DNA methylation episignature and comparative epigenomic profiling for Pitt-Hopkins syndrome caused by TCF4 variants.

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by pathogenic variants in TCF4, leading to intellectual disability, specific morphological features, and autonomic nervous system dysfunction. Epigenetic dysregulation has been implicated in PTHS, prompting the investigation of a DNA methylation (DNAm) "episignature" specific to PTHS for diagnostic purposes and variant reclassification and functional insights into the molecular pathophysiology of this disorder. A cohort of 67 individuals with genetically confirmed PTHS and three individuals with intellectual disability and a variant of uncertain significance (VUS) in TCF4 were studied. The DNAm episignature was developed with an Infinium Methylation EPIC BeadChip array analysis using peripheral blood cells. Support vector machine (SVM) modeling and clustering methods were employed to generate a DNAm classifier for PTHS. Validation was extended to an additional cohort of 11 individuals with PTHS. The episignature was assessed in relation to other neurodevelopmental disorders and its specificity was examined. A specific DNAm episignature for PTHS was established. The classifier exhibited high sensitivity for TCF4 haploinsufficiency and missense variants in the basic-helix-loop-helix domain. Notably, seven individuals with TCF4 variants exhibited negative episignatures, suggesting complexities related to mosaicism, genetic factors, and environmental influences. The episignature displayed degrees of overlap with other related disorders and biological pathways. This study defines a DNAm episignature for TCF4-related PTHS, enabling improved diagnostic accuracy and VUS reclassification. The finding that some cases scored negatively underscores the potential for multiple or nested episignatures and emphasizes the need for continued investigation to enhance specificity and coverage across PTHS-related variants.

Fatal gastrointestinal complications in Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder caused by mutations of the transcription factor 4 (Tcf4) gene. Individuals with PTHS often suffer from severe abdominal bloating and constipation. In this short communication, we discuss two individuals with PTHS who died unexpectedly due to gastrointestinal complications. We aim to increase awareness among healthcare professionals who care for individuals with PTHS, to ensure adequate screening and management of gastrointestinal symptoms in this population. Moreover, we discuss how fatal gastrointestinal complications may be related to PTHS and provide an overview of the literature.

Molecular Organization and Patterning of the Medulla Oblongata in Health and Disease.

The medulla oblongata, located in the hindbrain between the pons and the spinal cord, is an important relay center for critical sensory, proprioceptive, and motoric information. It is an evolutionarily highly conserved brain region, both structural and functional, and consists of a multitude of nuclei all involved in different aspects of basic but vital functions. Understanding the functional anatomy and developmental program of this structure can help elucidate potential role(s) of the medulla in neurological disorders. Here, we have described the early molecular patterning of the medulla during murine development, from the fundamental units that structure the very early medullary region into 5 rhombomeres (r7-r11) and 13 different longitudinal progenitor domains, to the neuronal clusters derived from these progenitors that ultimately make-up the different medullary nuclei. By doing so, we developed a schematic overview that can be used to predict the cell-fate of a progenitor group, or pinpoint the progenitor domain of origin of medullary nuclei. This schematic overview can further be used to help in the explanation of medulla-related symptoms of neurodevelopmental disorders, e.g., congenital central hypoventilation syndrome, Wold-Hirschhorn syndrome, Rett syndrome, and Pitt-Hopkins syndrome. Based on the genetic defects seen in these syndromes, we can use our model to predict which medullary nuclei might be affected, which can be used to quickly direct the research into these diseases to the likely affected nuclei.

Nkx2.9 Contributes to Mid-Hindbrain Patterning by Regulation of mdDA Neuronal Cell-Fate and Repression of a Hindbrain-Specific Cell-Fate.

Nkx2.9 is a member of the NK homeobox family and resembles Nkx2.2 both in homology and expression pattern. However, while Nkx2.2 is required for development of serotonergic neurons, the role of Nkx2.9 in the mid-hindbrain region is still ill-defined. We have previously shown that Nkx2.9 expression is downregulated upon loss of En1 during development. Here, we determined whether mdDA neurons require Nkx2.9 during their development. We show that Nkx2.9 is strongly expressed in the IsO and in the VZ and SVZ of the embryonic midbrain, and the majority of mdDA neurons expressed Nkx2.9 during their development. Although the expression of Dat and Cck are slightly affected during development, the overall development and cytoarchitecture of TH-expressing neurons is not affected in the adult Nkx2.9-depleted midbrain. Transcriptome analysis at E14.5 indicated that genes involved in mid- and hindbrain development are affected by Nkx2.9-ablation, such as Wnt8b and Tph2. Although the expression of Tph2 extends more rostral into the isthmic area in the Nkx2.9 mutants, the establishment of the IsO is not affected. Taken together, these data point to a minor role for Nkx2.9 in mid-hindbrain patterning by repressing a hindbrain-specific cell-fate in the IsO and by subtle regulation of mdDA neuronal subset specification.

