In situ hybridization for Sizn1 at E9.5, E12.5 and E14.5 whole mouse embryos. In E9.5 stage, Sizn1 is expressed in dorsal midline of the forebrain (A, B), the ventral rhombencephalon (A, C) and in primodial germ cells (A, D). In E12.5 stage, the expression of Sizn1 is now restricted to the ventral neural tube from the telencephalon through the rhombencephalon (E, lateral view and F, medial view). Expression is found in the lateral olfactory tract (LOT), pallium-subpallium boundary (PSB), amygdala, septum and zona limitans intrathalamica (ZLI). (G) The ZLI expression was confirmed by comparison with Shh-in situ hybridization, which is known to be expressed in the ZLI (Puelles and Rubenstein, 2003). While the pattern of E14.5 is generally similar to that at E12.5, there has been considerable expansion of the expression domains. In a mid coronal section of the whole brain (E14.5) shows strong expression in the amygdala, hypothalamus, ventral thalamus and striatum (H). A lateral (I) and medial (J) mid-sagittal view also shows extensive expression nearly circumferentially in the mesencephalon and rhombencephalon. Am=amygdala, d=diencepahlon, Hy=hypothalamus, m=mesencephalon, Pg=primodial germ cell, r=rhombencephalon, Sp=Septum, St=striatum and VT=ventral thalamus. Scale bar is adjusted to 500 µm in each image.

Sizn1 is expressed by Cajal-Retzius cell in the cerebral cortical molecular layer at E14.5 (A). Immunofluorescence for Reln, a marker of early Cajal-Retzius cells, decorates a row of cells in the molecular layer. Labeling with an anti-Sizn1 antibody shows a similar layer of cells and merging of these labels shows complete overlap. DAPI labeling highlights the tissue structure (scale bar = 20 µm). (B and C) Sizn1 is also co-labeled with Reln in the LOT and amygdala. Arrows and arrowhead highlight examples of co-labeled and non co-labeled cell, respectively.

Immunohistochemistry for Sizn1 at E13.5, E14.5, E15.5 and adult brains. Top left panel is low magnification image of A–F (Scale bar is 500 µm). (A) Sizn1 expression is found in the molecular layer of the developing cortical plate corresponding to Cajal-Reitzius cells (arrows and see Figure 3). (B) Scattered labeled cells are found at the pallial-subpallial boundary (PSB, arrows). (C) Scattered Sizn1 labeled cells are present in the cortical hem (arrows). Numerous labeled cells are found in the developing septal region (D) and striatum (F). (E) Labeled cells in the medial ganglionic eminence (MGE, arrows) are primarily found in the ventricular zone. Scale bar in image F is 100 µm and corresponded to images A–F. Schematic diagrams of E13.5 brain coronal section (G; Scale bar=500 µm)) and of E14.5 sagittal section (I; Scale bar=100µm) for Sizn1 expression pattern. (H) Coronal section of E15.5 embryonic caudal forebrain. Anterior (an) and posterior (po) DAPI labeled sections on the left and Sizn1 immunofluorescence on the right (green). Sizn1 expression is localized in the amygdala, habenula, hippocampus, hypothalamus, pallium-subpallium boundary and zona limitans intrathalamica (scale bar = 500 µm). (J and K) Immunostaining for Sizn1 in the adult brain with emphasis on the septal region (Scale bar=500 µm (I) and 100 µm (J)). Am=amygdala, Ha=habenula, Hip=hippocampus, Hy=hypothalamus, Sp=Septum, St=striatum, LOT=lateral olfactory tract, GE=gangilonic eminence, PSB=pallium-subpallium boundary and ZLI= zona limitans intrathalamica.

Sizn1 is expressed in neurons. Sizn1-expressing cell also express NeuN (scale bar = 20 µm).