Extraocular ectoderm triggers dorsal retinal fate during optic vesicle evagination in zebrafish.

Dorsal retinal fate is established early in eye development, via expression of spatially restricted dorsal-specific transcription factors in the optic vesicle; yet the events leading to initiation of dorsal fate are not clear. We hypothesized that induction of dorsal fate would require an extraocular signal arising from a neighboring tissue to pattern the prospective dorsal retina, however no such signal has been identified. We used the zebrafish embryo to determine the source, timing, and identity of the dorsal retina-inducing signal. Extensive cell movements occur during zebrafish optic vesicle morphogenesis, however the location of prospective dorsal cells within the early optic vesicle and their spatial relationship to early dorsal markers is currently unknown. Our mRNA expression and fate mapping analyses demonstrate that the dorsolateral optic vesicle is the earliest region to express dorsal specific markers, and cells from this domain contribute to the dorsal retinal pole at 24 hpf. We show that three bmp genes marking dorsal retina at 25 hpf are also expressed extraocularly before retinal patterning begins. We identified gdf6a as a dorsal initiation signal acting from the extraocular non-neural ectoderm during optic vesicle evagination. We find that bmp2b is involved in dorsal retina initiation, acting upstream of gdf6a. Together, this work has identified the nature and source of extraocular signals required to pattern the dorsal retina.

Proliferation and patterning are mediated independently in the dorsal spinal cord downstream of canonical Wnt signaling.

Canonical Wnt signaling can regulate proliferation and patterning in the developing spinal cord, but the relationship between these functions has remained elusive. It has been difficult to separate the distinct activities of Wnts because localized changes in proliferation could conceivably alter patterning, and gain and loss of function experiments have resulted in both types of defects. To resolve this issue we have investigated canonical Wnt signaling in the zebrafish spinal cord using multiple approaches. We demonstrate that Wnt signaling is required initially for proliferation throughout the entire spinal cord, and later for patterning dorsal progenitor domains. Furthermore, we find that spinal cord patterning is normal in embryos after cell division has been pharmacologically blocked. Finally, we determine the transcriptional mediators of Wnt signaling that are responsible for patterning and proliferation. We show that tcf7 gene knockdown results in dorsal patterning defects without decreasing the mitotic index in dorsal domains. In contrast, tcf3 gene knockdown results in a reduced mitotic index without affecting dorsal patterning. Together, our work demonstrates that proliferation and patterning in the developing spinal cord are separable events that are regulated independently by Wnt signaling.

Negative regulation of Vsx1 by its paralog Chx10/Vsx2 is conserved in the vertebrate retina.

Chx10/Vsx2 and Vsx1 are the only Paired-like CVC (Prd-L:CVC) homeobox genes in the mouse genome. Both are expressed in the retina and have important but distinct roles in retinal development. Mutations in Chx10/Vsx2 cause reduced retinal progenitor cell (RPC) proliferation and an absence of bipolar cells, while mutations in Vsx1 impair differentiation of cone bipolar cells. Given their structural similarities and importance in retinal development, we sought to determine if a regulatory interaction exists between these genes and whether inactivation of both genes blocks initiation of retinal development. We found that Chx10/Vsx2 binds to a specific sequence in the Vsx1 5'-intergenic region and represses the activity of a luciferase reporter under the control of the Vsx1 promoter. This is consistent with our observation that there is an inverse relationship between the levels of Chx10/Vsx2 and Vsx1 immunostaining within the bipolar cell class. Furthermore, Vsx1 mRNA is upregulated in the RPCs of Chx10/Vsx2 deficient mice and zebrafish embryos injected with a chx10/vsx2 morpholino. In mice deficient for both Chx10/Vsx2 and Vsx1 and zebrafish embryos co-injected with chx10/Vsx2 and vsx1 morpholinos, the changes in embryonic retinal development and marker expression are similar in magnitude to embryos with Chx10/Vsx2 loss of function only. From these studies, we propose that Vsx1 is a direct target of Chx10/Vsx2-mediated transcriptional repression. Although Vsx1 mRNA is upregulated in Chx10/Vsx2 deficient RPCs, Vsx1 does not genetically compensate for loss of Chx10/Vsx2, demonstrating that Prd-L:CVC genes, although important, are not absolutely required to initiate retinal development.

