Biology subjects

Stottmann, R.

Publications and source records attributed to Stottmann, R..

16 recordsLinked to original sources

bioRxiv2026

Synergistic Variants in C-terminal Binding Protein 1 and Alkaline Phosphatase Lead to Mandibular Hypoplasia Through Impaired Wnt Signaling: An Oligogenic Model

Craniofacial malformations account for one third of all congenital anomalies. Genetic factors play a vital role, yet the list of causal genes and their mechanisms are far from complete. As part of a larger effort to sequence patients with micrognathia and Pierre-Robin sequence, we identified two candidate pathogenic missense variants in C-terminal binding protein 1 (CTBP1) along with a heterozygous early stop missense variant in alkaline phosphatase (ALPL) in a proband with mandibular hypoplasia. Ctbp1 has been shown to regulate Wnt/{beta}-Catenin signaling but it has not yet been implicated in craniofacial development. Here we generated two orthologous variants of Ctbp1 mimicking the patient variants using genome editing in mice and explored the micrognathia phenotype in combination with a previously reported Alpl null allele. Ctbp1Q148H/G238S; Alplnull/Wt complex heterozygous mutants have smaller mandibles recapitulating the human mandibular hypoplasia. We identified that a reduction in cell proliferation and active {beta}-Catenin levels could possibly account for the micrognathia phenotype in the Ctbp1; Alpl complex heterozygous. These data uncover a novel role for Ctbp1 in craniofacial development and highlight the complex genetic and molecular signaling in the pathogenesis of craniofacial malformations.

genetics↗

bioRxiv2025

Gene replacement therapy for Piga GPI-anchor deficiency in the developing nervous system

Glycosylphosphatidylinositol (GPI) anchors are a class of post-translational modifications observed on over 150 proteins. Pathogenic variants in the GPI biosynthesis enzyme, PIGA, in humans are associated with several brain anomalies such as hypomyelination, cerebellar hypoplasia, ataxic gait, and can lead to premature mortality. We previously genetically deleted Piga from the embryonic mouse brain which led to early postnatal death and significant structural brain malformations similar to those observed in humans with PIGA variants. The current treatment options for PIGA patients only manage symptoms and provides palliative care, demonstrating a need for new therapeutic options. We employed an AAV9-mediated PIGA gene-replacement (AAV9-hPIGA) strategy to assess the efficacy of gene therapy in the brain. We show that a single intracerebroventricular treatment on the first day of life successfully rescued survival rates, structural brain anomalies, and neurological impairments. Additionally, we used mass spectrometry to identify and quantify GPI-anchored proteins in untreated and treated mutant mice. We found that AAV9- hPIGA treatment restored GPI-anchored protein levels in mutant animals. These investigations enhance our understanding of GPI-anchored protein production during brain development and contribute to the development of a more effective intervention for PIGA-related symptoms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/693709v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@32038dorg.highwire.dtl.DTLVardef@1875eb5org.highwire.dtl.DTLVardef@5abfe8org.highwire.dtl.DTLVardef@1ed5719_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIA single AAV9-hPIGA injection rescues Piga-related phenotypes including survival and structural brain defects C_LIO_LIAAV9-hPIGA treatment rescued long-term ataxia phenotypes in Piga mutants C_LIO_LINeuronal GPI-anchored protein levels in Piga mutants are increased with AAV9- hPIGA gene replacement C_LI

