Journal: Genes & Development

Article Title: Independent control of neurogenesis and dorsoventral patterning by NKX2-2

doi: 10.1101/gad.352886.125

Figure Lengend Snippet: In vitro generation of limb-level mouse motor neurons. ( A ) Schematic of ventral progenitor domains in the brachial (caudal cervical) spinal cord; at this level, p3 progenitors generate V3 interneurons (INs) instead of branchiovisceral motor neurons. ( B ) Summary of culture conditions that produce brachial motor neurons. ( C ) Immunofluorescence images of day 7 mouse cultures show that HOXC8 + somatic motor neurons (MNX1 + /ISL1 + ) are produced, some of which are FOXP1 + , indicating LMC-like identity. ( D ) Immunofluorescence images of day 5 cultures show that motor neurons (ISL1 + ) produced under these conditions are somatic (MNX1 + ). No expression of PHOX2A is observed. ( E ) RT-qPCR for Sim1 mRNA shows a >1000-fold expression increase in late stages of culture. ( F ) Summary of proportions of cells expressing NKX2-2, ISL1, and OLIG2 (normalized to the total number of cells expressing at least one of these three genes) over time.

Article Snippet: Primary antibodies, host species, and concentrations used in this study were as follows: ISL1 (goat, 1:5000, has 10% cross-reactivity to ISL2; Neuromics GT15051-100; RRID: AB_2126323), MNX1 (guinea pig, 1:100; from Jessell Laboratory), FOXP1 (mouse, 1:400; Santa Cruz Biotechnology sc-398811), NKX2-2 (mouse, 1:100; Developmental Studies Hybridoma Bank [DSHB] 74.5A5; RRID: AB_531794), BrdU (rat, 1:400; Abcam ab6326; RRID: AB_305426), OLIG2 (guinea pig, 1:100; from Jessell Laboratory), IRX3 (rabbit, 1:100; from Jessell Laboratory), and PAX6 (mouse, 1:100; DSHB supernatant).

Techniques: In Vitro, Immunofluorescence, Produced, Expressing, Quantitative RT-PCR

Journal: Investigative Ophthalmology & Visual Science

Article Title: Cortex-Restricted Deletion of Foxp1 Impairs Visual Responses by Disrupting Geniculocortical Connections in a Mouse Model of Autism

doi: 10.1167/iovs.67.2.50

Figure Lengend Snippet: Foxp1 -cKO mice exhibit reduced preference for the safe zone over the cliff. The schematic diagrams of the visual cliff test arena are shown in panels (A) (side view) and (B) (top view). (C–E) Statistics of visual cliff performance from six-week-old WT ( n = 11) and cKO mice ( n = 9). (C) Time spent in safe and cliff zones during the five-minute exploration. Foxp1 -cKO mice spent less time in the safe zone compared to WT controls. *** P < 0.001. (D) Foxp1 -cKO mice displayed a lower preference for the safe side compared to the control mice. (E) Histograms showing the number of transitions between the two zones, which was higher in cKO mice than in the control mice. *** P < 0.001.

Article Snippet: To generate forebrain-deletion Foxp1 animals, mice homozygous for the floxed- Foxp1 allele (stock no. 017699; Jackson Laboratories, Bar Harbor, ME, USA) were bred with Emx1 - Cre mice.

Techniques: Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Cortex-Restricted Deletion of Foxp1 Impairs Visual Responses by Disrupting Geniculocortical Connections in a Mouse Model of Autism

