Beverly A. Karpinski

Dysphagia and disrupted cranial nerve development in a mouse model of DiGeorge/22q11 Deletion Syndrome

ABSTRACT We assessed feeding-related developmental anomalies in the LgDel mouse model of chromosome 22q11 deletion syndrome (22q11DS), a common developmental disorder that frequently includes perinatal dysphagia – debilitating feeding, swallowing and nutrition difficulties from birth onward – within its phenotypic spectrum. LgDel pups gain significantly less weight during the first postnatal weeks, and have several signs of respiratory infections due to food aspiration. Most 22q11 genes are expressed in anlagen of craniofacial and brainstem regions critical for feeding and swallowing, and diminished expression in LgDel embryos apparently compromises development of these regions. Palate and jaw anomalies indicate divergent oro-facial morphogenesis. Altered expression and patterning of hindbrain transcriptional regulators, especially those related to retinoic acid (RA) signaling, prefigures these disruptions. Subsequently, gene expression, axon growth and sensory ganglion formation in the trigeminal (V), glossopharyngeal (IX) or vagus (X) cranial nerves (CNs) that innervate targets essential for feeding, swallowing and digestion are disrupted. Posterior CN IX and X ganglia anomalies primarily reflect diminished dosage of the 22q11DS candidate gene Tbx1. Genetic modification of RA signaling in LgDel embryos rescues the anterior CN V phenotype and returns expression levels or pattern of RA-sensitive genes to those in wild-type embryos. Thus, diminished 22q11 gene dosage, including but not limited to Tbx1, disrupts oro-facial and CN development by modifying RA-modulated anterior-posterior hindbrain differentiation. These disruptions likely contribute to dysphagia in infants and young children with 22q11DS.

Ranbp1, Deleted in DiGeorge/22q11.2 Deletion Syndrome, is a Microcephaly Gene That Selectively Disrupts Layer 2/3 Cortical Projection Neuron Generation

Ranbp1, a Ran GTPase-binding protein implicated in nuclear/cytoplasmic trafficking, is included within the DiGeorge/22q11.2 Deletion Syndrome (22q11.2 DS) critical region associated with behavioral impairments including autism and schizophrenia. Ranbp1 is highly expressed in the developing forebrain ventricular/subventricular zone but has no known obligate function during brain development. We assessed the role of Ranbp1 in a targeted mouse mutant. Ranbp1(-/-) mice are not recovered live at birth, and over 60% of Ranbp1(-/-) embryos are exencephalic. Non-exencephalic Ranbp1(-/-) embryos are microcephalic, and proliferation of cortical progenitors is altered. At E10.5, radial progenitors divide more slowly in the Ranpb1(-/-) dorsal pallium. At E14.5, basal, but not apical/radial glial progenitors, are compromised in the cortex. In both E10.5 apical and E14.5 basal progenitors, M phase of the cell cycle appears selectively retarded by loss of Ranpb1 function. Ranbp1(-/-)-dependent proliferative deficits substantially diminish the frequency of layer 2/3, but not layer 5/6 cortical projection neurons. Ranbp1(-/-) cortical phenotypes parallel less severe alterations in LgDel mice that carry a deletion parallel to many (but not all) 22q11.2 DS patients. Thus, Ranbp1 emerges as a microcephaly gene within the 22q11.2 deleted region that may contribute to altered cortical precursor proliferation and neurogenesis associated with broader 22q11.2 deletion.

Hard to swallow: Developmental biological insights into pediatric dysphagia.