Tcf4 Is Involved in Subset Specification of Mesodiencephalic Dopaminergic Neurons.

During development, mesodiencephalic dopaminergic (mdDA) neurons form into different molecular subsets. Knowledge of which factors contribute to the specification of these subsets is currently insufficient. In this study, we examined the role of Tcf4, a member of the E-box protein family, in mdDA neuronal development and subset specification. We show that Tcf4 is expressed throughout development, but is no longer detected in adult midbrain. Deletion of Tcf4 results in an initial increase in TH-expressing neurons at E11.5, but this normalizes at later embryonic stages. However, the caudal subset marker Nxph3 and rostral subset marker Ahd2 are affected at E14.5, indicating that Tcf4 is involved in correct differentiation of mdDA neuronal subsets. At P0, expression of these markers partially recovers, whereas expression of Th transcript and TH protein appears to be affected in lateral parts of the mdDA neuronal population. The initial increase in TH-expressing cells and delay in subset specification could be due to the increase in expression of the bHLH factor Ascl1, known for its role in mdDA neuronal differentiation, upon loss of Tcf4. Taken together, our data identified a minor role for Tcf4 in mdDA neuronal development and subset specification.

Acquisition of the Midbrain Dopaminergic Neuronal Identity.

The mesodiencephalic dopaminergic (mdDA) group of neurons comprises molecularly distinct subgroups, of which the substantia nigra (SN) and ventral tegmental area (VTA) are the best known, due to the selective degeneration of the SN during Parkinson's disease. However, although significant research has been conducted on the molecular build-up of these subsets, much is still unknown about how these subsets develop and which factors are involved in this process. In this review, we aim to describe the life of an mdDA neuron, from specification in the floor plate to differentiation into the different subsets. All mdDA neurons are born in the mesodiencephalic floor plate under the influence of both SHH-signaling, important for floor plate patterning, and WNT-signaling, involved in establishing the progenitor pool and the start of the specification of mdDA neurons. Furthermore, transcription factors, like Ngn2, Ascl1, Lmx1a, and En1, and epigenetic factors, like Ezh2, are important in the correct specification of dopamine (DA) progenitors. Later during development, mdDA neurons are further subdivided into different molecular subsets by, amongst others, Otx2, involved in the specification of subsets in the VTA, and En1, Pitx3, Lmx1a, and WNT-signaling, involved in the specification of subsets in the SN. Interestingly, factors involved in early specification in the floor plate can serve a dual function and can also be involved in subset specification. Besides the mdDA group of neurons, other systems in the embryo contain different subsets, like the immune system. Interestingly, many factors involved in the development of mdDA neurons are similarly involved in immune system development and vice versa. This indicates that similar mechanisms are used in the development of these systems, and that knowledge about the development of the immune system may hold clues for the factors involved in the development of mdDA neurons, which may be used in culture protocols for cell replacement therapies.

Tcf4 is required for correct brain development during embryogenesis.

Tcf4 has been linked to autism, schizophrenia, and Pitt-Hopkins Syndrome (PTHS) in humans, suggesting a role for Tcf4 in brain development and importantly cortical development. However, the mechanisms behind its role in disease and brain development are still elusive. We provide evidence that Tcf4 has a critical function in the differentiation of cortical regions, corpus callosum and anterior commissure formation, and development of the hippocampus during murine embryonic development. In the present study, we show that Tcf4 is expressed throughout the developing brain at the peak of neurogenesis. Deletion of Tcf4 results in mis-specification of the cortical neurons, malformation of the corpus callosum and anterior commissure, and hypoplasia of the hippocampus. Furthermore, the Tcf4 mutant shows an absence of midline remodeling, underlined by the loss of GFAP-expressing midline glia in the indusium griseum and callosal wedge and midline zipper glia in the telencephalic midline. RNA-sequencing on E14.5 cortex material shows that Tcf4 functions as a transcriptional activator and loss of Tcf4 results in downregulation of genes linked to neurogenesis and neuronal maturation. Furthermore, many genes that are differentially expressed after Tcf4 ablation are linked to other neurodevelopmental disorders. Taken together, we show that correct brain development and neuronal differentiation are severely affected in Tcf4 mutants, phenocopying morphological brain defects detected in PTHS patients. The presented data identifies new leads to understand the mechanisms behind brain and specifically cortical development and can provide novel insights in developmental mechanisms underlying human brain defects.

