[The application of lentiviral vectors for tissue-specific gene manipulations].

The trophectoderm (TE) ofblastocysts, the first epithelium established in mammalian development, 1) plays signaling, supportive, and patterning functions during pre-implantation development, 2) ensures embryo implantation into the uterine wall, and 3) gives rise to extra-embryonic tissues essential for embryo patterning and growth after implantation. We show that mouse TE, itself permissive to lentiviral (LV) infection, represents a robust non-permeable physical barrier to the virus particles, thereby shielding the cells of the inner cell mass (ICM) from viral infection. This LV feature will allow modulations of gene expression in a lineage-specific manner, thus having significant applications in mouse functional genetics.

Different levels of Hoxa2 are required for particular developmental processes.

Hoxa2 is required for a variety of developmental processes in the branchial arches and in the hindbrain. We have created a Hoxa2 allele that is about 45% as active in transcription as its wild-type counterpart. This allele, together with the Hoxa2 null and wild-type alleles, allowed the generation of embryos developing in the presence of different levels of Hoxa2 activity. Analysis of these embryos indicates that in general the hindbrain is more resistant to Hoxa2 deficiencies than the second branchial arch. Also, within the second arch, proximo-caudal areas are more sensitive than the rostro-distal. In the hindbrain, basic segmentation and patterning processes seem to occur normally at Hoxa2 levels as low as 20% of the normal. In addition, specific neuronal markers along the dorso-ventral axis of the hindbrain seem differentially affected by reduced Hoxa2 levels. These results provide new clues to understand the role of Hoxa2 in the different embryonic areas where it is required.

Differential expression of two different homeobox gene families during mouse tegument morphogenesis.

The expression of six genes belonging to two different homeobox gene families was studied during the embryonic and postnatal morphogenesis of head and body regions of the mouse integument. The first family included the Otx1 and Otx2 genes, both related to the orthodenticle Drosophila gene and the second was represented by four members of the Antennapedia class HOX genes: Hoxc8 and three Hoxd genes, d9, d11 and d13. In situ hybridizations with 35S labeled antisense RNA probes were performed on head serial frontonasal sections, as well as entire embryo and postnatal tail longitudinal sections. The expression of these genes shows a differential spatiotemporal pattern along the cephalo-caudal axis. In 12.5-day and 15.5-day embryos, the Otx2 gene expression is restricted to the nasal epithelium and its associated glands, while the Otx1 transcripts are present in both nasal and facial integuments, including nasal glands and hair vibrissa follicles. The Hoxc8 expression first appears in skin at 14.5 days of gestation in the sternal region and is extended at 16.5 days to the thoracic ventral and lumbar dorsal regions. The Hoxd9 and Hoxd11 genes are only expressed in the caudal skin from 14.5 days of gestation. The Hoxd13 transcripts are the last to appear, 2 days after birth, and are limited to the last epidermal cells to differentiate, i.e. those of the hair matrix of the caudal pelage hair follicles. Taken together, these observations strengthen the hypothesis that different homeobox gene families specify the regional identity of the skin in the cephalic and body regions.

Characterization of cDNAs encoding two chick retinoic acid receptor alpha isoforms and distribution of retinoic acid receptor alpha, beta and gamma transcripts during chick skin development.

The amino acid sequence of the retinoic acid receptors alpha, beta and gamma (RAR alpha, beta and gamma) can be divided into six functional domains (A-F), different isoforms arising from the presence of different A domains by differential splicing. In order to address the respective roles of the different RARs during skin morphogenesis in birds, cDNAs encoding two chick RAR alpha isoforms (alpha1 and alpha2) have been isolated. While the A1 and B-F domains of the RAR alpha are highly conserved across species, the chick A2 domain contains 50% specific amino acids. The three RAR alpha, beta and gamma genes display specific patterns of expression during chick skin morphogenesis. As in mouse, RAR alpha and gamma transcripts are present in both the dermis and epidermis during the first stages of skin appendage formation. Furthermore, Northern blot analysis suggests that different RAR alpha and gamma isoforms could be successively required during feather formation. The RAR gamma gene, continuously expressed in the epidermal cells in both chick and mouse, is thus likely to play a similar role in skin development in these two species. However, RAR alpha transcripts, only transiently detected during mouse skin development, still accumulate in epidermis during the later stages of chick skin differentiation. Furthermore, RAR beta transcripts, never detected during normal development in mouse skin, are actually present at the early stages of chick skin morphogenesis. Thus, our results suggest that the role of the three RAR in skin development has not been strictly conserved in the different classes of vertebrates.

Hox11 acts cell autonomously in spleen development and its absence results in altered cell fate of mesenchymal spleen precursors.

The genetic steps governing development of the spleen are largely unknown. Absence of Hox11 in mice results in asplenia, but it is unclear how Hox11 exerts its effect on spleen development. To more precisely define Hox11's role in spleen morphogenesis, we have examined the fate of the developing spleen in Hox11(-/-) mice. Perturbation of spleen development begins between dE13 and dE13.5. Cells of the spleen anlage persist past this developmental stage as an unorganized rudiment between the stomach and the pancreas. They fail to proliferate, and haematopoietic cells do not colonize the rudiment. At later stages of embryonic development, the cells can be observed in the mesenchyme of the pancreas, also an expression site of Hox11. In Hox11-/-<-->+/+ chimaeras, spleens were devoid of Hox11(-/-) cells, indicating that the genetic defect is cell autonomous and not due to failure of the organ anlage to attract and retain haematopoietic cells. In -/-<-->+/+ chimaeric embryos, Hox11(-/-) cells were initially present in the spleen anlage. However, at dE13, a reorganization of the spleen occurred in the chimaeras and Hox11(-/-) cells were subsequently excluded from the spleen, suggesting that a change in the affinity for one of the spleen cells had occurred. These observations demonstrate that spleen development consists of genetically separable steps and that absence of Hox11 arrests spleen development at an early stage. The formation of the spleen primordium before the entry of haematopoietic cells does not require the activity of Hox11. However, subsequent differentiation of spleen precursor cells is dependent on the Hox11 gene.

