UC Irvine

Approximately 3% of newborns worldwide are affected by birth defects, yet the causes for about 70-80% of these cases remain unidentified, despite various known contributing factors. Cornelia de Lange Syndrome (CdLS), a disorder characterized by congenital malformations across multiple organs and tissues, is caused by mutations in genes associated with the cohesin complex or its interacting proteins. Most CdLS cases are caused by haploinsufficiency for the NIPBL gene, which plays a role in the loading of the cohesin complex onto chromatin. Originally recognized for its critical role in sister chromatid cohesion, the cohesin complex is now known to also play a role in regulating global gene expression. Studies using cell lines derived from CdLS patients and Nipbl-haploinsufficient tissues from animal models indicate that subtle, significant, and widespread changes in gene expression precede birth defects. Given that these changes are generally modest (less than 2-fold), this suggests that the birth defects observed in CdLS and related animal models are likely not caused by alterations in any single gene. Rather, they are probably the result of cumulative changes in the expression of multiple genes. Thus, studies of CdLS and its animal models have shed light on the mechanisms underlying birth defects of unknown causes, that broad shifts in gene expression rather than specific mutations in individual genes may drive the development of birth defects. In the United States, congenital heart defects (CHDs) affect nearly 1% of all newborns. They are also the primary cause of birth defect-related infant mortality and morbidity. The causal origins of CHDs are largely unknown, with mutations in single genes accounting for fewer than 20% of cases. About 30% of individuals with the Nipbl-haploinsufficient form of CdLS present with CHDs. Studies in Nipbl+/- mice, which exhibit a similar prevalence of CHDs, have primarily identified atrial septal defects – among the most common types of CHDs observed in nonsyndromic cases in man. Nipbl+/- mice also display abnormal development in the ventricular septum and right ventricle. Intriguingly, these cardiac structures originate from a progenitor cell population known as the second heart field (SHF), which points to the SHF as a potential source of CHDs in Nipbl+/- mice. In Chapter 2, we explored the impact of Nipbl-haploinsufficiency within the SHF on the development of CHDs using light sheet fluorescence microscopy (LSFM) and two SHF-Cre recombinase-expressing mouse lines: Mef2c-Cre and Isl1 Cre/+. Remarkably, it was found that only Isl1 Cre/+ driven Nipbl-haploinsufficiency in the SHF led to CHDs. This occurrence was likely due to extensive Isl1-Cre recombination in non-cardiac tissues and/or a reduction in Isl1 expression, the latter resulting from knock-in mediated haploinsufficiency. In surprising contrast, mice rendered globally haploinsufficient for both Isl1 and Nipbl (Nipbl+/-; Isl1+/-) exhibited a wider range and higher frequency of CHDs compared to those haploinsufficient for either gene alone. RNA sequencing of E10.5 mouse hearts indicated that Nipbl-haploinsufficiency upregulates Isl1 expression, seemingly providing a protective effect against the development of severe CHDs, as evidenced by fewer and less severe CHDs in Nipbl+/- mice compared to their Nipbl+/-; Isl1+/- counterparts. Furthermore, we observed significant alterations in the expression of homeodomain genes such as Pitx1, Pax6, and Hoxc4. Nipbl+/-; Isl1+/- hearts did not upregulate these genes to the same level as Nipbl+/- hearts. These results introduce new quantitative risk factors for the development of CHDs in both CdLS and nonsyndromic cases. In addition to exhibiting ASDs, Nipbl+/- mice also demonstrate early abnormalities in other cardiac regions during embryonic development. By embryonic day 13.5 (E13.5), 70% of Nipbl+/- mice displayed delays in ventricular septal fusion, while three days earlier, at E10.5, all exhibited right ventricle hypoplasia. Even earlier, at E7.75 and E7.5, in situ hybridization experiments revealed diminished expression of two key transcription factors, Nkx2-5 and Mesp1, which play roles in the growth and differentiation of early heart progenitor cells. These suggests that cardiac development abnormalities might originate at the very onset of heart formation. Supporting this hypothesis, studies on nipbl-morphant zebrafish have shown that defects in the initial migration of cardiogenic mesoderm can be detected as early as 18 hours post-fertilization. These findings imply that, at least for CHDs, causative events may occur as early as gastrulation – the stage when the three primary germ layers form and the earliest progenitor cells of major tissues and organs begin to differentiate. To investigate the early developmental origins of CHDs associated with Nipbl-haploinsufficiency, in Chapter 3, we performed single-cell RNA-sequencing on both wildtype (WT) and Nipbl+/- mouse embryos during the late-gastrulation and early cardiac crescent stages. The results indicate that although Nipbl+/- embryos contain the same cell populations as WT embryos, they exhibit subtle yet significant misallocations of specific mesodermal cell populations. Our analysis confirmed that these discrepancies are not related to apoptosis, cell proliferation, global developmental delays, or changes in the overall structure of cell lineages. Rather this phenomenon appears to stem from altered cell fate decisions in mesoderm cells' differentiation toward cardiac or non-cardiac fates. As in previous studies, we observed that while most gene expression changes in Nipbl-deficient tissues at these stages were minor, certain key developmental genes, particularly Nanog, were significantly misexpressed. Specifically, Nipbl+/- embryos did not downregulate Nanog at the end of gastrulation – a process typically observed in all cells except germ cells. This persistent overexpression of Nanog leads to aberrant expression of many of its target genes across all germ layers. We additionally observed underexpression of Hox genes and overexpression of Nodal signaling genes, which play roles in anterior-posterior and left-right patterning, respectively. Based on these findings, we propose a model suggesting that birth defects in CdLS may arise from prolonged overexpression of Nanog and subsequent dysregulation of developmental pathways. These disruptions likely contribute to incorrect cell fate decisions and the misallocation of specific progenitor cell populations, ultimately leading to the developmental anomalies observed.