Fig. 6: Slit2 and Ntn1 are essential for proper ventricular septation and compartment boundary regulation.
From: A disrupted compartment boundary underlies abnormal cardiac patterning and congenital heart defects
a–e, Machine learning-based 3-D reconstructions of the embryonic heart and other tissues from histology at E14.5 were used for quantitative morphometric analysis of the embryonic heart (Extended Data Figs. 9 and 10). Compact myo, compact myocardium. d,e, A 3-D reconstruction of a membranous VSD (d) anteriorly and muscular VSD (e) posteriorly from a Tbx5del/+ mutant heart. f–i, Histology (f–h) and incidence (i) of muscular or membranous (arrowhead) VSDs of Slit2 mutants are shown. Membranous VSDs were enriched in Slit2−/− (n = 3/4, log(OR) = 5.0, P = 0.02 by a generalized linear model without adjustments for multiple comparisons) and Slit2+/− (n = 1/8, OR = 2.5, P = 0.02). The asterisk demarcates non-compacted IVS. j–m, Histology (j–l) and incidence (m) of muscular or membranous VSDs of Ntn1 mutants are shown. Membranous VSDs were enriched in Ntn1−/− (n = 6/7, log(OR) = 6.32, P = 0.0052 by a generalized linear model without adjustments for multiple comparisons) and Ntn1+/− (n = 1/9, OR = 3.17, P = 0.0052 by a generalized linear model without adjustments for multiple comparisons). n–r, Lightsheet imaging of Slit2−/− mutant (Tbx5CreERT2/+;Mef2cAHF-DreERT2;ROSA26Ai66/+;Slit2−/−) (n = 4) hearts at E14.5 showed a broadened distribution of the Tbx5+/Mef2cAHF+ lineage (tdTomato+ immunostaining) compared with controls (Tbx5CreERT2/+;Mef2cAHF-DreERT2;ROSA26Ai66/+;Slit2+/+) (n = 2) (n–q), as quantified by linear profiles of fluorescence signals (r). Gray shading represents the control signal, and blue represents the mutant signal. The significance of P < 0.05 was determined by Welch’s two-sided t-test at each position along the right–left axis. s–v, Lightsheet imaging of Ntn1−/− mutant (Tbx5CreERT2/+;Mef2cAHF-DreERT2;ROSA26Ai66/+;Ntn1−/−) (n = 6) hearts at E14.5 showed a leftward shift in positioning of the Tbx5+/Mef2cAHF+ lineage compared with controls (Tbx5CreERT2/+;Mef2cAHF-DreERT2;ROSA26Ai66/+;Ntn1+/+) (n = 4) (s–u), as quantified by linear profiles (v). The significance of P < 0.05 was determined by Welch’s two-sided t-test at each position along the right–left axis. Precise P values are available in source data. n,s, Cartoon depictions. Panels o(i), p(i), t(i) and u(i) are optical sections that are orthogonal views to o, p, t and u. q, For each sample, three linear profiles (basal, middle, apical) spanned the ventricular chambers from right to left from optical sections every 50 μm from anterior to posterior of the heart. Precise values of statistics for r and v can be found in source data. w, Cartoon depicting the summary of findings. Scale bars, 500 μm (a, o–u(i)), 200 μm (d–l). Panel w created with BioRender.com.
