Journal: Genes & Development

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

doi: 10.1101/gad.352886.125

Figure Lengend Snippet: NKX2-2 represses NEUROG2 in vpMNs in a HES-independent manner. ( A ) Schematic of human and mouse motor neurogenesis, as summarized in the table below . ( B ) Schematic of dorsoventral patterning in the mouse ventral spinal cord via Nkx2-2 -dependent repression of Olig2 , Pax6 , and Irx3 . ( C ) Differential gene expression analysis of bulk RNA-seq data from human vpMNs and pMNs. Each dot is a gene; genes that are significantly ( q -value < 0.01) upregulated/downregulated are colored dark gray. Genes of interest are labeled in red (significantly upregulated/downregulated) or blue (not significantly changed). ( D ) qPCR results comparing log 2 (fold change) between expression in human NKX2-2-KO OLIG2 + and WT NKX2-2 + /OLIG2 + cells. From left to right : (n.s.) P = 0.159, (n.s.) P = 0.725, (*) P = 0.0378, (***) P = 8.05 × 10 −4 , (***) P = 5.26 × 10 −4 , (***) P = 4.62 × 10 −4 .

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: Gene Expression, RNA Sequencing, Labeling, Expressing

Journal: Genes & Development

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

doi: 10.1101/gad.352886.125

Figure Lengend Snippet: Nkx-2-2 represses Neurog2 independently of its Tinman domain in mouse upper cervical spinal progenitors. ( A ) Differential gene expression analysis between non-DOX-treated and iNkx2-2 SDmut ( left ), iNkx2-2 WT ( middle ), or iNkx2-2 TNmut ( right ) cells on day 4. Each dot is a gene; genes that are significantly ( q -value < 0.01) upregulated/downregulated are colored dark gray. Genes of interest are labeled in red (significantly upregulated/downregulated) or blue (not significantly changed). ( B ) Schematic of ventral progenitor domains in the rostral cervical spinal cord, along with patterning genes, proneural genes, and neuronal fate for each lineage. (IN) Interneuron, (sMN) somatic motor neuron, (bvMN) branchiovisceral motor neuron. ( C ) Heat map representation of expression level changes in patterning genes, proneural genes, and immediate downstream targets, as well as somatic/branchiovisceral motor neuron genes, upon ectopic expression of WT or mutant Nkx2-2 . ( D ) Schematic summarizing dual functions of Nkx2-2 , both of which lead to Notch-independent Neurog2 repression. ( E ) H3K27ac (blue) and NKX2-2 (red) ChIP-seq tracks surrounding Neurog2 , Olig2 , and Pax6 (gene bodies are shown in gray below ). NKX2-2-bound sites that significantly ( q -value < 0.05) decrease in H3K27 acetylation in iNkx2-2 WT but not iNkx2-2 TNmut are highlighted in blue; those that decrease with both iNkx2-2 WT and iNkx2-2 TNmut are highlighted in red.

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: Gene Expression, Labeling, Expressing, Mutagenesis, ChIP-sequencing

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: Genes & Development

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

doi: 10.1101/gad.352886.125

Figure Lengend Snippet: Nkx2-2 delays motor neurogenesis in a TN-independent manner. ( A , left ) NEUROG2 and OLIG2 immunolabeling of day 4 cultures following 24 h of DAPT treatment alone or in combination with DOX (iNkx2-2 TNmut ). ( Right ) Quantified intensities taken from >200 nuclei across the entire embryoid body. (***) P = 7.23 × 10 −15 for NEUROG2, (***) P = 6.26 × 10 −59 for OLIG2. Two-sided t -test. ( B ) Immunolabeling of day 4 and 5 cultures for OLIG2, ISL1, and MNX1 after consecutive 24 h DOX (iNkx2-2 WT or iNkx2-2 TNmut ) and DAPT treatment. ( C ) Quantification from flow cytometry data shown in : pMN (OLIG2 + /ISL1 − ):MN (ISL1 + ) ratios and MN proportions in iNkx2-2 cultures relative to those in non-DOX-treated controls. Relative pMN:MN ratio: (***) P = 2.05 × 10 −4 for day 4, (***) P = 3.40 × 10 −5 for day 5. Relative MN proportion: (n.s.) P = 0.956 for day 4, (***) P = 5.28 × 10 −7 for day 5. Two-sided t -test; n = 3. ( D ) Proportion of late-born (after day 5) motor neurons following pulsed or sustained ectopic TN mutant Nkx2-2 expression. (***) P = 2.75 × 10 −5 for pulsed, (***) P = 1.13 × 10 −5 for sustained. Two-sided t -test; n = 3. ( E ) Birth curve of motor neurogenesis following pulsed or sustained ectopic TN mutant Nkx2-2 expression. ( F ) Flow cytometry analysis of iNkx2-2 TNmut motor neurons and control cultures that are born after 24 h of DAPT exposure (BrdU + and ISL1 + ). From left to right : (**) P = 0.015, (*) P = 0.482, (***) P = 2.75 × 10 −5 for pulsed, (***) P = 4.88 × 10 −4 , (*) P = 0.275, (***) P = 1.13 × 10 −5 for sustained. Two-sided t -test; n = 3.

