The autism risk genes MET and PLAUR differentially impact cortical development

Abstract

Candidate risk genes for autism spectrum disorder (ASD) have been identified, but the challenge of determining their contribution to pathogenesis remains. We previously identified two ASD risk genes encoding the receptor tyrosine kinase MET and the urokinase plasminogen activator receptor (PLAUR), which is thought to modulate availability of the MET ligand. We also reported a role for Met signaling in cortical interneuron development in vitro and a reduction of these neurons in uPAR (mouse ortholog of PLAUR) null mice, suggesting that disruption of either gene impacts cortical development similarly. Here, we modify this conclusion, reporting that interneuron numbers are unchanged in the neocortex of Met(fx/fx) / Dlx5/6(cre) mice, in which Met is ablated from cells arising from the ventral telencephalon (VTel). Consistent with this, Met transcript is not detected in the VTel during interneuron genesis and migration; furthermore, during the postnatal period of interneuron maturation, Met is co-expressed in glutamatergic projection neurons, but not interneurons. Low levels of Met protein are expressed in the VTel at E12.5 and E14.5, likely reflecting the arrival of Met containing corticofugal axons. Met expression, however, is induced in E12.5 VTel cells after 2 days in vitro, perhaps underlying discrepancies between observations in vitro and in Met(fx/fx) / Dlx5/6(cre) mice. We suggest that, in vivo, Met impacts the development of cortical projection neurons, whereas uPAR influences interneuron maturation. An altered balance between excitation and inhibition has been postulated as a biological mechanism for ASD; this imbalance could arise from different risk genes differentially affecting either or both elements.

Figure 1

Brightfield photomicrographs illustrate GAD-67 (A,B) and PV (C,D) immunoreactivity in coronal sections through the hippocampus of adult wild type (A,C) and Met^fx/fx/Dlx5/6^cre (B,D) mice. Quantitative analysis reveals no significant difference (p>0.05) in the number of GAD-67- (E) and PV- (F) immunoreactive cells in any subfield of the hippocampus examined. Black histograms, wild type; gray histograms, Met^fx/fx/Dlx5/6^cre. Scale bar = 500μm.

Figure 2

Brightfield photomicrographs illustrate GAD-67 (A,B) and PV (D,E) immunoreactivity in coronal sections through the parietal cortex of adult wild type (A,D) and Met^fx/fx/Dlx5/6^cre (B,E) mice. Quantitative analysis reveals no significant difference (p>0.05) in the number of GAD-67- (C) and PV- (F) immunoreactive cells in frontal, parietal or occipital cortex. Black histograms, wild type; gray histograms, Met^fx/fx/Dlx5/6^cre. Scale bar = 500μm.

Figure 3

Darkfield photomicrographs of coronal sections from wild type mice after processing for autoradiography and emulsion dipping. At E14.5 (A) Met transcript can be detected in the olfactory epithelium (oe), but is absent from the telencephalon (tel). Even as early as E12.5 (B) a prominent Met signal can be observed in multiple peripheral structures, including the ventral horn of the spinal cord (sc) and developing limb muscle (lm). By E16.5, Met transcript can be readily observed in the cortical plate (CP) at both rostral (C) and caudal (D) levels, but is absent from the ventral telencephalon (VTel) at both levels. At more caudal levels, the signal is particularly intense in the cortical plate laterally, while the subplate (SP) is more prominent medially. The boxed area in D is shown at higher magnification in E. Scale bar = 1mm (A–D), 250μm (E).

Figure 4

Confocal photomicrographs of coronal sections from the medial frontal cortex of postnatal day 14 wild type mice after processing for fluorescent in situ hybridization, illustrating double-labeling for Met and Gad1 (A–C), Met and Plp1 (D– F), Met and Slc1a2 (G–I), or Met and VGlut1 (J–L). Full arrows indicate cells with only a single label of Met, Gad1, Plp1, or Slc1a2. Arrowheads indicate cells that co-express Met and VGlut1 (L). While there is double labeling of Met and VGlut1, Met is not co-localized with Gad1, Plp1, or Slc1a2. Accumulated punctate labeling over cellular profiles is readily distinguishable from more diffuse background puncta, which is similar to background labeling using the sense strand. Scale bar in panel J represents 50μm.

Figure 5

Western blotting analysis of Met protein expression in the ventral telencephalon in wild type mice. Lane 1, E12.5; Lane 2, E14.5; Lane 3, E12.5 + 2 days in vitro (DIV); Lane 4, P7. Protein levels are low embryonically in vivo, although there is an increase in levels between E12.5 and E14.5. A more robust increase is observed when explants of E12.5 ventral telencephalon are maintained for 2 DIV. None-the-less, this expression is modest compared to that observed during the peak of Met expression at P7. A one-way ANOVA reveals a significant effect of the source of the tissue on the relative level of Met expression (p>0.05).