genetics↗

bioRxiv2025

Genetic Dissection of the role of Piga and Pgap2 in the embryonic mouse brain

Glycosylphosphatidylinositol (GPI) anchors are a post-translational modification of over 150 proteins. These GPI-anchored proteins are enriched in lipid rafts in the plasma membrane and serve a variety of functions including acting as receptors and cell adhesion molecules. Human pathogenic variants in GPI biosynthesis pathway enzymes are collectively called inherited GPI deficiencies and lead to several brain anomalies such as global developmental delay, hypomyelination, cerebellar hypoplasia, and premature death. PIGA is the first catalytic enzyme in the GPI anchor biosynthesis cascade, which creates the backbone of all GPI anchors, and PGAP2 is one of the last enzymes in the GPI-anchor biosynthesis pathway and modifies the protein-bound anchor for trafficking to the cell membrane. A Nestin-Cre mediated deletion of Piga in the mouse led to early postnatal death and significant structural brain malformations, similar to pathogenic human PIGA variants. While this model provides an opportunity to develop treatments for these neurological phenotypes, we still lack a deep understanding of GPI-anchor functions in brain development. Additionally, there are no studies on Pgap2 in the brain to date. We extended these studies with a series of genetic ablations to precisely determine the role of Piga and Pgap2 in the forebrain, oligodendrocytes, and cerebellum. We find Piga expression in the hindbrain is absolutely required for survival while Pgap2 ablations were much less deleterious. These studies broaden our knowledge on the brain region-specific requirements of GPI-anchor biosynthesis enzymes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/692975v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@18eb4feorg.highwire.dtl.DTLVardef@cb152org.highwire.dtl.DTLVardef@14d7e84org.highwire.dtl.DTLVardef@ad2d9a_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementIn this study, we examine the roles of the GPI-anchor biosynthesis enzymes Piga and Pgap2 to understand the poorly understood mechanisms underlying brain anomalies seen in the inherited GPI deficiencies. Using Cre/lox conditional mouse genetic models, we deleted Piga and Pgap2 from various brain regions, including the forebrain, oligodendrocytes, and cerebellum, and observed brain abnormalities. We find that Piga ablations mimic many brain phenotypes seen in human inherited GPI deficiencies, while Pgap2 ablations do not grossly disrupt neurodevelopment. Additionally, we demonstrate that Piga expression is essential in the cerebellum for survival. These findings reveal mechanisms underlying several inherited GPI deficiency brain phenotypes and will inform targeted therapeutic strategies.

genetics↗

bioRxiv2025

Cse1l Regulates Neural Crest Cell Survival and is Critical for Craniofacial and Cardiac Development

Human congenital anomalies account for twice the mortality of childhood cancer. Despite advancements in genome sequencing and transgenic mouse models that have aided in understanding their pathogenesis, significant gaps remain. Through a forward genetics approach, we previously discovered the hypo-morphic anteater allele of Cse1l which displayed variable craniofacial phenotypes. To circumvent the variability seen in this model, we generated a conditional allele of Cse1l and genetically ablated it in the dorsal midline giving rise to portions of the nervous system and the cranial neural crest cells using the Wnt1-Cre2 driver. Our analysis revealed that Wnt1-Cre2; Cse1lCRISPR/flox embryos exhibited severe malformations in the forebrain, midbrain, and hindbrain, accompanied by a dramatic hypoplasia of the frontonasal, maxillary, and mandibular processes, and the second pharyngeal arch. Wnt1-Cre2; Cse1lCRISPR/flox embryos were embryonic lethal by E11.5 likely due to defects in the ventricular myocardium. Wnt1-Cre2; Cse1lCRISPR/flox embryos exhibited consistently increased apoptosis at E9.5 in the affected tissues along with an increase in p53 expression. These data together show a previously unknown critical function of CSE1L in neural crest cell survival during development. Summary StatementCse1l is critical for neural crest cell survival and genetic ablation of Cse1l in neural crest cells resulted in dramatic apoptosis with increase in p53 expression.

developmental biology↗

bioRxiv2024

Differential contribution of P73+ Cajal-Retzius cells and Reelin to cortical morphogenesis

Cajal-Retzius cells (CRs) are a peculiar neuronal type within the developing mammalian cerebral cortex. One of their best documented feature is the robust secretion of Reln, a glycoprotein essential for the establishment of cortical layers through the control of radial migration of glutamatergic neurons. We previously identified Gmnc as a critical fate determinant for P73+ CRs subtypes from the hem, septum and thalamic eminence. In Gmnc-/- mutants, P73+ CRs are initially produced, cover the telencephalic vesicle but undergo massive apoptosis resulting in their complete depletion at mid-corticogenesis. Here we investigated the consequence of such a CRs depletion on dorsal cortex lamination and hippocampal morphogenesis. We found preplate splitting occurs normally in Gmnc-/- mutants but is followed by defective radial migration arrest in the dorsal cortex, altered cellular organization in the lateral cortex, aberrant hippocampal progenitor proliferation resulting in abnormal CA1 folding and lack of vasculature development in the hippocampal fissure. We then performed conditional Reln deletion in P73+ CRs to evaluate its relative contribution and found that only radial migration defects were recapitulated. We concluded that at mid-corticogenesis, CRs-derived Reln is required for radial migration arrest and additionally identified Reln-independent functions for CRs in the control of hippocampal progenitor proliferation and vessel remodelling.

neuroscience↗

bioRxiv2024

Genetic Analysis and Functional Assessment of a TGFBR2 Variant in Micrognathia and Cleft Palate

