Wing Yip Tam

Foxp4 is essential in maintenance of purkinje cell dendritic arborization in the mouse cerebellum

Purkinje cells (PCs) are one of the principal neurons in the cerebellar cortex that play a central role in the coordination of fine-tuning body movement and balance. To acquire normal cerebellum function, PCs develop extensive dendritic arbors that establish synaptic connections with the parallel fibers of granule cells to form the proper neuronal circuitry. Therefore, dendritic arborization of PCs is an important developmental step to construct the mature neural network in the cerebellum. However, the genetic control of this process is not fully understood. In this study, Foxp4, a forkhead transcription factor that is expressed specifically in migrating and mature PCs of cerebellum from embryonic stages to adulthood, was knocked down by small interfering RNA (siRNA) in organotypic cerebellar slice culture. When Foxp4 expression was knocked down at postnatal day 5 (P5), no abnormalities for early dendritic remodeling of PCs were observed. However, when Foxp4 was knocked down in P10 cerebellar slices, the organization of PC dendritic arbors was highly impaired, leaving hypoplastic but non-apoptotic cell bodies. The radial alignment of Bergmann glial fibers that associated with PC dendrites was also lost. These results suggest that Foxp4 is dispensable for the early PC dendrite outgrowth, but is essential for the maintenance of PC dendritic arborization and subsequent association with Bergmann glial fibers.

Tracking Down the Migration of Mouse Neural Crest Cells

During early embryonic development, cell migration is one of the most important morphogenetic processes. Neural crest cells arise from the dorsal part of the neural tube and migrate along different pathways to numerous locations where they differentiate into a variety of tissues. In the mouse, studies of neural crest cell migration have been difficult partly because of the absence of specific markers which can label neural crest cells throughout their migration from their origin to the site of differentiation. Nevertheless, the use of different experimental strategies involving extrinsic, intrinsic or genetic cell markers has already led to a good understanding of this migration. In our studies, extrinsic markers such as wheat germ agglutinin-gold conjugates and DiI and genetic markers including Hoxb2-lacZ and green fluorescent protein have been employed in tracing migrating neural crest cells. The labelling procedures and the strength and weaknesses of the tracing methods are reviewed herein.

Lhx1/5 control dendritogenesis and spine morphogenesis of Purkinje cells via regulation of Espin

In the cerebellar cortex, Purkinje cells (PCs) receive signals from different inputs through their extensively branched dendrites and serve as an integration centre. Defects in the dendritic development of PCs thus disrupt cerebellar circuitry and cause ataxia. Here we report that specific inactivation of both Lhx1 and Lhx5 in postnatal PCs results in ataxic mutant mice with abnormal dendritic development. The PCs in the mutants have reduced expression of Espin, an F-actin cytoskeleton regulator. We show that Espin expression is transcriptionally activated by Lhx1/5. Downregulation of Espin leads to F-actin mislocalization, thereby impairing dendritogenesis and dendritic spine maturation in the PCs. The mutant PCs therefore fail to form proper synapses and show aberrant electrophysiological properties. By overexpressing Espin, we can successfully rescue the defects in the mutant PCs. Our findings suggest that Lhx1/5, through regulating Espin expression, control dendritogenesis and spine morphogenesis in postnatal PCs.

Transmembrane 6 superfamily 1 (Tm6sf1) is a novel lysosomal transmembrane protein

The lysosome is a membrane-bound organelle involved in the turnover of various intracellular and extracellular macromolecules. These are degraded by acidic hydrolases in the lumen of lysosome. The lysosomal membrane is important not only in retaining the acidic hydrolases to protect cells against cytosolic proteolysis, but it also facilitates protein trafficking though organelle fusion. In this study, we report on a novel lysosomal membrane protein transmembrane 6 superfamily 1 (Tm6sf1). Expression of Tm6sf1-DsRed fusion proteins in HEK293A cells displayed punctate or ringlike vesicles, which colocalized with conventional lysosome markers including LAMP1/2, RAB7, and Rnf167. Using fluorescence time-lapse live cell imaging, we demonstrated the fusion of Tm6sf1 vesicles with lysosomes and the integration of Tm6sf1 into the lysosomal membrane. We also examined the expression of Tm6sf1 in mouse tissues and found immunopositive signals in major organs such as the cerebellum, kidney, and intestine. These data suggest that Tm6sf1 is a widely expressed lysosomal transmembrane protein and can be used as a novel marker of lysosome.

Early Sacral Neural Crest Migration in Dominant megacolon Mouse Embryos

Hirschsprung’s disease, or congenital megacolon, is the most common gastrointestinal motility disorder in newborns. The prominent feature of Hirschsprung’s disease is an abnormal dilatation of the distal colon resulting from a regional absence or reduction of enteric ganglion cells. It has been known that all intrinsic enteric ganglion cells are derived from neural crest cells, which migrate along defined pathways from the neural tube (embryonic central nervous system) to the gut during embryonic development. Recent studies on avian embryos have also indicated that neural crest cells at the sacral level contribute a significant number of enteric neurons to the hindgut, the region of the gut where aganglionosis is usually detected in Hirschsprung’s disease. In the present study, we aimed to identify anomalies in the early migration of sacral neural crest cells in the Dominant megacolon(Dom) mouse mutant, a model for Hirschsprung’s disease. A combination of whole embryo culture, in situ cell labeling and histochemical staining was used to follow the early sacral neural crest cell migration. In the wild-type embryos, when sacral neural crest cells caudal to the 24th somite were labeled at embryonic day 10.0 (E10.0), labeled cells were found in the mesenchyme on the two sides of neural tube and many of them resided in the region of dorsal root ganglia at E11.0. Some of them were also found in the region around the dorsal aorta. In embryos heterozygous and homozygous for Dom, similar distribution and migratory pattern were found, indicating that the early migration of sacral neural crest cells was not affected in the mutant. Our results hence implicated that anomalies in the early sacral crest cell migration are unlikely to be a cause of aganglionosis in the hindgut of the Dom mutant.

Abnormalities of Interstitial Cells of Cajal in Dominant Megacolon Mice

Interstitial cells of Cajal (ICCs) in the gastrointestinal tract are a group of cells interacting with enteric neurons and smooth muscle cells. They serve as mediators of neurotransmission and pacemakers of the peristaltic movement of the gut. Previous investigations on patients with Hirschsprung’s disease and mice with aganglionic megacolon showed contrasting results on the relationship between the abnormalities of ICCs and enteric neurons. In order to ascertain whether the development of ICCs is also perturbed in the aganglionic segment of the gut, we used Dominant megacolon (Dom) mice as an animal model and an antibody specific to c-kit (a tyrosine kinase receptor expressed in ICCs) to examine the spatial distribution of ICCs in the developing and adult colon. Our results showed that in the wild-type adult colon, ICCs were bipolar in shape in the smooth muscle layer and dense networks of ICCs were observed in the myenteric and deep muscular plexuses, whereas in the aganglionic colon of Dom heterozygous mice, no ICCs could be found in the smooth muscle layer and disrupted patterns of ICC networks were observed in the myenteric and deep muscular plexuses. On 14.5 days of development, the levels of expression of c-kit and its ligand (stem cell factor) were similar in the wild-type, heterozygous and homozygous Dom embryos. However, by 18.5 days, the numbers of ICCs were greatly reduced in the myenteric and submucosal plexuses of the terminal colon of the heterozygous embryos comparing with the wild-type counterparts. Based on our results, we postulate that the abnormal development of ICCs may disrupt the neurotransmission and pacemaker activity of the gut, leading to interrupted peristalsis.