doi: 10.1167/iovs.67.2.50

Figure Lengend Snippet: Selective deletion of Foxp1 in cortical V1 neurons along the visual pathway. (A) FOXP1 immunofluorescence staining ( red ) on the coronal sections of six-week-old mouse brains. Nuclei were stained with DAPI ( blue ). The WT mice showed intense FOXP1 staining that was almost absent in the Foxp1 -cKO mouse brains. (a) Magnified view of the square area in the left panel . Arrows indicate cells that express FOXP1. II-VI, cortical layers; Scale bar : 100 µm ( left panel ) and 50 µm ( right insert ). (B) Retinal sections were analyzed by FOXP1 immunofluorescence staining. Foxp1 ( red ) was preserved in the GCL of both WT and cKO mouse eyes. (a) Magnified images of RGC cells in the retina. Arrows indicate FOXP1-positive cells. Scale bar : 50 µm ( left panel ) and 10 µm ( right insert ). INL, inner nuclear layer; ONL, outer nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer. (C) FOXP1 immunofluorescence in the thalamus. (a) High-magnification views of the square regions in the left panel. FOXP1 signals were present in the dLGN of WT and Foxp1 -cKO mice. Scale bar : 200 µm ( left panel ) and 50 µm ( right insert ). (D – F) Quantitative analysis of the number of FOXP1 + cells in V1 (D) , GCL of the retina (E) , and dLGN of the thalamus (F) . N = 3 animals per genotype. Ns, no significant difference. *** P < 0.001.

Article Snippet: To generate forebrain-deletion Foxp1 animals, mice homozygous for the floxed- Foxp1 allele (stock no. 017699; Jackson Laboratories, Bar Harbor, ME, USA) were bred with Emx1 - Cre mice.

Techniques: Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Cortex-Restricted Deletion of Foxp1 Impairs Visual Responses by Disrupting Geniculocortical Connections in a Mouse Model of Autism

doi: 10.1167/iovs.67.2.50

Figure Lengend Snippet: Foxp1 -cKO mice display impaired FVEPs responses. (A) Representative FVEPs traces were recorded from the WT ( n = 14) and Foxp1 -cKO mice ( n = 13) at six weeks of age. The two positive and two negative alternating peaks of FVEPs are designated as P1, P2, N1, and N2. (B) Statistical analysis of the amplitude of N1-P1 wave, *** P < 0.001. (C , D) Quantification analysis of the implicit time of N1 and P1 waves, respectively. Ns, no significant difference. (E) Statistical analysis of amplitudes of N2-P2 wave, *** P < 0.05. (F , G) Statistical analysis of the implicit time of N2 and P2 waves, respectively. Ns, no significant difference. Foxp1 -cKO mice showed a significant reduction in N1-P1 and N2-P2 amplitude and slightly postponed the N1 wave compared to WT controls. ** P < 0.01.

Article Snippet: To generate forebrain-deletion Foxp1 animals, mice homozygous for the floxed- Foxp1 allele (stock no. 017699; Jackson Laboratories, Bar Harbor, ME, USA) were bred with Emx1 - Cre mice.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Cortex-Restricted Deletion of Foxp1 Impairs Visual Responses by Disrupting Geniculocortical Connections in a Mouse Model of Autism

doi: 10.1167/iovs.67.2.50

Figure Lengend Snippet: Foxp1 -cKO mice exhibit normal ERG. (A – C) Representative ERG waveforms recorded under scotopic conditions from the WT ( blue traces , n = 10) and Foxp1 -cKO mice ( red traces , n = 11) at the indicated stimulus intensities. (D – F) Quantitative analysis of scotopic ERG b-wave amplitudes at light intensities of 0.01 log cd·s/m 2 (D) , a- and b-wave amplitudes at light intensities of 3.0 log cd·s/m 2 (E) and 10.0 log cd·s/m 2 (F) in WT and KO mice. (G) Representative ERG traces recorded under photopic conditions at a light intensity of 3.0 log cd·s/m 2 . (H) Statistical analysis of the a- and b-wave amplitudes based on the ERG trace results shown in (G) . Ns, no significant difference.

Article Snippet: To generate forebrain-deletion Foxp1 animals, mice homozygous for the floxed- Foxp1 allele (stock no. 017699; Jackson Laboratories, Bar Harbor, ME, USA) were bred with Emx1 - Cre mice.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Cortex-Restricted Deletion of Foxp1 Impairs Visual Responses by Disrupting Geniculocortical Connections in a Mouse Model of Autism

doi: 10.1167/iovs.67.2.50

Figure Lengend Snippet: Loss of Foxp1 does not alter light-induced neuronal activation in retina and LGN. (A) C-Fos immunostaining in the retina of six-week-old WT and cKO mice after light stimulation. Nuclei were counterstained with DAPI ( blue ). (a , b) High-magnification views of the square regions in the left panel . Arrows mark c-Fos + cells. Scale bar : 50 µm ( left panel ) and 10 µm ( right insert ). INL, inner nuclear layer; ONL, outer nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer. (B) Immunostaining of c-Fos in the LGN. (a – c) Magnified view of square area in the left panel . Arrows designate c-Fos + cells. Scale bar : 200 µm ( left panel ) and 10 µm ( right insert ). (C , D) Quantitative analysis of the number of c-Fos + cells in distinct regions as calculated from A and B , respectively. N = 4 animals per genotype. Ns, no significant difference.

