Izabela Kowalczyk

All Publications

• Balancing WNT signalling in early forebrain development: The role of LRP4 as a modulator of LRP6 function FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY Geng, S., Paul, F., Kowalczyk, I., Raimundo, S., Sporbert, A., Mamo, T., Hammes, A. 2023; 11: 1173688

  Abstract

  The specification of the forebrain relies on the precise regulation of WNT/ß-catenin signalling to support neuronal progenitor cell expansion, patterning, and morphogenesis. Imbalances in WNT signalling activity in the early neuroepithelium lead to congenital disorders, such as neural tube defects (NTDs). LDL receptor-related protein (LRP) family members, including the well-studied receptors LRP5 and LRP6, play critical roles in modulating WNT signalling capacity through tightly regulated interactions with their co-receptor Frizzled, WNT ligands, inhibitors and intracellular WNT pathway components. However, little is known about the function of LRP4 as a potential modulator of WNT signalling in the central nervous system. In this study, we investigated the role of LRP4 in the regulation of WNT signalling during early mouse forebrain development. Our results demonstrate that LRP4 can modulate LRP5- and LRP6-mediated WNT signalling in the developing forebrain prior to the onset of neurogenesis at embryonic stage 9.5 and is therefore essential for accurate neural tube morphogenesis. Specifically, LRP4 functions as a genetic modifier for impaired mitotic activity and forebrain hypoplasia, but not for NTDs in LRP6-deficient mutants. In vivo and in vitro data provide evidence that LRP4 is a key player in fine-tuning WNT signalling capacity and mitotic activity of mouse neuronal progenitors and of human retinal pigment epithelial (hTERT RPE-1) cells. Our data demonstrate the crucial roles of LRP4 and LRP6 in regulating WNT signalling and forebrain development and highlight the need to consider the interaction between different signalling pathways to understand the underlying mechanisms of disease. The findings have significant implications for our mechanistic understanding of how LRPs participate in controlling WNT signalling.

  View details for DOI 10.3389/fcell.2023.1173688

  View details for Web of Science ID 000975645400001

  View details for PubMedID 37091972

  View details for PubMedCentralID PMC10119419

• Identification of disease-relevant modulators of the SHH pathway in the developing brain DEVELOPMENT Mecklenburg, N., Kowalczyk, I., Witte, F., Goerne, J., Laier, A., Mamo, T. M., Gonschior, H., Lehmann, M., Richter, M., Sporbert, A., Purfuerst, B., Huebner, N., Hammes, A. 2021; 148 (17)

  Abstract

  Pathogenic gene variants in humans that affect the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown modifier genes. To identify such modifiers, we established novel congenic mouse models. LRP2-deficient C57BL/6N mice suffer from heart outflow tract defects and holoprosencephaly caused by impaired SHH activity. These defects are fully rescued on a FVB/N background, indicating a strong influence of modifier genes. Applying comparative transcriptomics, we identified Pttg1 and Ulk4 as candidate modifiers upregulated in the rescue strain. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. In addition, we characterized ULK4 and PTTG1 as previously unidentified components of primary cilia in the neuroepithelium. The identification of genes that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart is likely relevant to understanding the variability in human congenital disorders.

  View details for DOI 10.1242/dev.199307

  View details for Web of Science ID 000696114300003

  View details for PubMedID 34463328

• Neural tube closure requires the endocytic receptor Lrp2 and its functional interaction with intracellular scaffolds DEVELOPMENT Kowalczyk, I., Lee, C., Schuster, E., Hoeren, J., Trivigno, V., Riedel, L., Gorne, J., Wallingford, J. B., Hammes, A., Feistel, K. 2021; 148 (2)

  Abstract

  Pathogenic mutations in the endocytic receptor LRP2 in humans are associated with severe neural tube closure defects (NTDs) such as anencephaly and spina bifida. Here, we have combined analysis of neural tube closure in mouse and in the African Clawed Frog Xenopus laevis to elucidate the etiology of Lrp2-related NTDs. Lrp2 loss of function impaired neuroepithelial morphogenesis, culminating in NTDs that impeded anterior neural plate folding and neural tube closure in both model organisms. Loss of Lrp2 severely affected apical constriction as well as proper localization of the core planar cell polarity (PCP) protein Vangl2, demonstrating a highly conserved role of the receptor in these processes, which are essential for neural tube formation. In addition, we identified a novel functional interaction of Lrp2 with the intracellular adaptor proteins Shroom3 and Gipc1 in the developing forebrain. Our data suggest that, during neurulation, motifs within the intracellular domain of Lrp2 function as a hub that orchestrates endocytic membrane removal for efficient apical constriction, as well as PCP component trafficking in a temporospatial manner.

