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.2019 Jan 1;29(1):189-201.
doi: 10.1093/cercor/bhx318.

Developmental Connectivity and Molecular Phenotypes of Unique Cortical Projection Neurons that Express a Synapse-Associated Receptor Tyrosine Kinase

Affiliations

Developmental Connectivity and Molecular Phenotypes of Unique Cortical Projection Neurons that Express a Synapse-Associated Receptor Tyrosine Kinase

Ryan J Kast et al. Cereb Cortex..

Abstract

The complex circuitry and cell-type diversity of the cerebral cortex are required for its high-level functions. The mechanisms underlying the diversification of cortical neurons during prenatal development have received substantial attention, but understanding of neuronal heterogeneity is more limited during later periods of cortical circuit maturation. To address this knowledge gap, connectivity analysis and molecular phenotyping of cortical neuron subtypes that express the developing synapse-enriched MET receptor tyrosine kinase were performed. Experiments used a MetGFP transgenic mouse line, combined with coexpression analysis of class-specific molecular markers and retrograde connectivity mapping. The results reveal that MET is expressed by a minor subset of subcerebral and a larger number of intratelencephalic projection neurons. Remarkably, MET is excluded from most layer 6 corticothalamic neurons. These findings are particularly relevant for understanding the maturation of discrete cortical circuits, given converging evidence that MET influences dendritic elaboration and glutamatergic synapse maturation. The data suggest that classically defined cortical projection classes can be further subdivided based on molecular characteristics that likely influence synaptic maturation and circuit wiring. Additionally, given that MET is classified as a high confidence autism risk gene, the data suggest that projection neuron subpopulations may be differentially vulnerable to disorder-associated genetic variation.

