Distinct timing of neurogenesis of ipsilateral and contralateral retinal ganglion cells.

In higher vertebrates, the circuit formed by retinal ganglion cells (RGCs) projecting ipsilaterally (iRGCs) or contralaterally (cRGCs) to the brain permits binocular vision and depth perception. iRGCs and cRGCs differ in their position within the retina and in expression of transcription, guidance and activity-related factors. To parse whether these two populations also differ in the timing of their genesis, a feature of distinct neural subtypes and associated projections, we used newer birthdating methods and cell subtype specific markers to determine birthdate and cell cycle exit more precisely than previously. In the ventrotemporal (VT) retina, i- and cRGCs intermingle and neurogenesis in this zone lags behind RGC production in the rest of the retina where only cRGCs are positioned. In addition, within the VT retina, i- and cRGC populations are born at distinct times: neurogenesis of iRGCs surges at E13, and cRGCs arise as early as E14, not later in embryogenesis as reported. Moreover, in the ventral ciliary margin zone (CMZ), which contains progenitors that give rise to some iRGCs in ventral neural retina (Marcucci et al., 2016), cell cycle exit is slower than in other retinal regions in which progenitors give rise only to cRGCs. Further, when the cell cycle regulator Cyclin D2 is missing, cell cycle length in the CMZ is further reduced, mirroring the reduction of both i- and cRGCs in the Cyclin D2 mutant. These results strengthen the view that differential regulation of cell cycle dynamics at the progenitor level is associated with specific RGC fates and laterality of axonal projection.

Ipsilateral and Contralateral Retinal Ganglion Cells Express Distinct Genes during Decussation at the Optic Chiasm.

The increasing availability of transcriptomic technologies within the last decade has facilitated high-throughput identification of gene expression differences that define distinct cell types as well as the molecular pathways that drive their specification. The retinal projection neurons, retinal ganglion cells (RGCs), can be categorized into distinct morphological and functional subtypes and by the laterality of their projections. Here, we present a method for purifying the sparse population of ipsilaterally projecting RGCs in mouse retina from their contralaterally projecting counterparts during embryonic development through rapid retrograde labeling followed by fluorescence-activated cell sorting. Through microarray analysis, we uncovered the distinct molecular signatures that define and distinguish ipsilateral and contralateral RGCs during the critical period of axonal outgrowth and decussation, with more than 300 genes differentially expressed within these two cell populations. Among the differentially expressed genes confirmed through in vivo expression validation, several genes that mark "immaturity" are expressed within postmitotic ipsilateral RGCs. Moreover, at least one complementary pair, Igf1 and Igfbp5, is upregulated in contralateral or ipsilateral RGCs, respectively, and may represent signaling pathways that determine ipsilateral versus contralateral RGC identity. Importantly, the cell cycle regulator cyclin D2 is highly expressed in peripheral ventral retina with a dynamic expression pattern that peaks during the period of ipsilateral RGC production. Thus, the molecular signatures of ipsilateral and contralateral RGCs and the mechanisms that regulate their differentiation are more diverse than previously expected.

The Ciliary Margin Zone of the Mammalian Retina Generates Retinal Ganglion Cells.

The retina of lower vertebrates grows continuously by integrating new neurons generated from progenitors in the ciliary margin zone (CMZ). Whether the mammalian CMZ provides the neural retina with retinal cells is controversial. Live imaging of embryonic retina expressing eGFP in the CMZ shows that cells migrate laterally from the CMZ to the neural retina where differentiated retinal ganglion cells (RGCs) reside. Because Cyclin D2, a cell-cycle regulator, is enriched in ventral CMZ, we analyzed Cyclin D2-/- mice to test whether the CMZ is a source of retinal cells. Neurogenesis is diminished in Cyclin D2 mutants, leading to a reduction of RGCs in the ventral retina. In line with these findings, in the albino retina, the decreased production of ipsilateral RGCs is correlated with fewer Cyclin D2+ cells. Together, these results implicate the mammalian CMZ as a neurogenic site that produces RGCs and whose proper generation depends on Cyclin D2 activity.

Exuberant growth and synapse formation of olfactory sensory neuron axonal arborizations.

Neural connections in the adult nervous system are established with a high degree of precision. Several examples throughout the nervous system indicate that this precision is achieved by first establishing an initial exuberant immature pattern of connectivity that is then sculpted into the adult pattern via pruning. This often emerges as an activity-dependent process. In the olfactory system, sensory axons from neurons expressing the same odorant receptor project with high precision to specific glomerular structures in the olfactory bulb. This process undergoes maturation-dependent refinements that are not fully understood. Due to technical impediments that have made it difficult to focus on single axons, it is unknown whether olfactory sensory projections are established in an exuberant fashion. Here we developed a novel technique of electroporation that allowed us to simultaneously label single olfactory sensory neuron (OSN) axonal arbors and their presynaptic specializations. Using this method we were able to incorporate plasmids into OSNs at an immature stage, thereby allowing a time-course study of axonal arbor development and synapse formation in single olfactory sensory axons. We observed that the number of branch points, the total branch length, and the number and density of presynaptic specializations peaked at postnatal day 8 and decreased afterwards. Our data demonstrate that olfactory sensory axons develop in an exuberant way, both in terms of branch growth and synaptic composition. We hypothesize that exuberant branches and synapses are eliminated to achieve the mature pattern in a process likely to be regulated by neural activity.

