Expression patterns of NEUROD1 and miR-19b in the developing chick pallium and their spatial distribution relative to PH3-positive cells.

(A) Screening of NEUROD1 (IHC) and miR-19b (RNA-ISH) expression within chicken pallium at different stages (A) A cross-section of HH24 chicken pallium showing NeuroD1 and miR-19b with a merged image in the inset. Scale bar-500µm, Inset (100µm), n=3 (B) miR-19b and NeuroD1 expression dynamics in wulst (Red inset) and DVR (Yellow inset) region at stage HH29 chicken pallium. Scale bar-500µm, Inset (250µm), n=3 (C) a similar comparison of miR-19b and NeuroD1 expression at stage HH29 chicken pallium. Scale bar - 800µm, inset (250µm), n=3 (D) Distribution of PH3 and NeuroD1-positive cells at all above-mentioned stages domain of PH3 positive cells is marked by green arrows wherein NeuroD1 is represented by red arrows. The intensity plot shows mutually exclusive expression domains of NeuroD1 and miR-19b. Insets depict magnified images. n=3, Scale bar: 800µm. White arrowheads show NeuroD1 positive cells in SVZ. A.U.= arbitrary unit; VZ= ventricular zone, SVZ= sub-ventricular zone.

miR-19b regulates E2f8 expression and cell-cycle progression.

(A) Venn diagram showing miR-19b targets predicted by miRDB and TargetScan in chicken (Gallus gallus) and gene ontology analysis for predicted targets. (B) List of functionally validated targets. The red square indicates the role in the mentioned process/pathway. Potential targets and associated processes highlighted in magenta. (C) Sensor-assay for in vitro demonstration of miR-19b binding to its target region of E2f8. Respective 3’UTRs fused with the coding sequence of mCherry were subjected to miR-19b manipulation. mCherry intensity was further quantified and plotted. Scale bar: 100µm, Plot bar: mean ± s.e.m., N=3 wells, t-test performed, **p≤0.01 and ****p≤0.0001. A.U.= arbitrary unit. (D) represent the conservation of mi19b binding sequences in E2f8 3’ UTR across all species. Effect of miR-19b manipulation on E2f8 (RNA-ISH) (E) and (F), scale bar-300µm. (G) Flow cytometry experiment to analyze cell-cycle distribution of DF1 cells upon manipulation of miR-19b. Go/G1, S and G2/M are presented in population group named as P6, P7 and P8, respectively. Representative images of EdU labeling assay(H) and PH3 immunohistochemistry (I)to demonstrate effect of miR-19b manipulation on cell proliferation. Scale bar: 200µm (H), 50µm (I). n=3, Plot bar: mean ± s.e.m.; A.U.= arbitrary unit. ns= not significant; VZ= ventricular zone, SVZ= sub-ventricular zone. t-test was performed, *p≤0.1, **p≤0.01 and ***p≤0.001, ns = non-significant.

Expression Patterns of Fezf2 and Mef2c in the Embryonic Chick Pallium and the Effects of CDKN1A Overexpression and miR-19b Loss of Function

(A) Expression patterns of Fezf2 and NeuroD1 in the SVZ and Mef2c in the dorsal pallium layer at HH28. The inset highlights double-positive cells for Fezf2 and NeuroD1. Scale bar = 300 µm; 25 µm (inset). n = 3. (B) Fezf2 expression is restricted to the APH, while Mef2c expression extends into the mesopallium (Msp). The inset shows magnified views, and the intensity plot depicts spatial expression profiles of Fezf2 and Mef2c. Scale bar=150µm; n=3; DP= Dorsal Pallium. (C) Effect of ectopic CDKN1A expression on Fezf2(ISH) and NeuroD1(IHC) expression. Electroporation of CDKN1A-overexpressing and control constructs demonstrates altered expression of Fezf2. Magnified regions highlight colocalization of Fezf2 and NeuroD1 (circled regions). Bar graphs display signal intensity quantification; n = 4. Scale bar: 300µm (D) Impact of CDKN1A overexpression on Mef2c expression (IHC). White arrows indicate double- positive cells for Mef2c, and bar graphs quantify their abundance. n=3. (E) Loss of miR-19b affects Fezf2 expression. Signal intensity of Fezf2 per unit electroporated region is quantified and plotted. Bar graph plotted for quantification. n=4, Scale bar: 150µm; Plot bar: mean ± s.e.m. A.U.= arbitrary unit. ns= not significant; VZ= ventricular zone, SVZ= sub-ventricular zone; t-test was performed; *p≤0.1, **p≤0.01 and ***p≤0.001; ns = non-significant.

miR-19b Mediated Regulation of NeuroD1 and Its Impact on Patterning in the Developing Chick Pallium

(A) mRNA in situ hybridization (ISH) screening of miR-19b and NeuroD1 immunostaining in the chick pallium at HH29. The intensity plot highlights mutually exclusive expression domains of NeuroD1 and miR-19b. Insets show magnified views of the expression patterns. Scale bar = 100µm; 30µm (insets), n = 3. (B) Conservation analysis of miR-19b binding sequences in the NeuroD1 3′ UTR across species. (C) Sensor assay demonstrating in vitro binding of miR-19b to the target region of NeuroD1. The 3′ UTR of NeuroD1 was fused with mCherry and subjected to miR-19b manipulation. mCherry intensity was quantified and plotted. Scale bar = 100 µm, with n = 3 wells. A.U. = arbitrary units. (D, E) miR-19b regulates NeuroD1 expression in in vivo as shown by immunostaining in gain-of-function (D) and loss-of-function (E) experiments. Scale bar = 100 µm, n=4. (F) Expression patterns of NeuroD1 and Mef2c in the chick pallium at HH29. Scale bar = 500µm. (G, H) NeuroD1 loss-of-function experiment using NeuroD1-DBD-EnR electroporation shows loss of Mef2c ISH expression. (G) and IHC expression (H). The yellow asterisk (control) and white asterisk (test) mark the Mef2c mRNA expression domains. Scale bar:100µm, n=3 (I-J) NeuroD1 gain-of-function led to ectopic Mef2c mRNA (I) and Proteins (J) shown by ISH and immunostaining (arrowheads show ectopic expression of Mef2c). n=3, scale bar: 100µm. (K) Effect of miR-19b overexpression and Sponge-19b expression on Mef2c expression. Yellow asterisk is showing Mef2c ISH in control and white asterisk show decreased Mef2c expression using ISH; n=3; Scale bar:100µm; Plot bar: mean ± s.e.m. A.U.= arbitrary unit. ns= not significant; VZ= ventricular zone, SVZ= sub-ventricular zone; Msp = Mesopallium; t-test was performed; *p≤0.1, **p≤0.01 and ***p≤0.001; ns = non-significant.