Cairine Logan

Validating In Utero Electroporation for the Rapid Analysis of Gene Regulatory Elements in the Murine Telencephalon

With the ultimate goal of understanding how genetic modules have evolved in the telencephalon, we set out to modernize the functional analysis of cross‐species cis‐regulatory elements in mouse. In utero electroporation is rapidly replacing transgenesis as the method of choice for gain‐ and loss‐of‐function studies in the murine telencephalon, but the application of this technique to the analysis of transcriptional regulation has yet to be fully explored and exploited. To empirically define the developmental stages required to target specific populations of neurons in the dorsal telencephalon, or pallium, which gives rise to the neocortex in mouse, we performed a temporal and spatial analysis of the migratory properties of electroporated versus birth‐dated cells. Next, we compared the activities of two known Ngn2 enhancers via transgenesis and in utero electroporation, demonstrating that the latter technique more faithfully reports the endogenous telencephalic expression pattern observed in an Ngn2lacZ knock‐in line. Finally, we used this approach to test the telencephalic activities of a series of deletion constructs comprised of the zebrafish ER81 upstream regulatory region, allowing us to identify a previously uncharacterized enhancer that displays cross‐species activity in the murine piriform cortex and lateral neocortex, yet not in more medial domains of the forebrain. Taken together, our data supports the contention that in utero technology can be exploited to rapidly examine the architecture and evolution of pallial‐specific cis‐regulatory elements. Developmental Dynamics 236:1273–1286, 2007.

Expression and function of bone morphogenetic proteins in the development of the embryonic endocardial cushions.

Bone morphogenetic proteins (BMPs) are considered to be significant factors in the morphogenesis of the endocardial cushions of the developing embryonic heart. Previous studies have suggested that they are involved in the epithelial–mesenchymal transformation and migration of the cells forming the cushions, or in triggering an apoptotic cascade in a sub-population of cushion cells. We investigated the expression and function of BMP2 and BMP4 proteins in the developing heart of the chick and mouse embryos. In the chick, by immunocytochemistry, we find expression of BMP2 protein in the endocardial cushions of the outflow tract (OT) and atrio-ventricular (AV) regions at embryonic days (ED) 5–6, as well as in adjacent myocardial layers. Immunoblotting indicated that such expression persisted through ED 4–7, but peaked at ED4–5 in the OT and 5–6 in the AV cushions. This temporal sequence correlated with the peaks of apoptotic cell death found previously in the OT and AV cushions of the chick embryo. At equivalent stages in mouse, no such expression of BMP2 was found in the cushions, although expression was detected in adjacent myocardial layers. In the case of BMP4, in both chick and mouse, expression was found only in the myocardia and not in the cushions. Furthermore, BMP-specific receptors were found in the cushions, but not the myocardia, in both the AV and OT regions of the chick embryo. These results provide circumstantial evidence to support the contention that BMPs, originating from the myocardium, could be significant in the induction of apoptosis in chick embryo cushion cells, and confirms that there is species-specific variation in the expression pattern of BMP proteins, as had been predicted from previous studies of mRNA expression. Culture media conditioned by the growth of tissues from various regions of the developing heart were tested for their ability to induce apoptosis in cushion cells in culture. It was found that medium derived from the myocardia induced significant levels of cell death in the cushion cells, and that BMP4 could be detected in such media; however, retroviral over-expression of constitutively active (CA) and dominant-negative (DN) isoforms of BMP-specific receptors 1A and 1B (BMPR-1A and BMPR-1B) in cultured cells of the AV cushions did not alter levels of apoptosis or cell proliferation. Similar over-expression in cultured endocardial cells resulted in a significant change in cell shape, from endothelial to fibroblastic, with BMPR-1A CA and BMPR-1B DN, suggesting an influence of these receptors on cell transformation and/or cell migration. Taken together, these results provide support for the contention that BMP2 and BMP4 are important factors in the phenotypic transformational events involved in the morphogenesis of the chick embryo endocardial cushions, and could be involved in the induction of apoptosis in the cushion cells.