Cleft lip and cleft palate are among the most common congenital anomalies and are the result of incomplete fusion of embryonic craniofacial processes or palatal shelves, respectively. We know that genetics play a large role in these anomalies but the list of known causal genes is far from complete. As part of a larger sequencing effort of patients with micrognathia and cleft palate we identified a candidate variant in transforming growth factor beta receptor 2 (TGFBR2) which is rare, changing a highly conserved amino acid, and predicted to be pathogenic by a number of metrics. The family history and population genetics would suggest this specific variant would be incompletely penetrant, but this gene has been convincingly implicated in craniofacial development. In order to test the hypothesis this might be a causal variant, we used genome editing to create the orthologous variant in a new mouse model. Surprisingly, Tgfbr2V387M mice did not exhibit craniofacial anomalies or have reduced survival suggesting this is, in fact, not a causal variant for cleft palate/ micrognathia. The discrepancy between in silico predictions and mouse phenotypes highlights the complexity of translating human genetic findings to mouse models. We expect these findings will aid in interpretation of future variants seen in TGFBR2 from ongoing sequencing of patients with congenital craniofacial anomalies.

developmental biology↗

bioRxiv2023

SMPD4 mediated sphingolipid metabolism regulates brain and primary cilia development

Genetic variants in multiple sphingolipid biosynthesis genes cause human brain disorders. A recent study collected patients from twelve unrelated families with variants in the gene SMPD4, a neutral sphingomyelinase which metabolizes sphingomyelin into ceramide at an early stage of the biosynthesis pathway. These patients have severe developmental brain malformations including microcephaly and cerebellar hypoplasia. However, the mechanism of SMPD4 was not known and we pursued a new mouse model. We hypothesized that the role of SMPD4 in producing ceramide is important for making primary cilia, a crucial organelle mediating cellular signaling. We found that the mouse model has cerebellar hypoplasia due to failure of Purkinje cell development. Human induced pluripotent stem cells exhibit neural progenitor cell death and have shortened primary cilia which is rescued by adding exogenous ceramide. SMPD4 production of ceramide is crucial for human brain development.

developmental biology↗

bioRxiv2022

Dkk1 inhibition normalizes limb phenotypes in a mouse model of Fzd2 associated omodysplasia Robinow syndromes

FRIZZLED-2 (FZD2) is a transmembrane Wnt ligand receptor. We previously identified a pathogenic human FZD2 variant, encoding for a protein with a premature stop and loss of 17 amino acids. This includes a portion of the consensus DISHEVELLED binding sequence required for Wnt signal transduction. Patients with this variant exhibited FZD2-associated autosomal dominant Robinow Syndrome. To model this variant, we utilized zygote microinjection and i-GONAD-based CRISPR/Cas9-mediated genome editing to generate an allelic series in the mouse. Embryos mosaic for humanized Fzd2W553* knock-in exhibited cleft palate and shortened limbs, consistent with FZD2W548* patient phenotypes. We also generated two germline mouse alleles with small deletions, Fzd2D3 and Fzd2D4. Homozygotes for each allele survive embryonic development at normal ratios but exhibit a highly penetrant cleft palate phenotype, shortened limbs compared to wild-type and perinatal lethality. Fzd2D4 craniofacial tissues indicated decreased canonical WNT signaling. In utero treatment with IIIC3a (DKK inhibitor) normalized the limb lengths in Fzd2D4 homozygotes. The in vivo replication represents an approach to further investigate the mechanism of FZD2 phenotypes and validates the utility of CRISPR knock-in mice as a tool for demonstrating pathogenicity of human genetic variants. We also present evidence for a potential therapeutic intervention.

developmental biology↗

bioRxiv2020

A novel hypomorphic allele of Spag17 causes primary ciliary dyskinesia phenotypes in mice

Primary ciliary dyskinesia (PCD) is a human condition of dysfunctional motile cilia characterized by recurrent lung infection, infertility, organ laterality defects, and partially penetrant hydrocephalus. We recovered a mouse mutant from a forward genetic screen that developed all the phenotypes of PCD. Whole exome sequencing identified this primary ciliary dyskinesia only (Pcdo) allele to be a nonsense mutation (c.5236A>T) in the Spag17 coding sequence creating a premature stop codon at position 1746 (K1746*). The Pcdo variant abolished different isoforms of SPAG17 in the Pcdo mutant testis but not in the brain. Our data indicate differential requirements for SPAG17 in different motile cilia cell types. SPAG17 is required for proper development of the sperm flagellum, and is essential for either development or stability of the C1 microtubule structure within cilia, but not the brain ependymal cilia. We identified changes in ependymal cilia beating frequency but these did not apparently alter lateral ventricle cerebrospinal fluid (CSF) flow. Aqueductal (Aq) stenosis resulted in significantly slower and abnormally directed CSF flow and we suggest this is the root cause of the hydrocephalus. The Spag17Pcdo homozygous mutant mice are generally viable to adulthood, but have a significantly shortened life span with chronic morbidity. Our data indicate that the c.5236A>T Pcdo variant is a hypomorphic allele of Spag17 gene that causes phenotypes related to motile, but not primary, cilia. Spag17Pcdo is a novel and useful model for elucidating the molecular mechanisms underlying development of PCD in the mouse.

developmental biology↗

bioRxiv2019

Glycosylphosphatidylinositol Biosynthesis and Remodeling are Required for Neural Crest Cell, Cardiac and Neural Development.