Expression analyzes of early factors in midbrain differentiation programs.

Mesodiencephalic dopaminergic (mdDA) neurons are born in the ventricular zone (VZ) of the midbrain between E10 and E12. Although these neurons all express specific DA markers like Th and Pitx3, they are subdivided into distinct subsets, each depending on a unique set of transcription factors and signaling cascades for their differentiation. How a neural progenitor commits to an mdDA neuronal cell-fate and how the specification into the different subsets is determined remains unclear. To gain more insight into the development and specification of these neurons we have previously conducted a genome-wide expression analysis, in which dissected midbrain material (E10.5-E13.5) was compared to the adult mdDA region (Chakrabarty et al., 2012). In the present study, we have compared the genome-wide expression analysis including PITX3-GFP sorted (E12.5-E15.5) neurons to available expression data to search for genes specifically expressed in the midbrain during early stages of mdDA differentiation. We have divided these genes into 3 groups: (I) genes upregulated throughout differentiation (Mest, NeuroD1, and Tcf12), (II) genes upregulated during early stages of differentiation (Hes5, and Tcf3), and (III) genes upregulated during late stages of differentiation (Enc1). Here, we show the expression profile of these genes in the embryonic midbrain during development and adult stage and compared that to the appearance of mdDA neurons via co-staining for TH. With this analysis we have identified 6 novel factors that may play a role during cell-fate commitment of neural progenitors or later during differentiation of the mdDA group of neurons.

Tcf12 Is Involved in Early Cell-Fate Determination and Subset Specification of Midbrain Dopamine Neurons.

The basic helix-loop-helix (bHLH) protein family has previously been shown to be involved in the development of mesodiencephalic dopaminergic (mdDA) neurons in the murine midbrain. Specifically, Ngn2 and Mash1 are known to have a role in the specification of neural progenitors in the ventricular zone (VZ) of the midbrain towards an mdDA neuronal cell-fate. Furthermore, other members of the bHLH protein family, the E-box factors, are expressed in the developing midbrain and are thought to have a role in neuronal differentiation. Here we show that the E-box factor Tcf12 is implicated in early and late development of mdDA neurons. Tcf12 is expressed in the midbrain and in young TH-expressing mdDA neurons throughout development. Tcf12lox/lox;En1cre/+ embryos, that lose Tcf12 at ~embryonic day (E)9 throughout the En1 expression domain, have a changed spatial expression of Lmx1a and Nurr1 and a consistent loss of rostral TH expression. Expression of the subset marker Ahd2 is initially delayed, but recovers during development, eventually showing an ~10% increase in AHD2-expressing cells at postnatal day (P)30. Tcf12lox/lox;Pitx3cre/+ embryos, that lose Tcf12 at ~E12 in post-mitotic mdDA neurons, show no effect on the amount of TH-expressing neurons in the developing midbrain. However, similar as to Tcf12lox/lox;En1cre/+ embryos, subset specification is delayed during development. Taken together, we have identified Tcf12 as a novel factor in mdDA neuronal development. It serves a dual function; one in early cell-fate commitment of neural progenitors and one late in subset specification.

Mest/Peg1 Is Essential for the Development and Maintenance of a SNc Neuronal Subset.

Mesodiencephalic dopaminergic (mdDA) neurons originate at the floor plate and floor plate-basal plate boundary of the midbrain ventricular zone. During development mdDA neurons are specified by a unique set of transcription factors and signaling cascades, to form the different molecular subsets of the mdDA neuronal population. In a time series micro-array study performed previously, mesoderm specific transcript (Mest) was found to be one of the most upregulated genes during early mdDA neuronal development. Here, we show that Mest transcript is expressed in the midbrain throughout development and becomes restricted to the substantia nigra (SNc) at late stages. In Mest KO animals mdDA neurons are progressively lost in the adult, mostly affecting the SNc, reflected by a 50% decrease of TH protein and DA release in the striatum and a reduction of climbing behavior. Analysis of Lrp6 KO embryos suggest a subtle opposite phenotype to the Mest KO, hinting toward the possibility that specific loss of mdDA neurons in Mest ablated animals could be due to affected WNT-signaling. Interestingly, the mdDA neuronal region affected by the loss of Mest remains relatively unaffected in Pitx3 mutants, suggesting that both genes are essential for the development and/or maintenance of different mdDA neuronal subsets within the SNc. Overall, the neuroanatomical and phenotypical consequences detected upon the loss of Mest, resemble the loss of SNc neurons and loss of movement control as seen in Parkinson's Disease (PD), suggesting that the Mest mouse model may be used as a model-system for PD.