Article Snippet: To generate forebrain-deletion Foxp1 animals, mice homozygous for the floxed- Foxp1 allele (stock no. 017699; Jackson Laboratories, Bar Harbor, ME, USA) were bred with Emx1 - Cre mice.

Techniques: Activation Assay, Immunostaining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Cortex-Restricted Deletion of Foxp1 Impairs Visual Responses by Disrupting Geniculocortical Connections in a Mouse Model of Autism

doi: 10.1167/iovs.67.2.50

Figure Lengend Snippet: Foxp1 deficiency diminishes light-induced neuronal activation in V1. (A) Representative images of c-Fos immunostaining ( red ) in the V1 cortex of six-week-old WT and cKO mice after light stimulation. Nuclei were counterstained with DAPI ( blue ). II-VI, two to six layers of the visual cortex. Arrows indicate cells that express c-Fos. Scale bar : 200 µm ( left panel ) and 10 µm ( right insert ). (B) Statistics of the number of c-Fos + cells in distinct regions as calculated from A . Compared to the WT littermates, the cKO mice had a reduced number of c-Fos + cells in the specific layers of V1. N = 4 animals per genotype. *** P < 0.001.

Article Snippet: To generate forebrain-deletion Foxp1 animals, mice homozygous for the floxed- Foxp1 allele (stock no. 017699; Jackson Laboratories, Bar Harbor, ME, USA) were bred with Emx1 - Cre mice.

Techniques: Activation Assay, Immunostaining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Cortex-Restricted Deletion of Foxp1 Impairs Visual Responses by Disrupting Geniculocortical Connections in a Mouse Model of Autism

doi: 10.1167/iovs.67.2.50

Figure Lengend Snippet: Foxp1 -cKO mice exhibit decreased geniculocortical projections in layer IV of V1. (A , B) Immunostaining of VGluT2 ( red ) and 5-HTT ( green ) in brain sections from P7 WT and Foxp1 -cKO mice. Arrows indicate dense geniculocortical projections in layer IV of V1 from the WT brains. The VGluT2- and 5-HTT- staining were nearly lost in the cKO mice. S1, primary somatosensory cortex. Scale bar : 250 µm. (C , D) Quantifications of the normalized fluorescence intensities of VGluT2 and 5-HTT in layer IV of cKO mice relative to WT (set to 1). N = 4 animals per genotype. *** P < 0.001. (E) VGluT2 immunostaining ( red ) of geniculocortical axons on the coronal sections of six-week-old mouse brains. (a) High-magnification views of the square regions in the left panel. Scale bar : 200 µm ( left panel ) and 50 µm ( right insert ). (F) The normalized fluorescence intensity of VGluT2 in layer IV of cKO mice relative to WT. N = 4 animals per genotype. *** P < 0.001. (G) Brain coronal sections of WT and Foxp1 -cKO mice at six weeks of age were co-immunostained with VGluT2 ( green ) and PSD95 ( red ). (a) Magnified view of boxed area in the left panel. Arrows designate VGluT2/PSD-95 co-localized puncta. Scale bar : 20 µm ( left panel ) and 5 µm ( right insert ). (H) Quantitative analysis of the puncta number of VGluT2 + PSD-95 + geniculocortical synapses per 100 µm 2 in layer IV of V1. N = 5 brains per genotype. * P < 0.01.

Article Snippet: To generate forebrain-deletion Foxp1 animals, mice homozygous for the floxed- Foxp1 allele (stock no. 017699; Jackson Laboratories, Bar Harbor, ME, USA) were bred with Emx1 - Cre mice.

