Smad1/Smad5 signaling in limb ectoderm functions redundantly and is required for interdigital programmed cell death

Bone morphogenetic proteins (BMPs) are secreted signals that regulate apical ectodermal ridge (AER) functions and interdigital programmed cell death (PCD) of developing limb. However the identities of the intracellular mediators of these signals are unknown. To investigate the role of Smad proteins in BMP-regulated AER functions in limb development, we inactivated Smad1 and Smad5 selectively in AER and ventral ectoderm of developing limb, using Smad1 or/and Smad5 floxed alleles and an En1(Cre/+) knock-in allele. Single inactivation of either Smad1 or Smad5 did not result in limb abnormalities. However, the Smad1/Smad5 double mutants exhibited syndactyly due to a reduction in interdigital PCD and an increase in interdigital cell proliferation. Cell tracing experiments in the Smad1/Smad5 double mutants showed that ventral ectoderm became thicker and the descendents of ventral En1(Cre/+) expressing ectodermal cells were located at dorsal interdigital regions. At the molecular level, Fgf8 expression was prolonged in the interdigital ectoderm of embryonic day (E) 13 Smad1/Smad5 double mutants, suggesting that the ectopic Fgf8 expression may serve as a survival signal for interdigital epithelial and mesenchymal cells. Our result suggests that Smad1 and Smad5 are required and function redundantly as intracellular mediators for BMP signaling in the AER and ventral ectoderm. Smad1/Smad5 signaling in the AER and ventral ectoderm regulates interdigital tissue regression of developing limb. Our mutants with defects in interdigital PCD could also serve as a valuable model for investigation of PCD regulation machinery.     

Transmission of Panulirus argus virus 1 (PaV1) and its effect on the survival of juvenile Caribbean spiny lobster

The Caribbean spiny lobster Panulirus argus, an important fisheries species, is host to Panulirus argus virus 1 (PaV1), a lethal, unclassified virus--the first found in any species of lobster--prevalent in juvenile lobsters. We describe a series of laboratory experiments aimed at assessing the likely modes of disease transmission, determining the survival of lobsters relative to each transmission pathway and identifying potential alternate hosts. Given evidence for lower prevalence of PaV1 in large lobsters, the effect of lobster size on susceptibility was also examined. Results demonstrated that PaV1 can be transmitted to juvenile lobsters via inoculation, ingestion of diseased tissue, contact with diseased lobsters and--among the smallest juveniles--through water over distances of a few meters. Contact and waterborne transmission, the most likely modes of transmission in the wild, were less efficient than inoculation or ingestion. Nevertheless, about half of the smallest lobsters in contact and waterborne trials contracted the disease and died within 3 mo. Other decapods that co-occur with P. argus (e.g. spotted lobster P. guttatus, stone crab Menippe mercenaria, channel crab Mithrax spinosissimus) did not acquire the disease after inoculation with PaV1-infected hemolymph. Our results confirmed that PaV1 is highly infectious and lethal to juvenile P. argus, particularly early benthic juveniles in the wild, and, hence, is a threat to mariculture.     

Cthrc1 is a positive regulator of osteoblastic bone formation

Background

Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.

Methodology/Principal Findings

We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.

Conclusions/Significance

Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.     

Recent innovations in tissue-specific gene modifications in the mouse

Annotating the functions of individual genes in in vivo contexts has become the primary task of mouse genetics in the post-genome era. In addition to conventional approaches using transgenic technologies and gene targeting, the recent development of conditional gene modification techniques has opened novel opportunities for elucidating gene function at the level of the whole mouse to individual tissues or cell types. Tissue-specific gene modifications in the mouse have been made possible using site-specific DNA recombinases and conditional alleles. Recent innovations in this basic technology have facilitated new types of experiments, revealing novel insights into mammalian embryology. In this review, we focus on these recent innovations and new technical issues that impact the success of these conditional gene modification approaches.     

Normal and Abnormal Development in the Arabidopsis Vegetative Shoot Apex

Vegetative development in the Arabidopsis shoot apex follows both sequential and repetitive steps. Early in development, the young vegetative meristem is flat and has a rectangular shape with bilateral symmetry. The first pair of leaf primordia is radially symmetrical and is initiated on opposite sides of the meristem. As development proceeds, the meristem changes first to a bilaterally symmetrical trapezoid and then to a radially symmetrical dome. Vegetative development from the domed meristem continues as leaves are initiated in a repetitive manner. Abnormal development of the vegetative shoot apex is described for a number of mutants. The mutants we describe fall into at least three classes: (1) lesions in the shoot apex that do not show an apparent alteration in the shoot apical meristem, (2) lesions in the apical meristem that also (directly or indirectly) alter leaf primordia, and (3) lesions in the apical meristem that alter meristem size and leaf number but not leaf morphology. These mutations provide tools both to genetically analyze vegetative development of the shoot apex and to learn how vegetative development influences floral development.     

Cell-Autonomous Requirement for Rx Function in the Mammalian Retina and Posterior Pituitary

Rx is a paired-like homeobox gene that is required for vertebrate eye formation. Mice lacking Rx function do not develop eyes or the posterior pituitary. To determine whether Rx is required cell autonomously in these tissues, we generated embryonic chimeras consisting of wild type and Rx−/− cells. We found that in the eye, Rx-deficient cells cannot participate in the formation of the neuroretina, retina pigment epithelium and the distal part of the optic stalk. In addition, in the ventral forebrain, Rx function is required cell autonomously for the formation of the posterior pituitary. Interestingly, Rx−/− and wild type cells segregate before the morphogenesis of these two tissues begins. Our observations suggest that Rx function is not only required for the morphogenesis of the retina and posterior pituitary, but also prior to morphogenesis, for the sorting out of cells to form distinct fields of retinal/pituitary cells.     

Human-animal chimeras in biomedical research

Chimeras are individuals with tissues derived from more than one zygote. Interspecific chimeras have tissues derived from different species. The biological consequences of human-animal chimeras have become an issue of ethical debate. Ironically, human-animal chimeras with human blood, neurons, germ cells, and other tissues have been generated for decades. This has facilitated human biological studies and therapeutic strategies for disease.