High-throughput microarray detection of vomeronasal receptor gene expression in rodents.

We performed comprehensive data mining to explore the vomeronasal receptor (V1R and V2R) repertoires in mouse and rat using the mm5 and rn3 genome, respectively. This bioinformatic analysis was followed by investigation of gene expression using a custom designed high-density oligonucleotide array containing all of these receptors and other selected genes of interest. This array enabled us to detect the specific expression of V1R and V2Rs which were previously identified solely based on computational prediction from gene sequence data, thereby establishing that these genes are indeed part of the vomeronasal system, especially the V2Rs. One hundred sixty-eight V1Rs and 98 V2Rs were detected to be highly enriched in mouse vomeronasal organ (VNO), and 108 V1Rs and 87 V2Rs in rat VNO. We monitored the expression profile of mouse VR genes in other non-VNO tissues with the result that some VR genes were re-designated as VR-like genes based on their non-olfactory expression pattern. Temporal expression profiles for mouse VR genes were characterized and their patterns were classified, revealing the developmental dynamics of these so-called pheromone receptors. We found numerous patterns of temporal expression which indicate possible behavior-related functions. The uneven composition of VR genes in certain patterns suggests a functional differentiation between the two types of VR genes. We found the coherence between VR genes and transcription factors in terms of their temporal expression patterns. In situ hybridization experiments were performed to evaluate the cell number change over time for selected receptor genes.

Sequential onset of presynaptic molecules during olfactory sensory neuron maturation.

Differentiated olfactory sensory neurons express specific presynaptic proteins, including enzymes involved in neurotransmitter transport and proteins involved in the trafficking and release of synaptic vesicles. Studying the regulation of these presynaptic proteins will help to elucidate the presynaptic differentiation process that ultimately leads to synapse formation. It has been postulated that the formation of a synapse between the axons of the sensory neurons and the dendrites of second order neurons in the olfactory bulb is a critical step in the processes of sensory neuron maturation. One approach to study the relationship between synaptogenesis and sensory neuron maturation is to examine the expression patterns of synaptic molecules through the olfactory neuron lineage. To this end we designed specific in situ hybridization probes to target messengers for proteins involved in presynaptic vesicle release. Our findings show that, as they mature, mouse olfactory neurons sequentially express specific presynaptic genes. Furthermore, the different patterns of expression of these presynaptic genes suggest the existence of discrete steps in presynaptic development: genes encoding proteins involved in scaffolding show an early onset of expression, whereas expression of genes encoding proteins involved in the regulation of vesicle release starts later. In particular, the signature molecule for glutamatergic neurons vesicle glutamate transporter 2 shows the latest onset of expression. In addition, contact with the targets in the olfactory bulb is not controlling presynaptic protein gene expression, suggesting that olfactory sensory neurons follow an intrinsic program of development.

The expression of the mitochondrial gene MT-ND4 is downregulated in cystic fibrosis.

Cystic fibrosis (CF) is a disease produced by mutations in the CFTR channel. We have previously reported that the CFTR chloride transport activity indirectly regulates the differential expression of several genes, including SRC and MUC1. Here we report that MT-ND4, a mitochondrial gene encoding a subunit of the mitochondrial Complex I (mtCx-I), is also a CFTR-dependent gene. A reduced expression of MT-ND4 was observed in CFDE cells (derived from a CF patient) when compared to CFDE cells ectopically expressing wild-type CFTR. The differential expression of MT-ND4 in CF was confirmed by RT-PCR. In situ hybridizations of deparaffinized human lung tissue slices derived from wt-CFTR or CF patients also showed downregulation of ND4 in CF. In addition, the CFTR chloride transport inhibitors glibenclamide and CFTR(inh)-172 also reduced MT-ND4 expression in CFDE cells ectopically expressing wt CFTR. These results suggest that the CFTR chloride transport activity indirectly up-regulates MT-ND4 expression.

Tyrosine kinase c-Src constitutes a bridge between cystic fibrosis transmembrane regulator channel failure and MUC1 overexpression in cystic fibrosis.

Cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) chloride channel, is associated in the respiratory system with the accumulation of mucus and impaired lung function. The role of the CFTR channel in the regulation of the intracellular pathways that determine the overexpression of mucin genes is unknown. Using differential display, we have observed the differential expression of several mRNAs that may correspond to putative CFTR-dependent genes. One of these mRNAs was further characterized, and it corresponds to the tyrosine kinase c-Src. Additional results suggest that c-Src is a central element in the pathway connecting the CFTR channel with MUC1 overexpression and that the overexpression of mucins is a primary response to CFTR malfunction in cystic fibrosis, which occurs even in the absence of bacterial infection.