  View details for DOI 10.1242/dev.195008

  View details for Web of Science ID 000613906000019

  View details for PubMedID 33500317

  View details for PubMedCentralID PMC7860117

• Myt1 and Myth1l transcription factors limit proliferation in GBM cells by repressing YAP1 expression BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS Melhuish, T. A., Kowalczyk, I., Manukyan, A., Zhang, Y., Shah, A., Abounader, R., Wotton, D. 2018; 1861 (11): 983-995

  Abstract

  Myelin transcription factor 1 (Myt1) and Myt1l (Myt1-like) are zinc finger transcription factors that regulate neuronal differentiation. Reduced Myt1l expression has been implicated in glioblastoma (GBM), and the related St18 was originally identified as a potential tumor suppressor for breast cancer. We previously analyzed changes in gene expression in a human GBM cell line with re-expression of either Myt1 or Myt1l. This revealed largely overlapping gene expression changes, suggesting similar function in these cells. Here we show that re-expression of Myt1 or Myt1l reduces proliferation in two different GBM cell lines, activates gene expression programs associated with neuronal differentiation, and limits expression of proliferative and epithelial to mesenchymal transition gene-sets. Consistent with this, expression of both MYT1 and MYT1L is lower in more aggressive glioma sub-types. Examination of the gene expression changes in cells expressing Myt1 or Myt1l suggests that both repress expression of the YAP1 transcriptional coactivator, which functions primarily in the Hippo signaling pathway. Expression of YAP1 and its target genes is reduced in Myt-expressing cells, and there is an inverse correlation between YAP1 and MYT1/MYT1L expression in human brain cancer datasets. Proliferation of GBM cell lines is reduced by lowering YAP1 expression and increased with YAP1 over-expression, which overcomes the anti-proliferative effect of Myt1/Myt1l expression. Finally we show that reducing YAP1 expression in a GBM cell line slows the growth of orthotopic tumor xenografts. Together, our data suggest that Myt1 and Myt1l directly repress expression of YAP1, a protein which promotes proliferation and GBM growth.

  View details for DOI 10.1016/j.bbagrm.2018.10.005

  View details for Web of Science ID 000449445700001

  View details for PubMedID 30312684

  View details for PubMedCentralID PMC6203443

• Analysis of transcriptional activity by the Myt1 and Myt1l transcription factors JOURNAL OF CELLULAR BIOCHEMISTRY Manukyan, A., Kowalczyk, I., Melhuish, T. A., Lemiesz, A., Wotton, D. 2018; 119 (6): 4644-4655

  Abstract

  Myt1 and Myt1l (Myelin transcription factor 1, and Myt1-like) are members of a small family of closely related zinc finger transcription factors, characterized by two clusters of C2HC zinc fingers. Both are widely expressed during early embryogenesis, but are largely restricted to expression within the brain in the adult. Myt1l, as part of a three transcription factor mix, can reprogram fibroblasts to neurons and plays a role in maintaining neuronal identity. Previous analyses have indicated roles in both transcriptional activation and repression and suggested that Myt1 and Myt1l may have opposing functions in gene expression. We show that when targeted to DNA via multiple copies of the consensus Myt1/Myt1l binding site Myt1 represses transcription, whereas Myt1l activates. By targeting via a heterologous DNA binding domain we mapped an activation function in Myt1l to an amino-terminal region that is poorly conserved in Myt1. However, genome wide analyses of the effects of Myt1 and Myt1l expression in a glioblastoma cell line suggest that the two proteins have largely similar effects on endogenous gene expression. Transcriptional repression is likely mediated by binding to DNA via the known consensus site, whereas this site is not associated with the transcriptional start sites of genes with higher expression in the presence of Myt1 or Myt1l. This work suggests that these two proteins function similarly, despite differences observed in analyses based on synthetic reporter constructs.

  View details for DOI 10.1002/jcb.26636

  View details for Web of Science ID 000430667200001

  View details for PubMedID 29291346