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Figures

Figure 1.
Figure 1.
The MetGFP BAC transgenic mouse is a high-fidelity reporter of endogenousMet expression in the developing cortex. (A) In situ hybridization reveals endogenousMet transcript pattern in coronal sections of P0, P7, and P14 wild-type mouse forebrain. (B) GFP immunocytochemistry demonstrates the unique expression pattern of the GFP reporter in coronal sections of P0, P7, and P14 MetGFP mouse forebrain. Note the labeling of callosal, commissural, and corticofugal axons by GFP, reminiscent of endogenous MET protein transport during development. (C) Multiplexed fluorescence in situ hybridization shows colocalization ofGFP andMet transcripts in the posteromedial barrel field of the somatosensory cortex at P14. Yellow circles denote cells that coexpressMet andGFP transcripts. Scale bar = 50 μm.
Figure 2.
Figure 2.
Met co-expression with transcriptional regulators of cortical neuron subtype specification. (A) GFP immunocytochemistry at P0 shows laminar distribution of GFP-expressing cells (green). Asterisks denote GFP-expressing neurons in the subplate at this age. (A’) High magnification of layer 5 inset shows colocalization of GFP with Satb2 (cyan, white arrowheads), but not Ctip2 (magenta) at P0. (A”) High magnification of layer 6 inset reveals patterns of colocalization of GFP with Satb2 and Ctip2 at P0. (B) GFP immunocytochemistry shows laminar distribution of GFP-expressing cells at P7. Asterisks denote GFP-expressing neurons in the subplate at this age. (B’) High magnification of layer 5 inset illustrates colocalization of GFP with Satb2 and Ctip2 (white arrows) at P7. (B”) High magnification of layer 6 inset illustrates colocalization of GFP with Satb2 and Ctip2 at P7. (C) GFP immunocytochemistry at P14 shows laminar distribution of GFP-expressing cells. Note the sparse distribution of GFP-expressing neurons in the subplate. (C’) High magnification of layer 5 inset illustrates colocalization of GFP with Satb2 and Ctip2 at P14. (C”) High magnification of layer 6 inset illustrates colocalization of GFP with Satb2 and Ctip2 at P14. (D) Quantification of the percentage of layer 5 Satb2+ or Ctip2+ neurons that coexpressed GFP at P0, P7, and P14. (E) Quantification of the layer 6 Satb2+ or Ctip2+ neurons that coexpressed GFP at P0, P7, and P14. Error bars represent SEM. Scale bars: 100 μmA,B, and C; 20 μmA’,A”,B’,B”,C’, andC”.
Figure 3.
Figure 3.
Met expression in layer 6 is enriched in corticocortical neurons throughout development. (A) Retrograde tracing of layer 6 corticothalamic neurons by injection of CTB into the VPm thalamus, combined with GFP (green) immunocytochemistry. Boxed area is shown as higher magnification images (2-channel and 1-channel images, respectively), and reveals that GFP expression (white arrows) is excluded from CTB-labeled (magenta) corticothalamic neurons. (B) Retrograde labeling of layer 6 ipsilateral corticocortical neurons by CTB injection into primary motor cortex. White asterisk denotes CTB-labeled, GFP− corticocortical neuron in layer 6B. White box is shown at higher magnification in 2- and 1-channel images. Note colocalization (white arrowheads) of CTB and GFP in primary somatosensory cortex. (C) Quantification of the percentages of retrogradely traced layer 6 corticothalamic and corticocortical neurons in primary somatosensory cortex that express GFP at P14. (D) GFP, ppCCK, and PCP4 immunocytochemistry at P14 shows colocalization of GFP and ppCCK, but not GFP and PCP4 in layer 6 of the primary somatosensory cortex. (E) Quantification of the percentages of layer 6 ppCCK and PCP4 expressing layer 6 neurons that coexpress GFP. (F) Quantification of the percentage of layer 6 GFP neurons that coexpress PCP4 or ppCCK. (G) Ntsr1-cre; tdTomato (magenta) and GFP (green) immunocytochemistry reveal minimal colocalization of GFP and tdTomato-labeled corticothalamic neurons at P0, P7, and P14. (H) Quantification of the percentage of layer 6 GFP neurons that express Ntsr1-cre driven tdTomato. (I) Quantification of the percentage of Ntsr1-cre; tdTomato expressing neurons that coexpress GFP. Error bars represent SEM. Scale bars: 50 μmA,B low-magnification images; 20 μmA,B high-magnification images,D, andG.
Figure 4.
Figure 4.
Met is expressed in subsets of layer 5 intratelencephalic and PT neurons. (A) Retrograde tracing of PT projection neurons by injection of CTB (magenta) into the rostral cerebral peduncle combined with GFP immunocytochemistry (green). White box is shown at higher magnification in 2- and 1-channel images, revealing that a subset of PT neurons express GFP (arrowheads). (B) Retrograde tracing of intratelencephalic neurons by injection of CTB (magenta) into ipsilateral motor cortex combined with GFP immunocytochemistry (green). White box is shown at higher magnification in 2- and 1-channel images, revealing that a subset of layer 5 ipsilateral corticocortical neurons express GFP (green). (C) Retrograde tracing of layer 5 corticothalamic neurons by injection of CTB (magenta) into the VPm thalamus combined with GFP immunocytochemistry (green). White box is shown at higher magnification in 2- and 1-channel images, revealing colocalization in layer 5 of primary somatosensory cortex (arrowheads). (D) Quantification of the percentages of PT and intratelencephalic neurons in primary somatosensory cortex that express GFP. Error bars represent SEM. Scale bars: 50 μm low-magnification images inA,B, andC; 20 μm high-magnification images inA,B, andC.
Figure 5.
Figure 5.
Met is expressed in subsets of ipsilateral and contralateral layer 2/3 projection neurons. (A) Retrograde tracing of callosal projection neurons by injection of CTB (magenta) into the contralateral somatosensory cortex combined with GFP immunocytochemistry (green). White box is shown at higher magnification in 2- and 1-channel images, revealing that a subset of layer 2/3 contralateral projection neurons express GFP (arrowheads). (B) Retrograde tracing of ipsilateral projection neurons by injection of CTB (magenta) into the ipsilateral motor cortex combined with GFP immunocytochemistry (green). White box is shown at higher magnification in 2- and 1-channel images, revealing that a subset of layer 2/3 contralateral projection neurons express GFP (arrowheads). (C) Quantification of the percentages of contralateral and ipsilateral projection neurons that express GFP. Error bars represent SEM. Scale bars: 50 μm low-magnification images inA andB; 20 μm high-magnification images inA andB.
Figure 6.
Figure 6.
Summary ofMet expression across cortical projection neuron classes. (A) Line drawing modified with permission from Franklin and Paxinos (2008) mouse stereotaxic atlas illustrating the projection populations analyzed by retrograde tracing and molecular characterization. (B) Quantitative summary of the percentage of each layer- and projection-defined neuron population observed to be Met-expressing.
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