Loss of Baz1b in mice causes perinatal lethality, growth failure, and variable multi-system outcomes.

BAZ1B is one of 25-27 coding genes deleted in canonical Williams syndrome, a multi-system disorder causing slow growth, vascular stenosis, and gastrointestinal complaints, including constipation. BAZ1B is involved in (among other processes) chromatin organization, DNA damage repair, and mitosis, suggesting reduced BAZ1B may contribute to Williams syndrome symptoms. In mice, loss of Baz1b causes early neonatal death. 89.6% of Baz1b-/- mice die within 24 h of birth without vascular anomalies or congenital heart disease (except for patent ductus arteriosus). Some (<50%) Baz1b-/- were noted to have prolonged neonatal cyanosis, patent ductus arteriosus, or reduced lung aeration, and none developed a milk spot. Meanwhile, 35.5% of Baz1b+/- mice die over the first three weeks after birth. Surviving Baz1b heterozygotes grow slowly (with variable severity). 66.7% of Baz1b+/- mice develop bowel dilation, compared to 37.8% of wild-type mice, but small bowel and colon transit studies were normal. Additionally, enteric neuron density appeared normal in Baz1b-/- mice except in distal colon myenteric plexus, where neuron density was modestly elevated. Combined with several rare phenotypes (agnathia, microphthalmia, bowel dilation) recovered, our work confirms the importance of BAZ1B in survival and growth and suggests that reduced copy number of BAZ1B may contribute to the variability in Williams syndrome phenotypes.

Baz1b Dosage Influences Cardiovascular Function, Predisposing to Dilated Cardiomyopathy.

BAZ1B is one of several genes deleted in Williams-Beuren Syndrome (WBS), a complex, multisystem genetic condition that occurs in ~1 in 8000 live births. Also known as Williams Syndrome Transcription Factor (WSTF), BAZ1B is thought to be essential for neural crest migration. To evaluate the impact of Baz1b loss of function, we evaluated the "knockout first" allele of Baz1btm2a(KOMP)Wtsi . Quantitative PCR revealed markedly reduced, but not absent, expression of Baz1b, suggesting that Baz1btm2a(KOMP)Wtsi mutants are knockdowns rather than knockouts. Homozygous Baz1btm2a(KOMP)Wtsi mutant mice die just hours after birth, and both homozygous mutants and heterozygotes are smaller than age-matched wildtype littermates. Survival analyses conducted on 388 Baz1btm2a(KOMP)Wtsi mice revealed that heterozygotes and homozygous mutants are approximately three and sixteen times more likely to die than wildtype mice, respectively [hazard ratio for death in Baz1b+/- : 3.04 (95% CI, 1.83-5.06), p<0.0001; hazard ratio for death in Baz1b-/- : 15.83 (95% CI, 8.54-29.37); p<0.0001]. Furthermore, a linear mixed effects model for the weights of wildtype and heterozygous mice over a 29-day period showed a significant difference in size based on genotype (mean: WT 7.97 g, Baz1b+/- 6.56 g, p<0.0001). Because neural crest lineages contribute to cardiac development, structure, and function, we hypothesized that early sudden death and failure to thrive in mutant mice may be at least partially attributable to cardiac abnormalities. To evaluate any morphologic and functional abnormalities, we performed microCT and echocardiography. MicroCT analysis of the hearts from P0 pups did not reveal congenital heart disease typical of neural crest defects (e.g. tetralogy of Fallot, truncus arteriosus, double outlet right ventricle, or interrupted aortic arch). Echocardiograms, performed at 1-month to align with the growth analysis timeline, revealed mildly decreased ejection fraction (EF, median: WT 64%, Baz1b+/- 56%, p<0.01) and fractional shortening (FS, median: WT 34%, Baz1b+/- 29%, p<0.01), increased left ventricular internal dimension at diastole (LViDd) normalized to animal size (median: WT 0.22 mm/g, Baz1b+/- 0.27 mm/g, p<0.05), and unchanged left ventricular posterior wall dimension at diastole (LVPWd) normalized to body size (median: WT 0.041 mm/g, Baz1b+/- 0.048 mm/g, p=0.19) in Baz1b+/- when compared to wildtype. However, Baz1b+/- LVPWd is significantly smaller than WT when body size is not considered (median: WT 0.63 mm, Baz1b+/- 0.62 mm, p<0.01), suggesting a relationship between cardiac function and mutant animal growth (all tests for genotype in n=14 WT and n=14 Baz1b+/- by Mann-Whitney U Test). Taken together, our data suggest that Baz1b+/- mice exhibit a dilated cardiomyopathy and that dosage for this gene may contribute to early death, decreased somatic growth, and cardiac abnormalities in Baz1b mutant mice. Additional analyses in older mice and with mutants generated using the conditional Baz1btm2a(KOMP)Wtsi allele will allow us to better explore the mechanisms of both the growth failure and cardiomyopathy phenotypes in this model.

Sequencing Reveals miRNAs Enriched in the Developing Mouse Enteric Nervous System.

The enteric nervous system (ENS) is an essential network of neurons and glia in the bowel wall. Defects in ENS development can result in Hirschsprung disease (HSCR), a life-threatening condition characterized by severe constipation, abdominal distention, bilious vomiting, and failure to thrive. A growing body of literature connects HSCR to alterations in miRNA expression, but there are limited data on the normal miRNA landscape in the developing ENS. We sequenced small RNAs (smRNA-seq) and messenger RNAs (mRNA-seq) from ENS precursor cells of mid-gestation Ednrb-EGFP mice and compared them to aggregated RNA from all other cells in the developing bowel. Our smRNA-seq results identified 73 miRNAs that were significantly enriched and highly expressed in the developing ENS, with miR-9, miR-27b, miR-124, miR-137, and miR-488 as our top 5 miRNAs that are conserved in humans. However, contrary to prior reports, our follow-up analyses of miR-137 showed that loss of Mir137 in Nestin-cre, Wnt1-cre, Sox10-cre, or Baf53b-cre lineage cells had no effect on mouse survival or ENS development. Our data provide important context for future studies of miRNAs in HSCR and other ENS diseases and highlight open questions about facility-specific factors in development.