Dandy-Walker Phenotype with Brainstem Involvement: 2 Distinct Subgroups with Different Prognosis.

BACKGROUND AND PURPOSE: Although cardinal imaging features for the diagnostic criteria of the Dandy-Walker phenotype have been recently defined, there is a large range of unreported malformations among these patients. The brainstem, in particular, deserves careful attention because malformations in this region have potentially important implications for clinical outcomes. In this article, we offer detailed information on the association of brainstem dysgenesis in a large, multicentric cohort of patients with the Dandy-Walker phenotype, defining different subtypes of involvement and their potential clinical impact. MATERIALS AND METHODS: In this established multicenter cohort of 329 patients with the Dandy-Walker phenotype, we include and retrospectively review the MR imaging studies and clinical records of 73 subjects with additional brainstem malformations. Detailed evaluation of the different patterns of brainstem involvement and their potential clinical implications, along with comparisons between posterior fossa measurements for the diagnosis of the Dandy-Walker phenotype, was performed among the different subgroups of patients with brainstem involvement. RESULTS: There were 2 major forms of brainstem involvement in patients with Dandy-Walker phenotype including the following: 1) the mild form with anteroposterior disproportions of the brainstem structures "only" (57/73; 78%), most frequently with pontine hypoplasia (44/57; 77%), and 2) the severe form with patients with tegmental dysplasia with folding, bumps, and/or clefts (16/73; 22%). Patients with severe forms of brainstem malformation had significantly increased rates of massive ventriculomegaly, additional malformations involving the corpus callosum and gray matter, and interhemispheric cysts. Clinically, patients with the severe form had significantly increased rates of bulbar dysfunction, seizures, and mortality. CONCLUSIONS: Additional brainstem malformations in patients with the Dandy-Walker phenotype can be divided into 2 major subgroups: mild and severe. The severe form, though less prevalent, has characteristic imaging features, including tegmental folding, bumps, and clefts, and is directly associated with a more severe clinical presentation and increased mortality.

Refining the Neuroimaging Definition of the Dandy-Walker Phenotype.

BACKGROUND AND PURPOSE: The traditionally described Dandy-Walker malformation comprises a range of cerebellar and posterior fossa abnormalities with variable clinical severity. We aimed to establish updated imaging criteria for Dandy-Walker malformation on the basis of cerebellar development. MATERIALS AND METHODS: In this multicenter study, retrospective MR imaging examinations from fetuses and children previously diagnosed with Dandy-Walker malformation or vermian hypoplasia were re-evaluated, using the choroid plexus/tela choroidea location and the fastigial recess shape to differentiate Dandy-Walker malformation from vermian hypoplasia. Multiple additional measures of the posterior fossa and cerebellum were also obtained and compared between Dandy-Walker malformation and other diagnoses. RESULTS: Four hundred forty-six examinations were analyzed (174 fetal and 272 postnatal). The most common diagnoses were Dandy-Walker malformation (78%), vermian hypoplasia (14%), vermian hypoplasia with Blake pouch cyst (9%), and Blake pouch cyst (4%). Most measures were significant differentiators of Dandy-Walker malformation from non-Dandy-Walker malformation both pre- and postnatally (P < .01); the tegmentovermian and fastigial recess angles were the most significant quantitative measures. Posterior fossa perimeter and vascular injury evidence were not significant differentiators pre- or postnatally (P > .3). The superior posterior fossa angle, torcular location, and vermian height differentiated groups postnatally (P < .01), but not prenatally (P > .07). CONCLUSIONS: As confirmed by objective measures, the modern Dandy-Walker malformation phenotype is best defined by inferior predominant vermian hypoplasia, an enlarged tegmentovermian angle, inferolateral displacement of the tela choroidea/choroid plexus, an obtuse fastigial recess, and an unpaired caudal lobule. Posterior fossa size and torcular location should be eliminated from the diagnostic criteria. This refined phenotype may help guide future study of the numerous etiologies and varied clinical outcomes.

The ZIC gene family in development and disease.