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: Immunolabeling, Flow Cytometry, Mutagenesis, Expressing, Control

Journal: iScience

Article Title: The Drosophila Him gene is essential for adult muscle function and muscle stem cell maintenance

doi: 10.1016/j.isci.2026.114670

Figure Lengend Snippet: HimGFP design and expression in leg disc myoblasts and adult MuSCs (A) Schematic of the HimGFP “minigene” comprising 3.8 kb upstream genomic sequence, mGFP fused to the Him coding sequence via an -SSSS- linker, followed by the Him 3′ UTR. (B) Larval T2 leg discs labeled with anti-Mef2 and anti-GFP show co-expression of HimGFP and Mef2 (examples arrowed) in the associated myoblasts ( n = 4). Scale bars, 100 μm. (C) Sagittal thoracic section showing an adult MuSC (arrows) on the periphery of a DLM fiber (boxed area shown at higher magnification). The MuSC expresses HimGFP and canonical marker Zfh1, and stains with nuclear marker Hoechst ( n = 5). Scale bars, 100 μm. (D) Close-up of a sagittal thoracic section showing adult MuSCs (arrowed) on the periphery of a DLM fiber that express both HimGFP and Mef2 ( n = 3). Scale bars, 20 μm.

Article Snippet: Rabbit anti-Mef2 (1:1,000) , DSHB , Mef2.

Techniques: Expressing, Sequencing, Labeling, Marker

Journal: iScience

Article Title: The Drosophila Him gene is essential for adult muscle function and muscle stem cell maintenance

doi: 10.1016/j.isci.2026.114670

Figure Lengend Snippet: HimGFP design and expression in leg disc myoblasts and adult MuSCs (A) Schematic of the HimGFP “minigene” comprising 3.8 kb upstream genomic sequence, mGFP fused to the Him coding sequence via an -SSSS- linker, followed by the Him 3′ UTR. (B) Larval T2 leg discs labeled with anti-Mef2 and anti-GFP show co-expression of HimGFP and Mef2 (examples arrowed) in the associated myoblasts ( n = 4). Scale bars, 100 μm. (C) Sagittal thoracic section showing an adult MuSC (arrows) on the periphery of a DLM fiber (boxed area shown at higher magnification). The MuSC expresses HimGFP and canonical marker Zfh1, and stains with nuclear marker Hoechst ( n = 5). Scale bars, 100 μm. (D) Close-up of a sagittal thoracic section showing adult MuSCs (arrowed) on the periphery of a DLM fiber that express both HimGFP and Mef2 ( n = 3). Scale bars, 20 μm.

Article Snippet: Rabbit anti-Mef2 (1:1,000) , DSHB , Mef2.

Techniques: Expressing, Sequencing, Labeling, Marker

Journal: iScience

Article Title: The Drosophila Him gene is essential for adult muscle function and muscle stem cell maintenance

