INTRODUCTION
Holoprosencephaly (HPE) is understood to be a failure in the generation
of midline signals that normally instruct eye field and forebrain
division (Roessler et al., 1996; Belloni et al., 1996; Chiang et al.,
1996; Abramyan 2019). Five principal genes: SHH, ZIC2, SIX3 ,FGF8 and FGFR1 , are the most commonly detected genetic
factors responsible for HPE (Roessler et al., 2018b). While de
novo FGFR1 mutations match a specific syndromic set of brain and
limb findings, called Hartsfield syndrome (Simonis et al., 2013; Hong et
al., 2016), mutations in the first four HPE genes fit into a more
typical non-syndromic pattern in probands with a normal chromosome
analysis and microarray (Brown et al., 1998; Wallis et al., 1999; Hong
et al., 2018; Roessler and Muenke, 2010). Each of these five driver
genes is required in a conserved developmental program during
gastrulation for midline specification. The roles of several infrequent
minor genes, e.g. PTCH1, DISP1, GAS1, TGIF1, DLL1 , etc. are less
well understood (Roessler et al., 2018a). However, a common pathogenetic
HPE mechanism leading to diminished midline hedgehog signaling links the
major driver mutations into a conserved gene regulatory network (GRN,
Suppl. Fig.1).
Hedgehog (Hh) proteins secreted from the vertebrate prechordal plate and
ventral diencephalic midline are lipid-modified morphogens that play a
central role in patterning the early forebrain primordium (Ohkubo et
al., 2002; Cooper et al., 2003; Wilson and Houart, 2004; Bertrand and
Dahmane, 2006; Storm et al., 2006; Blassberg et al., 2016). Shh
functions by binding and inactivating Patched (e.g. human PTCH1 ,PTCH2 ), the major sterol-sensing and sterol-transporter related
receptor(s) for Hh ligands, which constitutively and
non-stoichiometrically suppress Smo activity by depriving this GPCR
receptor of cholesterol and/or related sterol ligands needed to
stabilize its active conformation (Taipale et al., 2002; Ingham et al.,
2011; Briscoe and Therond, 2013; Luchetti et al., 2016; Huang et al.,
2018; Hu and Song, 2019; Deshpande et al., 2019). SMO activates a
classical signal cascade that ultimately regulates the
post-translational state of Gli family transcription factors, which in
turn bind enhancers of target genes (Huangfu and Anderson, 2006). Hh
morphogen binding to Patched decreases its presence in the cilium
leading to its internalization and degradation. Activation of Smo in
vertebrates is correlated with its enrichment in the cilium (Huangfu and
Anderson, 2005; Corbit et al., 2005; Gigante et al., 2018) as well as
ultimately altering the processing of Gli transcription factors from
truncated repressors to full-length activators. The role of
non-classical GPCR signaling via SMO in human pathologies is less
clearly understood (Arensdorf et al., 2016; Qi et al., 2019). However,
the heptahelical domain is the frequent target for recurrent activating
mutations in cancer and somatic tumors (Xie et al., 1998; Lam et al.,
1999; Taipale et al., 2000; Ayers and Therond, 2010; Twigg et al.,
2016), while variation within the remaining domains characteristic of
the GPCR superfamily are less frequently observed (see Figure 1).
Mutagenesis screens in Drosophila and zebrafish have clearly
demonstrated that there is a functional consequence to variation in, and
cooperative interactions between, all of these domains (Nakano et al.,
2004; Zhao et al., 2007; Aanstad et al., 2009; Nachtergaele et al.,
2013). Furthermore, recent structural studies of various vertebrate Smo
molecules emphasize that both agonist and antagonist binding can alter
the three-dimensional structure(s) of Smo orthologs and shift the
orientation of all constituent domains (Byrne et al., 2016; Luchetti et
al., 2016; Huang et al., 2016; Huang et al., 2018; Byrne et al., 2018;
Zhang et al., 2018). Therefore, for completeness we chose to analyze all
variants detected in our series of HPE probands.
SHH signaling regulates the transcription of genes involved in
the specification of the ventral forebrain primordium, the resolution of
a primordial single eye field into paired optic vesicles, as well as
optic nerve and retinal development (Li et al., 1997; England et al.,
2006; Sanek et al., 2009; Xavier et al., 2016). This early function ofSHH in the splitting of the eye field into two lateral optic
primordia followed by induction of optic stalk tissue at the expense of
neural retina is key to our understanding of HPE and the dramatic
cyclopic phenotypes seen in both animal models and humans (Chiang et
al., 1996; Hammerschmidt et al., 1996; Hammerschmidt and McMahon, 1998;
Vinothkumar et al., 2008; Pillai-Kastoori et al., 2015). Our
understanding of these morphological and gene expression changes
influenced our approach to SMO bioassay development.
Here we report our functional testing in zebrafish of seven
non-synonymous variants of human SMO derived from HPE molecular
genomic and clinical data. Our studies reveal some of the inherent
difficulties of in vivo bioassay systems that depend on a lag in
experimental intervention (e.g. injection of the SMO gene) and
measurements of biological responses (e.g. marker gene changes, or
morphogenetic phenotypes). Given the complex GRN involved in hedgehog
signaling and its feedback wiring we now demonstrate that investigators
need to incorporate a clear understanding of these regulatory
relationships in assay development as well as in medical genetic
interpretation.