Skeletal Characterization of Smurf2-Deficient Mice and In Vitro Analysis of Smurf2-Deficient Chondrocytes.

Overexpression of Smad ubiquitin regulatory factor 2 (Smurf2) in chondrocytes was reported to cause spontaneous osteoarthritis (OA) in mice. However, it is unclear whether Smurf2 is involved in bone and cartilage homeostasis and if it is required for OA pathogenesis. Here we characterized age-related changes in the bone and articular cartilage of Smurf2-deficient (MT) mice by microCT and histology, and examined whether reduced Smurf2 expression affected the severity of OA upon surgical destabilization of the medial meniscus (DMM). Using immature articular chondrocytes (iMAC) from MT and wild-type (WT) mice, we also examined how Smurf2 deficiency affects chondrogenic and catabolic gene expressions and Smurf2 and Smurf1 proteins upon TGF-ß3 or IL-1ß treatment in culture. We found no differences in cortical, subchondral and trabecular bone between WT and MT in young (4 months) and old mice (16-24 months). The articular cartilage and age-related alterations between WT and MT were also similar. However, 2 months following DMM, young MT showed milder OA compared to WT (~70% vs ~30% normal or exhibiting only mild OA cartilage phenotype). The majority of the older WT and MT mice developed moderate/severe OA 2 months after DMM, but a higher subset of aged MT cartilage (27% vs. 9% WT) remained largely normal. Chondrogenic gene expression (Sox9, Col2, Acan) trended higher in MT iMACs than WT with/without TGF-ß3 treatment. IL-1ß treatment suppressed chondrgenic gene expression, but Sox9 expression in MT remained significantly higher than WT. Smurf2 protein in WT iMACs increased upon TGF-ß3 treatment and decreased upon IL-1ß treatment in a dose-dependent manner. Smurf1 protein elevated more in MT than WT upon TGF-ß3 treatment, suggesting a potential, but very mild compensatory effect. Overall, our data support a role of Smurf2 in regulating OA development but suggest that inhibiting Smurf2 alone may not be sufficient to prevent or consistently mitigate post-traumatic OA across a broad age range.

Canonical Wnt signaling is required for the maintenance of dorsal retinal identity.

Accurate retinotectal axon pathfinding depends upon the correct establishment of dorsal-ventral retinal polarity. We show that dorsal retinal gene expression is regulated by Wnt signaling in the dorsal retinal pigment epithelium (RPE). We find that a Wnt reporter transgene and Wnt pathway components are expressed in the dorsal RPE beginning at 14-16 hours post-fertilization. In the absence of Wnt signaling, tbx5 and Bmp genes initiate normal dorsal retinal expression but are not maintained. The expression of these genes is rescued by the downstream activation of Wnt signaling, and tbx5 is rescued by Bmp signaling. Furthermore, activation of Wnt signaling cannot rescue tbx5 in the absence of Bmp signaling, suggesting that Wnt signaling maintains dorsal retinal gene expression by regulating Bmp signaling. We present a model in which dorsal RPE-derived Wnt activity maintains the expression of Bmp ligands in the dorsal retina, thus coordinating the patterning of these two ocular tissues.

Expression pattern of zebrafish tcf7 suggests unexplored domains of Wnt/beta-catenin activity.

Tcf/Lef transcription factors play an important role in mediating canonical Wnt signaling. When bound by beta-catenin, Tcf/Lef proteins either activate or de-repress gene transcription. In zebrafish, four members have been identified: Lef1, Tcf3, Tcf3b, and Tcf4. Here, we report the cloning and expression of the tcf7 gene. Forms of Tcf7 expressed in the embryo contain two highly conserved regions: an N-terminal beta-catenin binding domain and a C-terminal HMG domain. Tcf7 lacks a putative Groucho corepressor binding site, suggesting that, like Lef1, it functions as a transcriptional activator. We isolated three C-terminal splice variants of tcf7 corresponding to human B, C, and D isoforms. tcf7 expression overlaps with lef1 expression maternally, in the tail bud, fin buds, and paraxial mesoderm, and we expect that the two genes function redundantly in those areas. tcf7 is also expressed in nonoverlapping areas such as the prechordal mesoderm, dorsal retina, and median fin fold, suggesting unique functions.