Mesodiencephalic dopaminergic neuronal differentiation does not involve GLI2A-mediated SHH-signaling and is under the direct influence of canonical WNT signaling.

Sonic Hedgehog (SHH) and WNT proteins are key regulators in many developmental processes, like embryonic patterning and brain development. In the brain, SHH is expressed in a gradient starting in the floor plate (FP) progressing ventrally in the midbrain, where it is thought to be involved in the development and specification of mesodiencephalic dopaminergic (mdDA) neurons. GLI2A-mediated SHH-signaling induces the expression of Gli1, which is inhibited when cells start expressing SHH themselves. To determine whether mdDA neurons receive GLI2A-mediated SHH-signaling during differentiation, we used a BAC-transgenic mouse model expressing eGFP under the control of the Gli1 promoter. This mouse-model allowed for mapping of GLI2A-mediated SHH-signaling temporal and spatial in the mouse midbrain. Since mdDA neurons are born from E10.5, peaking at E11.0-E12.0, we examined Gli1-eGFP embryos at E11.5, E12.5, and E13.5, indicating whether Gli1 was induced before or during mdDA development and differentiation. Our data indicate that GLI2A-mediated SHH-signaling is not involved in mdDA neuronal differentiation. However, it appears to be involved in the differentiation of neurons which make up a subset of the red nucleus (RN). In order to detect whether mdDA neuronal differentiation may be under the control of canonical WNT-signaling, we used a transgenic mouse-line expressing LacZ under the influence of stable ß-catenin. Here, we show that TH+ neurons of the midbrain receive canonical WNT-signaling during differentiation. Therefore, we suggest that early SHH-signaling is indirectly involved in mdDA development through early patterning of the midbrain area, whereas canonical WNT-signaling is directly involved in the differentiation of the mdDA neuronal population.

Epigenetic mechanisms in the development and maintenance of dopaminergic neurons.

Mesodiencephalic dopaminergic (mdDA) neurons are located in the ventral mesodiencephalon and are involved in psychiatric disorders and severely affected in neurodegenerative diseases such as Parkinson's disease. mdDA neuronal development has received much attention in the last 15 years and many transcription factors involved in mdDA specification have been discovered. More recently however, the impact of epigenetic regulation has come into focus, and it's emerging that the processes of histone modification and DNA methylation form the basis of genetic switches that operate during mdDA development. Here, we review the epigenetic control of mdDA development, maturation and maintenance. As we highlight, epigenetic mechanisms play a pivotal role in all of these processes and the knowledge gathered from studying epigenetics in these contexts may aid our understanding of mdDA-related pathologies.

LMX1B is part of a transcriptional complex with PSPC1 and PSF.

The LIM homeodomain transcription factor Lmx1b is essential for the development of the isthmic organizer and mesodiencephalic dopaminergic neurons. The uncoupling of Pitx3 and Th expression, in the Lmx1b null mutant, suggests that Lmx1b may act as a positional activator of the mdDA domain, eventually leading to properly differentiating mdDA neurons. In this study, we aimed to elucidate how Lmx1b functions mechanistically in this developmental process, by searching for molecular interactors of Lmx1b at the protein level. Initially, affinity-purification of LMX1B-HIS overexpressed protein in MN9D dopaminergic cells followed by mass-spectrometry analysis, resulted in the identification of PSPC1 protein as a possible binding partner of LMX1B. Subsequent immunoprecipitation experiments revealed an interaction between LMX1B and PSPC1 in a larger protein complex also containing PSF. This complex was observed in vitro and in vivo, and we hypothesize that, via PSF and PSPC1, LMX1B may be part of the previously identified Nurr1 transcriptional complex wherein interaction with the co-repressor PSF and the transcription factor Pitx3 is needed to drive expression of Nurr1 target genes in specifying the dopaminergic phenotype. Furthermore, we identified GRLF1, DHX9, MYO1C, HSP70 and TMPO as potential LMX1B interactors. DHX9 and GRLF1 are highly expressed in the developing mdDA neuronal field, and GRLF1 and MYO1C have both been linked to neurite outgrowth. The identification of these proteins suggests that Lmx1b may act directly in the transcriptional activation of Nurr1 target genes and be involved in other processes like neurite outgrowth as well.