doi: 10.1016/j.isci.2026.114670

Figure Lengend Snippet: Him is required for normal jump muscle morphology and function, and genetically interacts with Mef2 (A–H) Transverse cryosections of adult jump muscle labeled with anti-βPSintegrin (green) and Hoechst nuclear stain (blue). (A) Control ( yw;;nos-Cas9 ) jump muscle section ( n = 12) consists of two organized rows of muscle fibers arranged around a mid-line (white arrow). The WT morphology is disrupted in 100% of (B) Him 0 mutant ( n = 12), (C) Him 52 mutant ( n = 7), and (D) Him 0 /Him 52 heteroallelic mutant flies ( n = 7). Him mutants have “internal fibers” that do not contact the outer edge of the jump muscle (orange arrow in B). Both the HimGFP minigene and the Him duplication restore WT morphology in Him 0 (E,G) and Him 52 (F,H) mutants ( n = 8–11 per genotype). Scale bars, 50 μm. (I) Bar graph showing mean internal muscle fiber counts ±SEM of the genotypes in (A–H). Him mutants have significantly more internal fibers than WT. Both Him GFP and the Him duplication restore counts to WT values. (Kruskal-Wallis followed by post hoc Dunn’s test: p < 0.001, control versus Him 52 ∗∗∗ p < 0.001, control versus Him 0 ∗∗∗ p < 0.001, control versus Him 52 / Him 0 ∗∗∗ p < 0.001, control versus Him 52 ;; Him GFP p = 0.5985 (n.s.), control versus Him 52 ; ; Him Dup p = 0.2506 (n.s.), control versus Him 0 ;; Him GFP p = 0.2784 (n.s.), control versus Him 0 ;; Him Dup n = 0.2506 (n.s.). (J–L) Transverse cryosections of (J) control ( yw;;nos-Cas9 n = 11), (K) Him 0 mutant ( n = 6) and (L) Him 0 ; Mef2 22.21 /+ flies ( n = 11). Scale bars, 50 μm. The Mef2 22.21 allele rescues the Him mutant morphology toward WT. (M) Bar graph showing mean internal muscle fiber counts ±SEM of the genotypes in (K–M). Him 0 ;Mef2 22.21 /+ flies have significantly fewer internal fibers than Him 0 mutants, but more internal fibers than control flies (Kruskal-Wallis followed by post hoc Dunn’s test: p < 0.001, control versus Him0 ∗∗∗ p < 0.001, control versus Him0;Mef2 22.21 /+ ∗∗ p = 0.0012, Him 0 versus Him0;Mef2 22.21 /+ ∗ p = 0.0363). (N) Bar graph showing mean horizontal jump ±SEM, in response to a looming stimulus ( n = 15–20 flies per genotype). Him 0 and Him 52 adults jump significantly less distance than yw;;nosCas9 controls. HimGFP rescues each Him mutant to WT phenotype. (ANOVA followed by post hoc Dunnett’s test: ANOVA p < 0.0001, control versus Him 0 ∗ p = 0.0151, control versus Him 0 ;; Him GFP/+ non-significant (n.s.) p = 0.7185, control versus Him 52 ∗∗∗ p = 0.0001, control versus Him 52 ; Him GFP/+ n.s. p > 0.9999).

Article Snippet: Rabbit anti-Mef2 (1:1,000) , DSHB , Mef2.

Techniques: Labeling, Staining, Control, Mutagenesis

Journal: iScience

Article Title: The Drosophila Him gene is essential for adult muscle function and muscle stem cell maintenance

doi: 10.1016/j.isci.2026.114670

Figure Lengend Snippet: Him mutants have a reduced wing disc-associated myoblast pool (A–C) L3 wing imaginal discs were stained for Mhc (green), and F-actin (red). Premature muscle differentiation is not observed in (A) control ( w 1118 ) discs ( n = 17), or (B) Him 0 mutants ( n = 20). (C) 1,151-Gal4-driven UAS-Mef2 was used as a positive control ( n = 7). Scale bars, 50 μm. (D–F) L3 larval wing imaginal discs were stained for Ebd1 (green), which labels all myoblasts, and phospho-histone H3 (PH3, red), which labels mitotic cells. Scale bars, 50 μm. The myoblast pool size (outlined) was reduced in (E) Him 0 mutants ( n = 8) compared to (D) controls ( n = 9), and restored (F) when mutants were combined with Dp(1;3)DC343 ( n = 6). (G) Quantification of myoblast number for each genotype. (ANOVA followed by post hoc Dunnett’s test: ANOVA p = 0.0067, control versus Him 0 ∗∗ p = 0.0064, control versus rescue n.s. p = 0.9728). (H) Quantification of proliferating myoblasts shows the fraction of phospho-histone H3 (PH3) expressing cells was reduced in Him 0 mutants compared to controls, and restored when mutants were combined with Dp(1;3)DC343 . (ANOVA followed by post hoc Dunnett’s test: ANOVA p = 0.0022, versus control versus Him 0 ∗∗ p = 0.002, control versus rescue n.s. p = 0.9064).

Article Snippet: Rabbit anti-Mef2 (1:1,000) , DSHB , Mef2.

Techniques: Staining, Control, Positive Control, Expressing