The human ZIC gene family is comprised of five members encoding zinc-finger transcription factors, which are the vertebrate homologs of the Drosophila odd-paired gene. Mutations in ZIC genes in humans have recently been implicated in a wide variety of congenital malformations, including Dandy-Walker malformation, holoprosencephaly, neural tube defects, and heterotaxy. Mutant analysis of these genes in mice has underscored the conserved developmental roles of these genes. Further, this analysis has begun to elucidate the molecular and developmental mechanisms underlying these important birth defects.

Roof plate and dorsal spinal cord dl1 interneuron development in the dreher mutant mouse.

The establishment of neural circuits in the spinal cord depends on the differentiation of functionally distinct types of neurons in the embryonic neural tube. A number of genes have recently been shown to control the generation of dorsal interneurons through inductive signals provided by the roof plate. The roof plate is a transient signaling center on the dorsal midline of the neural tube that coordinates dorsal CNS development through the action of local peptide signals, primarily the bone morphogenic proteins (BMPs) and the Wingless-related genes (Wnts). The role of the roof plate has become evident through studies of mutations of genes in these gene families, and through several spontaneously occurring mouse mutants, including dreher(J) (dr(J)), all of which cause dorsal neural tube defects. We previously demonstrated that the roof plate is missing in the dreher mouse. Positional cloning of the dreher locus demonstrated that an inactivating point mutation in the LIM homeodomain (HD) transcription factor encoded by the Lmx1a gene, is responsible for the dreher(J) phenotype [Nature, 403 (2000) 764]. Here we report that Lmx1a is first expressed at E8.5 in a small number of cells in the lateral neural plate. As the neural tube closes, Lmx1a expression is restricted to the roof plate. In dr(J)/dr(J), although non-functional Lmx1a is correctly expressed at E8.5-E9.5, its expression is lost in the spinal cord roof plate by E10.5. Coincident with the loss of Lmx1a expression, Bmp expression fails, and the generation and differentiation of the dorsal-most spinal cord neurons, the dl1 interneurons, is abnormal. In dr(J)/dr(J) embryos, defects are evident in the number of dl1 progenitors, as well as in their migration to form the lateral and medial nuclei, and axon patterning, through mechanisms that apparently involve defects in early steps of neuronal polarity. Consistent with the general hypothesis that a failure of roof plate formation and function results in deficits in dorsal patterning of the neural tube, the dreher affects the generation and differentiation of the dl1 interneuron population.

The mouse Dreher gene Lmx1a controls formation of the roof plate in the vertebrate CNS.

In the vertebrate central nervous system (CNS), a cascade of signals that originates in the ectoderm adjacent to the neural tube is propagated by the roof plate to dorsalize the neural tube. Here we report that the phenotype of the spontaneous neurological mutant mouse dreher (dr) results from a failure of the roof plate to develop. Dorsalization of the neural tube is consequently affected: dorsal interneurons in the spinal cord and granule neurons in the cerebellar cortex are lost, and the dorsal vertebral neural arches fail to form. Positional cloning of dreher indicates that the LIM homeodomain protein, Lmx1a, is affected in three different alleles of dreher. Lmx1a is expressed in the roof plate along the neuraxis during development of the CNS. Thus, Lmx1a is required for development of the roof plate and, in turn, for specification of dorsal cell fates in the CNS and developing vertebrae.

Neurogenetics of the cerebellar system.

The development of the cerebellum occurs in four basic steps. During the first epoch, genes that mark the cerebellar territory are expressed in a restricted pattern along the anterioposterior axis of the embryo. In the second, an embryonic region termed the rhombic lip generates precursors of the granule cell population of the cerebellar cortex, and the lateral pontine nucleus and olivary nucleus of the brain stem. In the third period, the program of neurogenesis of the granule neuron gives rise to the formation of the fundamental layers of the cerebellum and to the pattern of foliation. Concomitantly, programs of gene expression define the principal neuronal classes, the granule cell and Purkinje cell, that will establish the cerebellar circuitry in the postnatal period. Understanding the molecular mechanisms underlying these steps of development is likely to yield important insights into malformations such as Joubert syndrome.