Expression and function of Bapx1 during chick limb development

The homeobox-containing transcription factor Bapx1 (also known as Nkx3.2) is crucial for development of the axial skeleton and parts of the chondrocranium. Here we describe the detailed expression of Bapx1 during chick limb development and show that in contrast to its expression in the axial skeleton, Bapx1 is expressed after the commitment to chondrogenesis. Bapx1 is initially expressed throughout the developing skeletal elements prior to the overt differentiation of the distinct chondrogenic layers. Once distinct layers (proliferating, prehypertrophic and hypertrophic) have formed, Bapx1 expression is restricted to the proliferating chondrocytes. Bapx1 transcripts are excluded from the articular cartilage. A second homeobox-containing transcription factor, Barx1, is expressed in a complementary fashion in the developing joint and articular cartilage. Interestingly, in vitro functional analyses showed that Bapx1 overexpression in micromass cultures increased both matrix production and nodule number suggesting that Bapx1 is sufficient to promote chondrogenesis in the limb. In contrast, Barx1 had the opposite effect on nodule number suggesting that it has an inhibitory effect on chondrogenic initiation consistent with its expression in the developing joint. A slight increase in matrix levels was also observed consistent with its expression in the articular chondrocytes. Finally, we show that Bapx1 is also expressed in the soft tissues such as the developing tendons, muscle sheaths and surrounding mesenchyme, and therefore may have additional as yet uncharacterized roles in limb morphogenesis.

Whole genome microarray analysis of chicken embryo facial prominences

The face is one of the three regions most frequently affected by congenital defects in humans. To understand the molecular mechanisms involved, it is necessary to have a more complete picture of gene expression in the embryo. Here, we use microarrays to profile expression in chicken facial prominences, post neural crest migration and before differentiation of mesenchymal cells. Chip‐wide analysis revealed that maxillary and mandibular prominences had similar expression profiles while the frontonasal mass chips were distinct. Of the 3094 genes that were differentially expressed in one or more regions of the face, a group of 56 genes was subsequently validated with quantitative polymerase chain reaction (QPCR) and a subset examined with in situ hybridization. Microarrays trends were consistent with the QPCR data for the majority of genes (81%). On the basis of QPCR and microarray data, groups of genes that characterize each of the facial prominences can be determined. Developmental Dynamics 239:574–591, 2010.

A BMP-mediated transcriptional cascade involving Cash1 and Tlx-3 specifies first-order relay sensory neurons in the developing hindbrain

The divergent homeobox-containing transcription factor, Tlx-3 (also known as Hox11L2/Rnx), is required for proper formation of first-order relay sensory neurons in the developing vertebrate brainstem. To date, however, the inductive signals and transcriptional regulatory cascade underlying their development are poorly understood. We previously isolated the chick Tlx-3 homologue and showed it is expressed early (i.e. beginning at HH15) in distinct subcomponents of both the trigeminal/solitary and vestibular nuclei. Here we show via in vivo rhombomere inversions that expression of Tlx-3 is under control of local environmental signals. Our RNA in situ analysis shows expression of the BMP-specific receptor, Bmpr-1b, correlates well with Tlx-3. Furthermore, manipulation of the BMP signaling pathway in vivo via electroporation of expression vectors encoding either BMP or NOGGIN coupled with MASH1 gain-of-function experiments demonstrate that a BMP-mediated transcriptional cascade involving Cash1 and Tlx-3 specifies first-order relay sensory neurons in the developing brainstem. Notably, high-level Noggin misexpression results in an increase in newly differentiated Tlx-3+ neurons that correlates with a corresponding increase in the number of Calretinin+ neurons in vestibular nuclei at later developmental stages strongly suggesting that Tlx-3, in addition to being required for proper formation of somatic as well as visceral sensory neurons in the trigeminal and solitary nuclei, respectively, is sufficient for proper formation of special somatic sensory neurons in vestibular nuclei.

Onset of Tlx‐3 expression in the chick cerebellar cortex correlates with the morphological development of fissures and delineates a posterior transverse boundary

Recent studies have shown that the mammalian cerebellar cortex can be subdivided into a reproducible array of zones and stripes. In particular, discontinuous patterns of gene expression together with mutational analysis suggest that there are at least four distinct transverse zones along the rostrocaudal axis in mouse: the anterior zone (lobules I–V), the central zone (lobules VI and VII), the posterior zone (lobules VIII and IX), and the nodular zone (lobule X). Here we show that the divergent homeobox‐containing transcription factor, Tlx‐ 3 (also known as Hox11L2 or Rnx) is transiently expressed in external granule cells in a distinct transverse domain of the developing chick cerebellar cortex. Expression is first detected at Hamburger and Hamilton (HH) stage 35. Interestingly, Tlx‐3 mRNA expression is initially confined to, and coincident with, the morphological development of fissures. Slightly later, at HH stage 38, expression extends throughout the developing external granular layer (EGL) of lobules I–IXab. Notably, no Tlx‐3 expression was detected in lobules IXc and X at any developmental time point examined. Expression is noticeably stronger in nonproliferating cells located in the deep layer of the EGL. Tlx‐3 expression is downregulated as granule cells migrate inward to form the internal granule layer and is undetectable shortly after birth. These results suggest that Tlx‐3 is expressed as granule cells become postmitotic and suggest that Tlx‐3 may play a role in the differentiation of distinct neuronal populations in the cerebellum. J. Comp. Neurol. 448:138–149, 2002.

