Ascl1 participates in Cajal-Retzius cell development in the neocortex.

Cajal-Retzius cells are essential pioneer neurons that guide neuronal migration in the developing neocortex. During development, Cajal-Retzius cells arise from distinct progenitor domains that line the margins of the dorsal telencephalon, or pallium. Here, we show that the proneural gene Ascl1 is expressed in Cajal-Retzius cell progenitors in the pallial septum, ventral pallium, and cortical hem. Using a short-term lineage trace, we demonstrate that it is primarily the Ascl1-expressing progenitors in the pallial septum and ventral pallium that differentiate into Cajal-Retzius cells. Accordingly, we found a small, albeit significant reduction in the number of Reelin(+) and Trp73(+) Cajal-Retzius cells in the Ascl1(-/-) neocortex. Conversely, using a gain-of-function approach, we found that Ascl1 induces the expression of both Reelin, a Cajal-Retzius marker, and Tbr1, a marker of pallial-derived neurons, in a subset of early-stage pallial progenitors, an activity that declines over developmental time. Taken together, our data indicate that the proneural gene Ascl1 is required and sufficient to promote the differentiation of a subset of Cajal-Retzius neurons during early neocortical development. Notably, this is the first study that reports a function for Ascl1 in the pallium, as this gene is best known for its role in specifying subpallial neuronal identities.

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.

Fast interactive integration of cross-sectional image datasets and surface data for morphometric analysis.

To investigate external facial morphology and cell proliferation patterns and their relationship with cleft lip malformation in mice, we need to compare samples of mice tissue photographs and surface reconstructions from micro-CT scans obtained from mouse embryos. Tissue samples obtained through digital photography are typically misaligned with respect to each other, which prevents further analysis. We have developed a system for fast interactive alignment of these image stacks for volume reconstruction and data visualization and analysis in 3D. The system is designed to work in multiprocessor environments and can utilize an arbitrary number of processors, cutting down significantly the turnaround time and allowing users to quickly process sets of hundreds of high resolution images using a combination of automated and interactive tools. Additional modules are used to reconstruct the shape of the original subject. Our system is interactive, fully scalable and can be applied to any photographic sliced dataset, regardless of subject and reduces significantly the processing time for stack alignment.

Plag1 and Plagl2 have overlapping and distinct functions in telencephalic development.

The Plag gene family has three members; Plagl1/Zac1, which is a tumor suppressor gene, and Plag1 and Plagl2, which are proto-oncogenes. All three genes are known to be expressed in embryonic neural progenitors, and Zac1 regulates proliferation, neuronal differentiation and migration in the developing neocortex. Here we examined the functions of Plag1 and Plagl2 in neocortical development. We first attempted, and were unable to generate, E12.5 Plag1;Plagl2 double mutants, indicating that at least one Plag1 or Plagl2 gene copy is required for embryonic survival. We therefore focused on single mutants, revealing a telencephalic patterning defect in E12.5 Plagl2 mutants and a proliferation/differentiation defect in Plag1 mutant neocortices. Specifically, the ventral pallium, a dorsal telencephalic territory, expands into the ventral telencephalon in Plagl2 mutants. In contrast, Plag1 mutants develop normal regional territories, but neocortical progenitors proliferate less and instead produce more neurons. Finally, in gain-of-function studies, both Plag1 and Plagl2 reduce neurogenesis and increase BrdU-uptake, indicative of enhanced proliferation, but while Plagl2 effects on proliferation are more immediate, Plag1 effects are delayed. Taken together, we found that the Plag proto-oncogenes genes are essential regulators of neocortical development and although Plag1 and Plagl2 functions are similar, they do not entirely overlap. This article has an associated First Person interview with the first author of the paper.

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

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.

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 animal's life span. Our data support multifunctional roles for Ltrk that parallel those of its mammalian counterparts.

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.

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.

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.

Gene expression is dynamically regulated in retinal progenitor cells prior to and during overt cellular differentiation.