Functional analysis of the weaver mutant GIRK2 K+ channel and rescue of weaver granule cells.

In the neurological mutant mouse weaver, granule cell precursors proliferate normally in the external germinal layer of the cerebellar cortex, but fail to differentiate. Granule neurons purified from weaver cerebella have greatly reduced G protein-activated inwardly rectifying K+ currents; instead, they display a constitutive Na+ conductance. Expression of the weaver GIRK2 channel in oocytes confirms that the mutation leads to constitutive activation, loss of monovalent cation selectivity, and increased sensitivity to three channel blockers. Pharmacological blockade of the Na+ influx in weaver granule cells restores their ability to differentiate normally. Thus, Na+ flux through the weaver GIRK2 channel underlies the failure of granule cell development in situ.

Impaired motor learning performance in cerebellar En-2 mutant mice.

Mice homozygous for a null mutation in their En-2 gene exhibit cerebellar neuroanatomical alterations including absence and misplacements of specific fissures and size reduction. The present study investigated cerebellar function by comparing the behavior of age-matched homozygous and heterozygous En-2 mutant and wild-type mice. Motor function of the mutants was found normal in several situations. Habituation to novelty in the open field was not significantly different in mutants. However, in a motor learning paradigm, the rotating rod, the performance of homozygous mutant mice improved significantly less than that of the heterozygous mice which were also significantly impaired compared to wild-type mice. Unlike other cerebellar mutants in which severe motor or sensory defects are obvious, the En-2 mouse model offers a unique tool to study the role of cerebellum in complex behavioral phenomena, including motor learning, without confounding effects.

A role for En-2 and other murine homologues of Drosophila segment polarity genes in regulating positional information in the developing cerebellum.

To gain insight into the molecular genetic basis of cerebellar patterning, the expression patterns of many vertebrate homologues of Drosophila segment polarity genes were examined during normal and abnormal cerebellar development, including members of the En, Wnt, Pax, Gli and Dvl gene families. Five of these genes were found to show transient, spatially restricted patterns of expression. Strikingly, expression of En-2, En-1, Wnt-7B and Pax-2 defined eleven similar sagittal domains at 17.5 dpc, reminiscent of the transient sagittal domains of expression of Purkinje cell markers which have been implicated in cerebellar afferent patterning. Postnatally, transient anterior/posterior differences in expression were observed for En-2, En-1, Gli and Wnt-7B dividing the cerebellum into anterior and posterior regions. The expression patterns of these genes were altered in cerebella of En-2 homozygous mutant mice, which show a cerebellar foliation patterning defect. Strikingly, four of the Wnt-7B expression domains that are adjacent to the En-2 domains are lost in En-2 mutant embryonic cerebella. These studies provide the first evidence of a potential network of regulatory genes that establish spatial cues in the developing cerebellum by dividing it into a grid of positional information required for patterning foliation and afferents. Taken together with previous gene expression studies, our data suggests that eleven sagittal domains and at least two anterior/posterior compartments are the basic elements of spatial information in the cerebellum.

DNA-protein interactions in the Caenorhabditis elegans embryo: oocyte and embryonic factors that bind to the promoter of the gut-specific ges-1 gene.