Pediatric dysphagia-feeding and swallowing difficulties that begin at birth, last throughout childhood, and continue into maturity--is one of the most common, least understood complications in children with developmental disorders. We argue that a major cause of pediatric dysphagia is altered hindbrain patterning during pre-natal development. Such changes can compromise craniofacial structures including oropharyngeal muscles and skeletal elements as well as motor and sensory circuits necessary for normal feeding and swallowing. Animal models of developmental disorders that include pediatric dysphagia in their phenotypic spectrum can provide mechanistic insight into pathogenesis of feeding and swallowing difficulties. A fairly common human genetic developmental disorder, DiGeorge/22q11.2 Deletion Syndrome (22q11DS) includes a substantial incidence of pediatric dysphagia in its phenotypic spectrum. Infant mice carrying a parallel deletion to 22q11DS patients have feeding and swallowing difficulties that approximate those seen in pediatric dysphagia. Altered hindbrain patterning, craniofacial malformations, and changes in cranial nerve growth prefigure these difficulties. Thus, in addition to craniofacial and pharyngeal anomalies that arise independently of altered neural development, pediatric dysphagia may result from disrupted hindbrain patterning and its impact on peripheral and central neural circuit development critical for feeding and swallowing. The mechanisms that disrupt hindbrain patterning and circuitry may provide a foundation to develop novel therapeutic approaches for improved clinical management of pediatric dysphagia.

Functional Divergence of the Nuclear Receptor NR2C1 as a Modulator of Pluripotentiality During Hominid Evolution.

Genes encoding nuclear receptors (NRs) are attractive as candidates for investigating the evolution of gene regulation because they (1) have a direct effect on gene expression and (2) modulate many cellular processes that underlie development. We employed a three-phase investigation linking NR molecular evolution among primates with direct experimental assessment of NR function. Phase 1 was an analysis of NR domain evolution and the results were used to guide the design of phase 2, a codon-model-based survey for alterations of natural selection within the hominids. By using a series of reliability and robustness analyses we selected a single gene, NR2C1, as the best candidate for experimental assessment. We carried out assays to determine whether changes between the ancestral and extant NR2C1s could have impacted stem cell pluripotency (phase 3). We evaluated human, chimpanzee, and ancestral NR2C1 for transcriptional modulation of Oct4 and Nanog (key regulators of pluripotency and cell lineage commitment), promoter activity for Pepck (a proxy for differentiation in numerous cell types), and average size of embryological stem cell colonies (a proxy for the self-renewal capacity of pluripotent cells). Results supported the signal for alteration of natural selection identified in phase 2. We suggest that adaptive evolution of gene regulation has impacted several aspects of pluripotentiality within primates. Our study illustrates that the combination of targeted evolutionary surveys and experimental analysis is an effective strategy for investigating the evolution of gene regulation with respect to developmental phenotypes.

A cellular and molecular mosaic establishes growth and differentiation states for cranial sensory neurons.

We compared apparent origins, cellular diversity and regulation of initial axon growth for differentiating cranial sensory neurons. We assessed the molecular and cellular composition of the developing olfactory and otic placodes, and cranial sensory ganglia to evaluate contributions of ectodermal placode versus neural crest at each site. Special sensory neuron populations-the olfactory and otic placodes, as well as those in vestibulo-acoustic ganglion- are entirely populated with cells expressing cranial placode-associated, rather than neural crest-associated markers. The remaining cranial sensory ganglia are a mosaic of cells that express placode-associated as well as neural crest-associated markers. We found two distinct populations of neural crest in the cranial ganglia: the first, as expected, is labeled by Wnt1:Cre mediated recombination. The second is not labeled by Wnt1:Cre recombination, and expresses both Sox10 and FoxD3. These populations-Wnt1:Cre recombined, and Sox10/Foxd3-expressing- are proliferatively distinct from one another. Together, the two neural crest-associated populations are substantially more proliferative than their placode-associated counterparts. Nevertheless, the apparently placode- and neural crest-associated populations are similarly sensitive to altered signaling that compromises cranial morphogenesis and differentiation. Acute disruption of either Fibroblast growth factor (Fgf) or Retinoic acid (RA) signaling alters axon growth and cell death, but does not preferentially target any of the three distinct populations. Apparently, mosaic derivation and diversity of precursors and early differentiating neurons, modulated uniformly by local signals, supports early cranial sensory neuron differentiation and growth.