Geometric Morphometrics and the Study of Development

Even though developmental biology seeks to provide developmental explanations for morphological variation, the quantification of morphological variation has been regarded as peripheral to the mechanistic study of development. In this chapter, we argue that this is now changing because the rapidly advancing knowledge of development in post-genomic biology is creating a need for more refined measurements of the morphological changes produced by genetic perturbations or treatments. This need, in turn, is driving the development of new morphometric methods that allow the rapid and meaningful integration of molecular, cellular and morphometric data. We predict that such integration will offer new ways of looking at development, which will lead to significant advances in the study of dysmorphology and also the relationship between the generation of variation through development and its transformation through evolutionary history.

Ltrk is differentially expressed in developing and adult neurons of the Lymnaea central nervous system.

The Trk receptor family plays diverse roles in both development and plasticity of the vertebrate nervous system. Ltrk is a related receptor that is expressed in the CNS of the mollusk Lymnaea, although little is known of its cellular distribution. This study provides three independent lines of evidence (based on RT‐PCR, in situ hybridization, and immunohistochemistry) that Ltrk is universally expressed by neurons and dorsal body cells of both the juvenile and the adult Lymnaea CNS. The highest level of expression by neuronal somata occurs in the late juvenile stage, whereas axon collaterals express high levels throughout the animals life span. Our data support multifunctional roles for Ltrk that parallel those of its mammalian counterparts. J. Comp. Neurol. 487:240–254, 2005.

Isolation and characterization of the leucine-rich proteoglycan nyctalopin gene (cNyx) from chick

We describe the isolation and molecular characterization of the chick ortholog of nyctalopin (NYX), the gene responsible for X-linked complete congenital stationary night blindness (CSNB1, also known as cCSNB). Chick Nyx (cNyx) comprises four exons spanning approximately 6.2 kb on Chromosome 1 and encodes a protein of 473 amino acids that shares 55% identity overall with its human counterpart. cNyx is expressed in both the developing and the fully differentiated retina. Transcripts are localized primarily to cells within the outer half of the inner nuclear layer (INL) and the ganglion cell layer (GCL), a pattern consistent with the principal electrophysiologic findings in CSNB1 that suggest a main defect in depolarizing ON-bipolar cells normally located in the outer half of the INL. Expression (albeit weaker) was also detected in the cerebrum and cerebellum and in non-neuronal tissues. Finally, we also report the identification of three novel splice variants, one of which predominates in the retina.

Hamartoma-like lesions in the mouse retina: an animal model of Pten hamartoma tumour syndrome

ABSTRACT PTEN hamartoma tumour syndrome (PHTS) is a heterogeneous group of rare, autosomal dominant disorders associated with PTEN germline mutations. PHTS patients routinely develop hamartomas, which are benign tissue overgrowths comprised of disorganized ‘normal’ cells. Efforts to generate PHTS animal models have been largely unsuccessful due to the early lethality of homozygous germline mutations in Pten, together with the lack of hamartoma formation in most conditional mutants generated to date. We report herein a novel PHTS mouse model that reproducibly forms hamartoma-like lesions in the central retina by postnatal day 21. Specifically, we generated a Pten conditional knockout (cKO) using a retinal-specific Pax6::Cre driver that leads to a nearly complete deletion of Pten in the peripheral retina but produces a mosaic of ‘wild-type’ and Pten cKO cells centrally. Structural defects were only observed in the mosaic central retina, including in Müller glia and in the outer and inner limiting membranes, suggesting that defective mechanical integrity partly underlies the hamartoma-like pathology. Finally, we used this newly developed model to test whether rapamycin, an mTOR inhibitor that is currently the only PHTS therapy, can block hamartoma growth. When administered in the early postnatal period, prior to hamartoma formation, rapamycin reduces hamartoma size, but also induces new morphological abnormalities in the Pten cKO retinal periphery. In contrast, administration of rapamycin after hamartoma initiation fails to reduce lesion size. We have thus generated and used an animal model of retinal PHTS to show that, although current therapies can reduce hamartoma formation, they might also induce new retinal dysmorphologies. This article has an associated First Person interview with the first author of the paper. Summary: The authors present the first PHTS animal model that successfully recapitulates hamartoma formation in the retina and can be used to assess drug therapies.