The glycosylphosphatidylinositol (GPI) anchor attaches nearly 150 proteins to the cell surface. Patients with pathogenic variants in GPI biosynthetic pathway genes display an array of phenotypes including seizures, developmental delay, dysmorphic facial features and cleft palate. There is virtually no mechanism to explain these phenotypes. we identified a novel mouse mutant (cleft lip/palate, edema and exencephaly; Clpex) with a hypomorphic mutation in Post-Glycophosphatidylinositol Attachment to Proteins-2 (Pgap2). Pgap2 is one of the final proteins in the GPI biosynthesis pathway and is required for anchor maturation. We found the Clpex mutation results in a global decrease in surface GPI expression. Surprisingly, Pgap2 showed tissue specific expression with enrichment in the affected tissues of the Clpex mutant. We found the phenotype in Clpex mutants is due to apoptosis of neural crest cells (NCCs) and the cranial neuroepithelium, as is observed in the GPI anchored Folate Receptor 1-/- mouse. We showed folinic acid supplementation in utero can rescue the cleft lip phenotype in Clpex. Finally, we generated a novel mouse model of NCC-specific total GPI deficiency in the Wnt1-Cre lineage. These mutants developed median cleft lip and palate demonstrating a cell autonomous role for GPI biosynthesis in NCC development.

developmental biology↗

bioRxiv2018

Gpr63 is a novel modifier of microcephaly in Ttc21b mouse mutants.

The primary cilium is a critical signaling center for proper embryonic development. Previous studies have demonstrated that mice lacking Ttc21b have impaired retrograde trafficking within the cilium and multiple organogenesis phenotypes, including microcephaly. Interestingly, the severity of the microcephaly in Ttc21baln/aln homozygous null mutants is considerably affected by the genetic background. Ttc21baln/aln mutants on an FVB/NJ background develop a forebrain significantly smaller than mutants on a C57BL/6J background. We performed a Quantitative Trait Locus (QTL) analysis to identify potential genetic modifiers and identified two regions linked to differential forebrain size: modifier of alien QTL1 (Moaq1) on chromosome 4 at 27.8 Mb and Moaq2 on chromosome 6 at 93.6 Mb. These QTLs were validated by constructing congenic strains. Further analysis of Moaq1 identified a brain specific orphan G-protein coupled receptor (GPCR), Gpr63, as a candidate gene. We identified a SNP between the FVB and B6 strains in Gpr63, which creates a missense mutation predicted to be deleterious in the FVB protein. We first demonstrated that Gpr63 can localize to the cilium and then used CRISPR-Cas9 genome editing to create FVB congenic mice with the B6 sequence of Gpr63 and a deletion allele leading to a truncation of the GPR63 C-terminal tail. These alleles genetically interact with Ttc21baln/aln, validating Gpr63 as a forebrain modifier of Ttc21b and strongly supporting Gpr63 as the variant causal gene (i.e., the quantitative trait gene, QTG) for Moaq1.

developmental biology↗

bioRxiv2018

Using human sequencing to guide craniofacial research.

A recent convergence of technological innovations has re-energized the ability to apply genetics to research in human craniofacial development. Next-generation exome and whole genome sequencing have dropped in price significantly making it relatively trivial to sequence and analyze patients and families with congenital craniofacial anomalies. A concurrent revolution in genome editing with the use of the CRISPR-Cas9 system enables the rapid generation of animal models, including mouse, which can precisely recapitulate human variants. Here we summarize the choices currently available to the research community. We illustrate this approach with the study of a family with a novel craniofacial syndrome with dominant inheritance pattern. The genomic analysis suggest a causal variant in AMOTL1 which we modeled in mice. We also made a novel deletion allele of Amotl1. Our results indicate that Amotl1 is not required in the mouse for survival to weaning. Mice carrying the variant identified in the human sequencing studies, however, do not survive to weaning in normal ratios. The cause of death is not understood for these mice complicating our conclusions about the pathogenicity in the index patient. Thus, we highlight some of the powerful opportunities and confounding factors confronting current craniofacial genetic research.

genetics↗

bioRxiv2018

Differential requirements of tubulin genes in mammalian forebrain development.

Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations results from de novo heterozygous mutations in tubulin genes. However, the absolute requirement for most of these genes in development and disease has not been previously tested in genetic, loss of function models. Here we present two novel pathogenic tubulin alleles: a human TUBA1A missense variant with a phenotype more severe than most tubulinopathies and a mouse ENU allele of Tuba1a. Furthermore, we directly test the requirement for Tuba1a, Tuba8, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tuba8, Tubb2a or Tubb2b does not lead to cortical malformation phenotypes or impair survival. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. Thus, despite their functional similarity, the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases.

developmental biology↗

bioRxiv2017

A de novo missense mutation in TUBA1A results in reduced neural progenitor survival and differentiation

Mutations in tubulins have been implicated in numerous human neurobiological disorders collectively known as \"tubulinopathies.\" We identified a patient with severe cortical dysgenesis and a novel de novo heterozygous missense mutation in Tubulin Alpha 1a (TUBA1A, c.1225 G>T). Induced pluripotent stem cells derived from this individual were differentiated into two dimensional neural rosette clusters to identify underlying mechanisms for the severe cortical dysgenesis phenotype. Patient-derived clones showed evidence of impaired neural progenitor survival and differentiation with abnormal neural rosette formation, increases in cell death, and fewer post-mitotic neurons. These features correlate with the drastically underdeveloped cortical tissues seen in the proband. This is the first experimental evidence in human tissue suggesting a mechanism underlying the role for TUBA1A in cortical development.\n\nSUMMARY STATEMENTVariants in tubulin genes often lead to severe congenital brain malformations. Here we identify a new mutation in TUBA1A and use iPSCS to show this alters proliferation, differentiation and survival of neural progenitors.

genetics↗

bioRxiv2017

Ttc21b is required in Bergmann glia during postnatal cerebellar development

Proper cerebellar development is dependent on tightly regulated proliferation, migration, and differentiation events. Disruptions in any of these leads to a range of cerebellar phenotypes from ataxia to childhood tumors. Animal models have shown proper regulation of sonic hedgehog (Shh) signaling is crucial for normal cerebellar architecture and increased signaling leads to cerebellar tumor formation. Primary cilia are known to be required for the proper regulation of multiple developmental signaling pathways, including Shh. Tetratricopeptide Repeat Domain 21B (Ttc21b) is required for proper primary cilia form and function and is primarily thought to restrict Shh signaling. Here we investigated a role for Ttc21b in cerebellar development. Surprisingly, Ttc21b ablation in Bergmann glia resulted in accumulation of ectopic granule cells in the lower/ posterior lobes of the cerebellum and a reduction in Shh signaling. Ttc21b ablation in just Purkinje cells resulted in a similar, phenotype seen in fewer cells, but across the entire extent of the cerebellum. These results suggest that Ttc21b expression is required for Bergmann glia structure and signaling in the developing cerebellum, and in some contexts, augments, rather than attenuates, Shh signaling.

genetics↗

bioRxiv2017

Copb2 Is Essential For Embryogenesis And Hypomorphic Mutations Cause Human Microcephaly

Primary microcephaly is a congenital brain malformation characterized by a head circumference less than three standard deviations below the mean for age and sex and results in moderate to severe mental deficiencies and decreased lifespan. We recently studied two children with primary microcephaly in an otherwise unaffected family. Exome sequencing identified an autosomal recessive mutation leading to an amino acid substitution in a WD40 domain of the highly conserved Coatomer Protein Complex, Subunit Beta 2 (COPB2). To study the role of Copb2 in neural development, we utilized genome editing technology to generate an allelic series in the mouse. Two independent null alleles revealed that Copb2 is essential for early stages of embryogenesis. Mice homozygous for the patient variant (Copb2R254C/R254C) appear to have a grossly normal phenotype, likely due to differences in corticogenesis between the two species. Strikingly, mice heterozygous for the patient mutation and a null allele (Copb2R254C/Znf) show a severe perinatal phenotype including low neonatal weight, significantly increased apoptosis in the brain, and death within the first week of life. Immunostaining of the Copb2R254C/Znf brain revealed a reduction in layer V (CTIP2+) neurons, while the overall cell density of the cortex is unchanged. Moreover, disruption of Copb2 in mouse neurospheres resulted in reduced proliferation. These results identify a general requirement for COPB2 in embryogenesis and a specific role in corticogenesis. We further demonstrate the utility of CRISPR-Cas9 generated mouse models in the study of potential pathogenicity of variants of potential clinical interest.

genetics↗