Foxd4 is essential for establishing neural cell fate and for neuronal differentiation

Many molecular factors required for later stages of neuronal differentiation have been identified; however, much less is known about the early events that regulate the initial establishment of the neuroectoderm. We have used an in vitro embryonic stem cell (ESC) differentiation model to investigate early events of neuronal differentiation and to define the role of mouse Foxd4, an ortholog of a forkhead‐family transcription factor central to Xenopus neural plate/neuroectodermal precursor development. We found that Foxd4 is a necessary regulator of the transition from pluripotent ESC to neuroectodermal stem cell, and its expression is necessary for neuronal differentiation. Mouse Foxd4 expression is not only limited to the neural plate but it is also expressed and apparently functions to regulate neurogenesis in the olfactory placode. These in vitro results suggest that mouse Foxd4 has a similar function to its Xenopus ortholog; this was confirmed by successfully substituting murine Foxd4 for its amphibian counterpart in overexpression experiments. Thus, Foxd4 appears to regulate the initial steps in establishing neuroectodermal precursors during initial development of the nervous system.

Testicular receptor 2, Nr2c1, is associated with stem cells in the developing olfactory epithelium and other cranial sensory and skeletal structures

Comparative genomic analysis of the nuclear receptor family suggests that the testicular receptor 2, Nr2c1, undergoes positive selection in the human-chimpanzee clade based upon a significant increase in nonsynonymous compared to synonymous substitutions. Previous in situ analyses of Nr2c1 lacked the temporal range and spatial resolution necessary to characterize cellular expression of this gene from early to mid gestation, when many nuclear receptors are key regulators of tissue specific stem or progenitor cells. Thus, we asked whether Nr2c1 protein is associated with stem cell populations in the mid-gestation mouse embryo. Nr2c1 is robustly expressed in the developing olfactory epithelium. Its expression in the olfactory epithelium shifts from multiple progenitor classes at early stages to primarily transit amplifying cells later in olfactory epithelium development. In the early developing central nervous system, Nr2c1 is limited to the anterior telencephalon/olfactory bulb anlagen, coincident with Nestin-positive neuroepithelial stem cells. Nr2c1 is also seen in additional cranial sensory specializations including cells surrounding the mystacial vibrissae, the retinal pigment epithelium and Scarpas ganglion. Nr2c1 was also detected in a subset of mesenchymal cells in developing teeth and cranial bones. The timing and distribution of embryonic expression suggests that Nr2c1 is primarily associated with the early genesis of mammalian cranial sensory neurons and craniofacial skeletal structures. Thus, Nr2c1 may be a candidate for mediating parallel adaptive changes in cranial neural sensory specializations such as the olfactory epithelium, retina and mystacial vibrissae and in non-neural craniofacial features including teeth.

Light/Electron Microscopy Correlative Workflow Involving Leica Stage Overview and Thermo Fisher/FEI MAPS Platforms Facilitate Neuronal Identification

Combing light microscopy (LM) and electron microscopy (EM) in Correlative Light Electron Microscopy workflows (re)capture it’s place among state of the art imaging technologies available on both light and electron microscopes. Each of the imaging modalities presents advantages and limitations, whereas with complementary approaches, one expects to overcome limitations and amplify advantages. Light microscopy (LM) (either confocal or wide field) is broadly used in biomedical research because of the convenience and less common because of the analytical capabilities offered by the photon. A wide variety of cellular labels are available along with reporting fluorescence proteins whose expression can be genetically promoted by wide variety of genes. Moreover, LM imaging can be employed in living specimens where it can provide time-resolved data sets with meaningful information on biological time scale. Although over the past decade a significant effort has been made to overcome the diffraction limitation imposed by the photon, LM is still a diffraction-limited technique. Scanning electron Microscopy (SEM) naturally provides biologically relevant resolution. Routinely, images with a resolution of a few nanometers can be obtained on many of the modern SEMs. This is the kind of resolution required for reliable characterization of cellular and subcellular morphology of normal and abnormal neuronal circuitry. The latter approach offers full spectrum of morphological characterization, but the cells and tissues need to be strongly fixed. Here we provide a simple workflow, using different LM imaging modalities to facilitate the identification of an individual neuron in trigeminal explant cell culture followed by SEM. The aim of the study is to evaluate the axonal growth and the morphology of growth cone.