The retina is comprised of one glial and six neuronal populations that are generated from a multipotent pool of retinal progenitor cells (RPCs) during development. To give rise to these different cell types, RPCs undergo temporal identity transitions, displaying distinct gene expression profiles at different stages of differentiation. Little, however, is known about temporal differences in RPC identities prior to the onset of overt cellular differentiation, during the period when a retinal identity is gradually acquired. Here we examined the sequential onset of expression of regional markers (i.e., homeodomain transcription factors) and cell fate determinants (i.e., basic-helix-loop-helix transcription factors and neurogenic genes) in RPCs from the earliest appearance of a morphologically-distinct retina. By performing a comparative analysis of the expression of a panel of 27 homeodomain, basic-helix-loop-helix and Notch pathway genes between embryonic day (E) 8.75 and postnatal day (P) 9, we identified six distinct RPC molecular profiles. At E8.75, the earliest stage assayed, murine RPCs expressed five homeodomain genes and a single neurogenic gene (Pax6, Six3, Six6, Rx, Otx2, Hes1). This early gene expression profile was remarkably similar to that of 'early' RPCs in the amphibian ciliary marginal zone (CMZ), where RPCs are compartmentalised according to developmental stage, and homologs of Pax6, Six3 and Rx are expressed in the 'early' stem cell zone. As development proceeds, expression of additional homeodomain, bHLH and neurogenic genes was gradually initiated in murine RPCs, allowing distinct genetic profiles to also be defined at E9.5, E10.5, E12.5, E15.5 and P0. In addition, RPCs in the postnatal ciliary margin, where retinal stem cells are retained throughout life, displayed a unique molecular signature, expressing all of the early-onset genes as well as several late-onset markers, indicative of a 'mixed' temporal identity. Taken together, the identification of temporal differences in gene expression in mammalian RPCs during pre-neurogenic developmental stages leads to new insights into how regional identities are progressively acquired during development, while comparisons at later stages highlight the dynamic nature of gene expression in temporally distinct RPC pools.

The early isoform of disabled-1 functions independently of Reelin-mediated tyrosine phosphorylation in chick retina.

The Reelin-Disabled-1 (Dab1) signaling pathway plays a key role in the positioning of neurons during brain development. Two alternatively spliced Dab1 isoforms have been identified in chick retina and brain: Dab1-E, expressed at early stages of development, and Dab1-L (commonly referred to as Dab1), expressed at later developmental stages. The well-studied Dab1-L serves as an adaptor protein linking Reelin signal to its downstream effectors; however, nothing is known regarding the role of Dab1-E. Here we show that Dab1-E is primarily expressed in proliferating retinal progenitor cells whereas Dab1-L is found exclusively in differentiated neuronal cells. In contrast to Dab1-L, which is tyrosine phosphorylated upon Reelin stimulation, Dab1-E is not tyrosine phosphorylated and may function independently of Reelin. Knockdown of Dab1-E in chick retina results in a significant reduction in the number of proliferating cells and promotes ganglion cell differentiation. Our results demonstrate a role for Dab1-E in the maintenance of the retinal progenitor pool and determination of cell fate.

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.

Dorsal versus ventral scales and the dorsoventral patterning of chick foot epidermis.

The dorsal and ventral scales of the chick foot can be distinguished morphologically and molecularly: the dorsal oblong overlapping scuta expressing both alpha and beta keratins, and the ventral roundish nonprotruding reticula expressing only alpha keratins. The question arises how En-1 and Lmx1, whose role in dorsoventral limb patterning has been well established, can affect skin morphogenesis, which occurs 8 to 12 days later. Forced expression of En-1 or of Lmx1 in the hindlimb have, respectively, as expected, a ventralizing or a dorsalizing effect on skin, leading to the formation of either reticula-type or scuta-type scales on both faces. In both cases, however, the scales are abnormal and even glabrous skin without any scales at all may form. The normal inductive interactions between dermis and epidermis are disturbed after En-1 or Lmx1 misexpression. Effectively, while Lmx1 endows the dermal precursors of the ventral region with scuta inducing ability, En-1 blocks the competence of the dorsal epidermis to build scuta.