, we measured its expression levels in the rat cortex from embryonic day 14 (e14) to postnatal day 30 (p30) using quantitative RT-PCR. Analysis of four biological replicates (number of embryos or pups) revealed that Shh mRNA level in the cortex was high at e14, but declined gradually as embryos developed, and became undetectable at birth (top graph in Fig. 1A). Notably, after birth and throughout postnatal development, Shh mRNA became detectable again and its level increased steadily as young rats matured, and reached a plateau between p21 and p30 (p21 versus p1, 7.6±2.1 versus 1, P=0.0259; p30 versus p1, 7.0±1.4 versus 1, P=0.0084; top graph in Fig. 1A). Data from RT-PCR analysis using a different PCR primer set (Materials and Methods) showed the same two-peaked pattern (Fig. S1A).

Fig. 1.

Fig. 1.

Shh mRNA and ShhN protein expression levels in rat brains during embryonic and postnatal development. (A) Quantitative RT-PCR assay of Shh mRNA level in rat cortex from embryonic day 14 (e14) to postnatal day 30 (p30). For comparison, mRNA levels of Bdnf and synaptic markers, bassoon and synaptophysin, were also assayed. The data were expressed as fold change from p1 and plotted against age (n=the number of rat embryos or pups per age). For e14 embryos, whole brains were used. Data of quantitative RT-PCR using a different Shh primer set are shown in Fig. S1A. (B) Shh mRNA level in postnatal rat hippocampus and cerebellum from p1 to p30. (C) Immunoblot with an antibody to N-terminal epitope of Shh. Lanes contain lysates from HEK cells transfected with wild-type full-length Shh (ShhFL), N-terminal fragment of Shh (ShhN), or vector. The ShhN antibody detects N-terminal fragment (∼19 kD) and full-length (∼45 kD) Shh. (D) Immunoblot with the ShhN antibody showing the ∼19 kD ShhN as the main Shh species in tissue extracts from e14 or e16 rat brains. A similar finding from mouse brain extracts using this antibody is shown in Fig. S1B. Also, a similar finding from rat brain extracts but using a different ShhN antibody is shown in Fig. S1C. (E) Immunoblot analysis of ShhN protein level in rat cortex from embryonic day 14 (e14) to postnatal day 30 (p30). ShhN level is high during embryonic development, declines to a nearly undetectable level at birth, and increases again during late postnatal development. Blot shown is one representative experiment; blots of additional experiments are shown in Fig. S1D. Histogram includes at least three experiments. (F,G) Immunoblot analysis showing that, similar to cortex, ShhN protein level in hippocampus (F) and cerebellum (G) increases during late postnatal development. Syp, synaptophysin. Error bars represent s.e.m. ***P0.001, **P0.01, *P0.05, unpaired t-test. The values of ShhN protein level represent the ShhN band intensities normalized to the actin band intensities.

For comparison, we measured mRNA levels of brain-derived neurotrophic factor (Bdnf) in the same set of cortical samples. Unlike Shh mRNA expression, Bdnf mRNA was undetectable before birth at all embryonic ages examined (middle graph in Fig. 1A). But similar to Shh mRNA expression, Bdnf mRNA level increased continuously during postnatal development and reached a peak level between p21 and p30 (middle graph in Fig. 1A). Knowing that synapses develop rapidly during postnatal development (Petralia et al., 1999; Sans et al., 2000), we measured mRNAs of two well-characterized synaptic proteins, bassoon and synaptophysin (Gundelfinger et al., 2016; Kwon and Chapman, 2011). As expected, bassoon and synaptophysin mRNA increased markedly from birth to p21 or p30 (bottom graph in Fig. 1A). Although relative Shh mRNA abundance in the postnatal cortex was lower compared with mRNA for Bdnf and synaptic markers, the temporal expression pattern of Shh mRNA was nearly identical to that of Bdnf and synaptic markers.

We examined two additional brain regions, the hippocampus and cerebellum, concentrating on postnatal ages. Similar to the cortex, Shh mRNA levels in both hippocampus and cerebellum increased steadily during postnatal development (Fig. 1B). At p30, the level of Shh mRNA was significantly higher than the level at birth (p30 versus p1 hippocampus, 4.2±0.7 versus 1, P=0.0465; p30 versus p1 cerebellum, 3.9±0.2 versus 1, P=0.0069; Fig. 1B).

We subsequently examined Shh protein level in the embryonic and postnatal cortex. We used a commercially available antibody to the N-terminal epitope of Shh (see Materials and Methods). We refer to this antibody as the ShhN antibody. We first evaluated the ShhN antibody by immunoblotting cell lysates from human embryonic kidney (HEK) cells that were transfected with the expression construct either for wild-type full-length Shh protein (ShhFL), or N-terminal fragment of Shh protein (ShhN) (Materials and Methods). As revealed in the blot shown in Fig. 1C, the antibody specifically recognized the expected ∼45 kD ShhFL and ∼19 kD ShhN (Lee et al., 1994). The lack of any Shh protein band from the vector-transfected cells validated the specificity of the ShhN antibody, as HEK cells do not produce endogenous Shh (Chen et al., 2002a,b). In brain tissue extracts, the antibody detected the ∼19 kD ShhN, not the ∼45 kD ShhFL (Fig. 1D; with a similar finding in mouse brain extracts, see Fig. S1B). A different ShhN antibody (5E1; Ericson et al., 1996; Petralia et al., 2011b) (Fig. S1C) similarly showed that the predominant form of Shh protein in brains is the ∼19 kD ShhN. Therefore, we focused on examining this Shh form in brains at different ages.

The temporal expression pattern of ShhN protein mirrored the pattern of Shh mRNA in the cortex: both were highly expressed at e14, gradually declined to a nearly undetectable level at birth, and steadily increased during postnatal development (Fig. 1E; Fig. S1D). Moreover, in postnatal hippocampus and cerebellum, again the protein was low in p1, but by p30, ShhN protein level was significantly higher than in p1 (p30 versus p1 hippocampus, 61±11.7 versus 7±3, P=0.037; p30 versus p1 cerebellum, 105.5±17.9 versus 17.5±6.9, P=0.0108; Fig. 1F,G). Together, these observations supported significant expression of Shh mRNA and ShhN protein in postnatal and young adult rat brain including the cortex, hippocampus and cerebellum. The closely correlated temporal expression pattern between mRNA and protein suggested that transcription regulation contributed at least in part to the level of Shh protein.

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