We describe an experimental system in which to investigate DNA-protein interactions in the early Caenorhabditis elegans embryo. A homogeneous population of developmentally blocked mid-proliferation stage embryos can be produced by exposure to the deoxynucleotide analog fluorodeoxyuridine. These blocked embryos remain viable for days and express a number of biochemical markers of early differentiation, for example, gut granules, the gut esterase ges-1, and two regulatory genes, mab-5 and hlh-1. Using the techniques of gel mobility shift and DNase I footprinting, we show that nuclear extracts prepared from these embryos contain factors that bind to the 5'-promoter sequences of the C. elegans gut-specific ges-1 gene. In particular, we examine a putative gut "activator" region, which was previously identified by deletion-transformation analysis and which contains two copies of a consensus GATA-factor binding sequence. Factors that bind to double-stranded oligonucleotides containing the ges-1 GATA sequences are present predominantly in nuclear extracts of embryos but are found neither in cytoplasmic nor in nuclear extracts of unfertilized oocytes. Two proteins, of 43 and 60 kDa, can be uv-crosslinked to double-stranded oligonucleotides containing the ges-1 GATA sequences. The sizes of these proteins correspond to the sizes expected for the elt-1 protein and for the skn-1 protein, two regulatory factors present in early C. elegans embryos and possible candidates for ges-1 control. However, we show that homozygous deficiency embryos (mDf7/mDf7 embryos and eDf19/eDf19 embryos, both of which lack the elt-1 gene, and nDf41/nDf41 embryos, which have no skn-1 gene), still express the ges-1 esterase. We conclude that neither the elt-1 gene nor the skn-1 gene is necessary zygotically for ges-1 expression. We suggest that neither the elt-1 protein nor the skn-1 protein interacts directly with the ges-1 gene and that the observed binding proteins must correspond to products of other genes. More generally, the present experimental system should allow the biochemical study of any gene expressed during early C. elegans embryogenesis.

Abnormal embryonic cerebellar development and patterning of postnatal foliation in two mouse Engrailed-2 mutants.

The cerebellum is an ideal system to study pattern formation in the central nervous system because of its simple cytoarchitecture and regular organization of folds and neural circuitry. Engrailed-2 (En-2) is expressed in a spatially restricted broad band around the mesencephalic-metencephalic junction, a region from which the cerebellum is derived. Mice homozygous for a targeted deletion of the En-2 homeobox, En-2hd, previously have been shown to have an altered adult cerebellar foliation pattern. To address whether the En-2hd allele was hypomorphic, we generated a putative null mutation that makes an N-terminal deletion (ntd). Mice homozygous for this new mutation, En-2ntd, display an identical cerebellar patterning defect, suggesting that both alleles represent null alleles. We also examined the developmental profile of En-2 homozygous mutant cerebellar foliation. This revealed a complex phenotype of general developmental delay and abnormal formation of specific fissures with the most severe morphological disruptions being limited to the posterior region of the cerebellum. The expression of two transgenes, which express lacZ in lobe-specific patterns in the cerebellum, also was found to be altered in En-2 homozygotes, suggesting possible lobe transformations. Finally, during embryogenesis there was a clear delay in fusion of the cerebellar rudiments at the midline by 15.5 d.p.c. This and the expression pattern of En-2 suggests that although cerebellar foliation is largely a postnatal process, the patterning of the cerebellum may begin during embryogenesis and that En-2 plays a critical role in this early process.

Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum.

We have raised an antiserum, designated alpha Enhb-1, to a portion of the mouse En-2 protein containing the homeodomain. The antiserum detects both the En-1 and En-2 proteins in mouse, chick and Xenopus embryos by Western blot analysis. Using whole-mount immunohistochemistry, combined in some cases with scanning electron microscopy, we have examined the distribution of the proteins in the early embryos of these species. The major features of expression were similar. The initial production of En protein occurred, just before or during the formation of the first somites, in a band of the anterior neural plate in the prospective mid/hindbrain region. Later in development En-1 protein accumulated in the ventral ectoderm of the developing mouse and chick limb buds, indicating that a dorsal-ventral polarity is present as soon as any limb bud swelling is apparent and that, at least in the mouse, this polarity is established independently of the apical ectodermal ridge. In all three species, alpha Enhb-1 bound to a subset of ventro-lateral differentiating neurons in the spinal cord and hindbrain and their pattern of birth in the mouse reflected the division of the hindbrain into rhombomeres. En-1 protein also accumulated in a lateral stripe of dermatome in the mouse and chick, indicating a dorsal-ventral subdivision of this tissue. The results show that En expression is a good marker for pattern formation in a variety of tissues and will be useful in experimental studies designed to characterize further these processes.