Techniques: Immunostaining, Staining, Fluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: Cortex-Restricted Deletion of Foxp1 Impairs Visual Responses by Disrupting Geniculocortical Connections in a Mouse Model of Autism

doi: 10.1167/iovs.67.2.50

Figure Lengend Snippet: Loss of Foxp1 changes the thickness of cortical layers in V1. (A – C) Representative images of laminar markers CUX1 (A) , CTIP2 (B) , and FOXP2 (C) immunostaining in brain coronal sections from six-week-old WT and cKO mice. Scale bar : 100 µm. (D – F) Quantitative analysis of the relative thickness in layer II-IV (D) , layer V (E) , and layer VI (F) of the VI cortex. N = 4-5 animals per genotype. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To generate forebrain-deletion Foxp1 animals, mice homozygous for the floxed- Foxp1 allele (stock no. 017699; Jackson Laboratories, Bar Harbor, ME, USA) were bred with Emx1 - Cre mice.

Techniques: Immunostaining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Cortex-Restricted Deletion of Foxp1 Impairs Visual Responses by Disrupting Geniculocortical Connections in a Mouse Model of Autism

doi: 10.1167/iovs.67.2.50

Figure Lengend Snippet: Morphological analysis of layer IV neurons in V1. (A) Representative Golgi-Cox-stained coronal brain sections from WT and Foxp1 -cKO mice at six weeks of age. Scale bar : 1 mm. (B) Reconstructions of the dendritic arborizations of layer IV neurons in V1 from WT and the cKO brains. Scale bar : 50 µm. (C – G) Statistical analyses of total dendrite length, tree length, surface area, segment number, and segment length of layer IV neurons in V1. N = 7 brains per genotype. For each animal, 20-31 neurons from four or five sections were analyzed. ** P < 0.01, *** P < 0.001. (H) Representative Golgi-staining micrographs of dendritic spines of the layer IV neurons. Scale bar : 2 µm. (I) Quantitative analysis of average spine density per 20 µm-long dendritic segment. The cKO mice had a decreased spine density compared to the controls. N = 4 brains per genotype. ** P < 0.01.

Article Snippet: To generate forebrain-deletion Foxp1 animals, mice homozygous for the floxed- Foxp1 allele (stock no. 017699; Jackson Laboratories, Bar Harbor, ME, USA) were bred with Emx1 - Cre mice.

Techniques: Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Cortex-Restricted Deletion of Foxp1 Impairs Visual Responses by Disrupting Geniculocortical Connections in a Mouse Model of Autism

doi: 10.1167/iovs.67.2.50

Figure Lengend Snippet: Ablation of Foxp1 impairs mitochondrial biogenesis and function. (A) Western blot analysis was performed in isolated cortical tissues from six-week-old WT and Foxp1 -cKO mice. GAPDH was used as the loading control. (B) Quantitative analysis of the relative protein expression levels of PGC-1α and TFAM in the V1 cortex, normalized to GAPDH. * P < 0.05, ** P < 0.01. (C) Immunoblots displaying the immunoreactive bands of FOXO1, COX IV and TOM20. (D) Quantitative analysis of the expression levels of FOXO1, COX IV, and TOM20, normalized to GAPDH. *** P < 0.001. (E) Mitochondrial membrane potential was assayed using the MitoTracker Red CMXROS probe. Primary cortical neuronal cultures were prepared from neonatal mouse brains and labeled with 20 nM Mito-Tracker Red CMXRos solution ( red ) after three days in culture. The cultured cells were then fixed and immunostained with βIII-tubulin ( green ). Scale bar : 20 µm. (F) Normalized fluorescence intensity of Mito-Tracker Red CMXRos in βIII-tubulin-positive neurons of KO mice relative to WT controls. N = 3 experiments, *** P < 0.001.

Article Snippet: To generate forebrain-deletion Foxp1 animals, mice homozygous for the floxed- Foxp1 allele (stock no. 017699; Jackson Laboratories, Bar Harbor, ME, USA) were bred with Emx1 - Cre mice.

Techniques: Western Blot, Isolation, Control, Expressing, Membrane, Labeling, Cell Culture, Fluorescence