Mills A et al. (2019), Wolf-Hirschhorn Syndrome-Associated Genes Are E...

XB-ART-55919

Front Physiol January 1, 2019; 10 431.

Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis.

Mills A , Bearce E , Cella R , Kim SW , Selig M , Lee S , Lowery LA .

Abstract
Wolf-Hirschhorn Syndrome (WHS) is a human developmental disorder arising from a hemizygous perturbation, typically a microdeletion, on the short arm of chromosome four. In addition to pronounced intellectual disability, seizures, and delayed growth, WHS presents with a characteristic facial dysmorphism and varying prevalence of microcephaly, micrognathia, cartilage malformation in the ear and nose, and facial asymmetries. These affected craniofacial tissues all derive from a shared embryonic precursor, the cranial neural crest (CNC), inviting the hypothesis that one or more WHS-affected genes may be critical regulators of neural crest development or migration. To explore this, we characterized expression of multiple genes within or immediately proximal to defined WHS critical regions, across the span of craniofacial development in the vertebrate model system Xenopus laevis. This subset of genes, whsc1, whsc2, letm1, and tacc3, are diverse in their currently-elucidated cellular functions; yet we find that their expression demonstrates shared tissue-specific enrichment within the anterior neural tube, migratory neural crest, and later craniofacial structures. We examine the ramifications of this by characterizing craniofacial development and neural crest migration following individual gene depletion. We observe that several WHS-associated genes significantly impact facial patterning, cartilage formation, neural crest motility in vivo and in vitro, and can separately contribute to forebrain scaling. Thus, we have determined that numerous genes within and surrounding the defined WHS critical regions potently impact craniofacial patterning, suggesting their role in WHS presentation may stem from essential functions during neural crest-derived tissue formation.

PubMed ID: 31031646
Article link: Front Physiol
Grant support: [+]
Genes referenced: fn1 letm1 tacc3 twist1
GO keywords: neural crest cell migration [+]
Morpholinos: letm1 tacc3 MO2

Disease Ontology terms: Wolf-Hirschhorn syndrome [+]

Article Images: [+] show captions

	Figure 1. WHS is typically caused by heterozygous microdeletion of numerous genes within 4p16.3. A segment of this region is illustrated here. A microdeletion that spans at least WHSC1, WHSC2, and LETM1 is currently assumed to be necessary for full WHS diagnostic presentation; children affected by the disorder often possess larger deletions that extend further telomeric and impact additional genes, such as TACC3.
	Figure 2. WHS related genes are expressed in the migrating neural crest cells during embryonic development. (A,F) Lateral views of whole mount in situ hybridizations for twist, a CNC-enriched transcription factor. Arrows indicate the pharyngeal arches (PA). (B–E,G–J) In situ hybridizations for whsc1, whsc2, letm1, and tacc3 demonstrate enrichment in CNCs that occupy the PAs (n = 20 per probe, per timepoint). Scalebar is 250 μm.





	Figure 8. Partial depletion of WHS-affected genes demonstrates numerous impacts on craniofacial development and neural crest migration. Tissues are denoted as affected (checked box) if phenotypes were significantly different from control (p ≤ 0.05); see individual figures for data distribution and statistics. (Abbreviations: PA, Pharyngeal Arch) ∗Denotes pre-migratory CNC (st. 16).
	FIGURE S1 \| Expression patterns for WHS related genes across early development. In situ hybridization utilized (A–F) antisense mRNA probe to whsc1, (G–L) full-length antisense mRNA probe to whsc2, (M–R) full-length antisense mRNA probe to letm1, and (S-X) 1 kb partial-length antisense mRNA probe to tacc3. Embryos shown at blastula stage (A,G,M,S), in dorsal view at stage 16–20 (B,H,N,T), in lateral view at stage 20–25 (C,I,O,U), detail of lateral anterior region at stage 35 (D,J,P,V), and in both lateral and dorsal views from stages 39–42 (E,K,Q,W and F,L,R,X). (Y) In situ hybridization probes generated against sense strands of WHS gene mRNAs, shown at stage 25. Brown coloration (S,T) is unbleached pigment, unrelated to in situ hybridization staining. Scalebar is 250 μm.bryos were anesthetized with benzocaine.
	FIGURE S2 \| Validation of WHS related MOs. (A,B) Gel of polymerase chain reaction (PCR) that shows injection of 10 ng MO targeted to whsc1 mRNA causes a greater than 80% reduction at 2 dpf. (C,D) Injection of 20 ng of a MO targeted against letm1 causes an 55% decrease in letm1 mRNA 2 dpf. (E,F) Western blot showing 10 ng injection of a MO targeted against whsc2 results in a greater than 50% reduction in Whsc2 protein by 2 dpf. (G,H) Western blot showing 40 ng of a MO targeted against tacc3 results in 22% reduction. Bar graphs (B,D,F,H) depict densitometry of gels (A,C) or blot (E,G) shown, but is consistent across triplicate results.
	FIGURE S3 \| Demonstration of measurement schemes for craniofacial morphology. Measurements for Figure 3 and Supplementary Figure S4 were performed as indicated. (A) Facial width was measured from the center of each eye. (B) Facial height was determined at the midline, from the top of the cement gland to the top of the eyes. (C) Midface angle was measured from the top of the cement gland to the center of each eye. (D) Facial area was measured as the space encapsulated within the perimeter of each eye. Adapted from Kennedy and Dickinson (2014).