CHOXC-8 and CHOXD-13 expression in embryonic chick skin and cutaneous appendage specification.

We studied the expression of two distantly clustered Hox genes which could, respectively, be involved in specification of dorsal feather- and foot scale-forming skin in the chick embryo: cHoxc-8, a median paralog, and cHoxd-13, located at the 5' extremity of the HoxD cluster. The cHoxc-8 transcripts are present at embryonic day 3.5 (E3.5) in the somitic cells, which give rise to the dorsal dermis by E5, and at E6.5-8.5 in the dorsal dermal and epidermal cells during the first stages of feather morphogenesis. The cHoxd-13 transcripts are present at E4.5-9.5 in the autopodial mesenchyme and at E10.5-12.5 in the plantar dermis during the initiation of reticulate scale morphogenesis. Both the cHoxc-8 and cHoxd-13 transcripts are no longer detectable after the anlagen stage of cutaneous appendage morphogenesis. Furthermore, heterotopic dermal-epidermal recombinations of dorsal, plantar, and apteric tissues revealed that the epidermal ability or inability to form feathers is already established by the time of skin formation. Retinoic acid (RA) treatment at E11 induces after 12 hr an inhibition of cHoxd-13 expression in the plantar dermis, followed by the formation of feather filaments on the reticulate scales. When E7.5 dorsal explants are treated with RA for 6 days, they form scale-like structures where the Hox transcripts are no more detectable. Protein analysis revealed that the plantar filaments, made up of feather beta-keratins, corresponded to a homeotic transformation, whereas the scale-like structures, composed also of feather beta-keratins, were teratoid. These results strengthen the hypothesis that different homeobox genes play a significant role in specifying the regional identity of the different epidermal territories.

BMP signaling is essential for development of skeletogenic and neurogenic cranial neural crest.

BMP signaling is essential for a wide variety of developmental processes. To evaluate the role of Bmp2/4 in cranial neural crest (CNC) formation or differentiation after its migration into the branchial arches, we used Xnoggin to block their activities in specific areas of the CNC in transgenic mice. This resulted in depletion of CNC cells from the targeted areas. As a consequence, the branchial arches normally populated by the affected neural crest cells were hypomorphic and their skeletal and neural derivatives failed to develop. In further analyses, we have identified Bmp2 as the factor required for production of migratory cranial neural crest. Its spatial and temporal expression patterns mirror CNC emergence and Bmp2 mutant embryos lack both branchial arches and detectable migratory CNC cells. Our results provide functional evidence for an essential role of BMP signaling in CNC development.

Characterization of cDNAs encoding the chick retinoic acid receptor gamma 2 and preferential distribution of retinoic acid receptor gamma transcripts during chick skin development.

Retinoic acid receptors alpha, beta and gamma (RAR alpha, beta and gamma) are ligand-inductible transcriptional activators which belong to the steroid/thyroid hormone receptor superfamily. At least two major isoforms (1 and 2) of each RAR arise by differential use of two promoters and alternative splicing. In mouse, the three RAR genes are expressed in stage- and tissue-specific patterns during embryonic development. In order to understand the role of the different RARs in chick, RAR gamma 2 cDNAs were isolated from an 8.5-day (stage 35 of Hamburger and Hamilton) chick embryo skin library. The deduced chick RAR gamma 2 amino acid sequence displays uncommon features such as 21 specific amino acid replacements, 12 of them being clustered in the amino-terminal region (domains A2 and B), and a truncated acidic carboxy-terminal region (F domain). However, the pattern of RAR gamma expression in chick embryo resembles that reported in mouse, particularly in skin where RAR gamma expression occurs in both the dermal and epidermal layers at the beginning of feather formation, and is subsequently restricted to the differentiating epidermal cells. Northern blot analysis suggests that different RAR gamma isoforms could be successively required during chick development.

Hoxa-2 restricts the chondrogenic domain and inhibits bone formation during development of the branchial area.

In Hoxa-2(-/- )embryos, the normal skeletal elements of the second branchial arch are replaced by a duplicated set of first arch elements. We show here that Hoxa-2 directs proper skeletal formation in the second arch by preventing chondrogenesis and intramembranous ossification. In normal embryos, Hoxa-2 is expressed throughout the second arch mesenchyme, but is excluded from the chondrogenic condensations. In the absence of Hoxa-2, chondrogenesis is activated ectopically within the rostral Hoxa-2 expression domain to form the mutant set of cartilages. In Hoxa-2(-/- )embryos the Sox9 expression domain is shifted into the normal Hoxa-2 domain. Misexpression of Sox9 in this area produces a phenotype resembling that of the Hoxa-2 mutants. These results indicate that Hoxa-2 acts at early stages of the chondrogenic pathway, upstream of Sox9 induction. We also show that Hoxa-2 inhibits dermal bone formation when misexpressed in its precursors. Furthermore, molecular analyses indicate that Cbfa1 is upregulated in the second branchial arches of Hoxa-2 mutant embryos suggesting that prevention of Cbfa1 induction might mediate Hoxa-2 inhibition of dermal bone formation during normal second arch development. The implications of these results on the patterning of the branchial area are discussed.