The Hedgehog (HH) pathway regulates a spectrum of developmental processes through the transcriptional mediation of GLI proteins. GLI repressors control tissue patterning by preventing sub-threshold activation of HH target genes, presumably even before HH induction, while lack of GLI repression activates most targets. Despite GLI repression being central to HH regulation, it is unknown when it first becomes established in HH-responsive tissues. Here, we investigate whether GLI3 prevents precocious gene expression during limb development. Contrary to current dogma, we find that GLI3 is inert prior to HH signaling. While GLI3 binds to most targets, loss of Gli3 does not increase target gene expression, enhancer acetylation or accessibility, as it does post-HH signaling. Furthermore, GLI repression is established independently of HH signaling, but after its onset. Collectively, these surprising results challenge current GLI pre-patterning models and demonstrate that GLI repression is not a default state for the HH pathway.

While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. These methods are applied to analysis of a pre-synaptic marker, bassoon, with two different post-synaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Using gephyrin as an inhibitory, post-synaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental nucleus (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation.

In the absence of Hedgehog (HH) signaling, GLI proteins are post-translationally modified within cilia into transcriptional repressors that subsequently prevent sub-threshold activation of HH target genes. GLI repression is presumably important for preventing precocious expression of target genes before the onset of HH pathway activation, a presumption that underlies the pre-patterning model of anterior-posterior limb polarity. Here, we report that GLI3 repressor is abundant and binds to target genes in early limb development. However, contrary to expectations, GLI3 repression neither regulates the activity of GLI enhancers nor expression of HH target genes as it does after HH signaling has been established. Within the cilia, the transition to active GLI repression is accompanied by increases in axonemal GLI3 localization, possibly signifying altered GLI3 processing. Together, our results demonstrate that GLI3 repression does not prevent precocious activation of HH target genes, or have a pre-patterning role in regulating anterior-posterior limb polarity.

The Hedgehog (HH) signaling pathway is primarily modulated by GLI transcriptional repression in the mouse limb. Previous studies have suggested a role for the BAF chromatin remodeling complex in mediating GLI repression. Consistent with this possibility, the core BAF complex protein SMARCC1 is present at most active limb enhancers including the majority of GLI enhancers. Despite this, we find that SMARCC1 maintains chromatin accessibility at GLI enhancers suggesting that it contributes to enhancer activation rather than helping to mediate GLI repression. Furthermore, SMARCC1 binds GLI-regulated enhancers independently of GLI3 and does not facilitate transcriptional repression of most GLI target genes. Finally, Smarcc1- and Shh- double knockout phenotypes suggest that SMARCC1 is not required to mediate constitutive GLI repression in HH mutants. We conclude that the BAF complex does not mediate GLI3 repression, which we propose instead utilizes alternative chromatin remodeling complexes.