	FIGURE S5 \| Half embryo knockdown can be utilized for analysis of brain morphology and neural crest cell migration in vivo. (A) At the 2-cell stage, a single blastomere is injected with WHS-associated gene MOs and exogenous eGFP mRNA. After neurulation (approx. stage 21), embryos are sorted based on left or right eGFP fluorescence, to determine side of depletion. To examine neural crest cell size, migration and morphology embryos were fixed between stage 25–30, and in situ hybridization was performed using twist anti-sense probe. To characterize brain morphology, embryos were raised to st. 47, and fixed and labeled with alpha-tubulin antibody, a neuronal marker, and Alexa-488 secondary. (B–D) Control MO does not significantly impact brain size, compared to non-injected hemispheres (a paired internal control).

References [+] :

Adelman, Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. 2012, Pubmed

	Figure 1. WHS is typically caused by heterozygous microdeletion of numerous genes within 4p16.3. A segment of this region is illustrated here. A microdeletion that spans at least WHSC1, WHSC2, and LETM1 is currently assumed to be necessary for full WHS diagnostic presentation; children affected by the disorder often possess larger deletions that extend further telomeric and impact additional genes, such as TACC3.
	Figure 2. WHS related genes are expressed in the migrating neural crest cells during embryonic development. (A,F) Lateral views of whole mount in situ hybridizations for twist, a CNC-enriched transcription factor. Arrows indicate the pharyngeal arches (PA). (B–E,G–J) In situ hybridizations for whsc1, whsc2, letm1, and tacc3 demonstrate enrichment in CNCs that occupy the PAs (n = 20 per probe, per timepoint). Scalebar is 250 μm.





	Figure 8. Partial depletion of WHS-affected genes demonstrates numerous impacts on craniofacial development and neural crest migration. Tissues are denoted as affected (checked box) if phenotypes were significantly different from control (p ≤ 0.05); see individual figures for data distribution and statistics. (Abbreviations: PA, Pharyngeal Arch) ∗Denotes pre-migratory CNC (st. 16).
	FIGURE S1 \| Expression patterns for WHS related genes across early development. In situ hybridization utilized (A–F) antisense mRNA probe to whsc1, (G–L) full-length antisense mRNA probe to whsc2, (M–R) full-length antisense mRNA probe to letm1, and (S-X) 1 kb partial-length antisense mRNA probe to tacc3. Embryos shown at blastula stage (A,G,M,S), in dorsal view at stage 16–20 (B,H,N,T), in lateral view at stage 20–25 (C,I,O,U), detail of lateral anterior region at stage 35 (D,J,P,V), and in both lateral and dorsal views from stages 39–42 (E,K,Q,W and F,L,R,X). (Y) In situ hybridization probes generated against sense strands of WHS gene mRNAs, shown at stage 25. Brown coloration (S,T) is unbleached pigment, unrelated to in situ hybridization staining. Scalebar is 250 μm.bryos were anesthetized with benzocaine.
	FIGURE S2 \| Validation of WHS related MOs. (A,B) Gel of polymerase chain reaction (PCR) that shows injection of 10 ng MO targeted to whsc1 mRNA causes a greater than 80% reduction at 2 dpf. (C,D) Injection of 20 ng of a MO targeted against letm1 causes an 55% decrease in letm1 mRNA 2 dpf. (E,F) Western blot showing 10 ng injection of a MO targeted against whsc2 results in a greater than 50% reduction in Whsc2 protein by 2 dpf. (G,H) Western blot showing 40 ng of a MO targeted against tacc3 results in 22% reduction. Bar graphs (B,D,F,H) depict densitometry of gels (A,C) or blot (E,G) shown, but is consistent across triplicate results.
	FIGURE S3 \| Demonstration of measurement schemes for craniofacial morphology. Measurements for Figure 3 and Supplementary Figure S4 were performed as indicated. (A) Facial width was measured from the center of each eye. (B) Facial height was determined at the midline, from the top of the cement gland to the top of the eyes. (C) Midface angle was measured from the top of the cement gland to the center of each eye. (D) Facial area was measured as the space encapsulated within the perimeter of each eye. Adapted from Kennedy and Dickinson (2014).

	FIGURE S5 \| Half embryo knockdown can be utilized for analysis of brain morphology and neural crest cell migration in vivo. (A) At the 2-cell stage, a single blastomere is injected with WHS-associated gene MOs and exogenous eGFP mRNA. After neurulation (approx. stage 21), embryos are sorted based on left or right eGFP fluorescence, to determine side of depletion. To examine neural crest cell size, migration and morphology embryos were fixed between stage 25–30, and in situ hybridization was performed using twist anti-sense probe. To characterize brain morphology, embryos were raised to st. 47, and fixed and labeled with alpha-tubulin antibody, a neuronal marker, and Alexa-488 secondary. (B–D) Control MO does not significantly impact brain size, compared to non-injected hemispheres (a paired internal control).