Lymphatic vasculature development.

Although the process of blood vasculature formation has been well documented, little is known about lymphatic vasculature development, despite its importance in normal and pathological conditions. The lack of specific lymphatic markers has hampered progress in this field. However, the recent identification of genes that participate in the formation of the lymphatic vasculature denotes the beginning of a new era in which better diagnoses and therapeutic treatment(s) of lymphatic disorders could become a reachable goal.

Severe immunodeficiency associated with a human immunodeficiency virus 1 NEF/3'-long terminal repeat transgene.

We have generated several transgenic mouse strains carrying a human immunodeficiency virus 1 (HIV-1) NEF/3' long terminal repeat (LTR) transgene under control of a T cell-specific promoter-enhancer element, showing a depletion of CD4+ T cells in the thymus and periphery. The immunological functions of the line with the most dramatic changes in lymphocyte populations, B6/338L, were analyzed in greater detail. The presence of the transgene in the heterozygous animal is associated with a dominant severe immunodeficiency. Older animals develop lymph-adenopathy and splenomegaly. CD4+CD8+ and CD4+CD8- single positive thymocytes already are depleted in these mice at the earliest stages in ontogeny, and peripheral T cells are reduced in frequency and present cell surface marker expression, which is characteristic for memory and activated T cells. The immunological response of B6/338L mice to several viral infections is also greatly impaired. Thus, the HIV-1 NEF/3' LTR as transgene in T cells can cause immunodeficiency and disease with striking similarities to a known retrovirus-induced immunodeficiency called murine AIDS (H. C. Morse III, S. K. Chattopadhyay, M. Makino, T. N. Frederickson, A. W. Hügin, and J. W. Hartley. 1992. AIDS. 6:607).

Regulation of class I MHC gene expression in the developing and mature CNS by neural activity.

To elucidate molecular mechanisms underlying activity-dependent synaptic remodeling in the developing mammalian visual system, we screened for genes whose expression in the lateral geniculate nucleus (LGN) is regulated by spontaneously generated action potentials present prior to vision. Activity blockade did not alter expression in the LGN of 32 known genes. Differential mRNA display, however, revealed a decrease in mRNAs encoding class I major histocompatibility complex antigens (class I MHC). Postnatally, visually driven activity can regulate class I MHC in the LGN during the final remodeling of retinal ganglion cell axon terminals. Moreover, in the mature hippocampus, class I MHC mRNA levels are increased by kainic acid-induced seizures. Normal expression of class I MHC mRNA is correlated with times and regions of synaptic plasticity, and immunohistochemistry confirms that class I MHC is present in specific subsets of CNS neurons. Finally, beta2-microglobulin, a cosubunit of class I MHC, and CD3zeta, a component of a receptor complex for class I MHC, are also expressed by CNS neurons. These observations indicate that class I MHC molecules, classically thought to mediate cell-cell interactions exclusively in immune function, may play a novel role in neuronal signaling and activity-dependent changes in synaptic connectivity.

Plasma from human mothers of fetuses with severe arthrogryposis multiplex congenita causes deformities in mice.

Arthrogryposis multiplex congenita (AMC) is characterized by fixed joint contractures and other deformities, sometimes resulting in fetal death. The cause is unknown in most cases, but some women with fetuses affected by severe AMC have serum antibodies that inhibit fetal acetylcholine receptor (AChR) function, and antibodies to fetal antigens might play a pathogenic role in other congenital disorders. To investigate this possibility, we have established a model by injecting pregnant mice with plasma from four anti-AChR antibody-positive women whose fetuses had severe AMC. We found that human antibodies can be transferred efficiently to the mouse fetus during the last few days of fetal life. Many of the fetuses of dams injected with AMC maternal plasmas or Ig were stillborn and showed fixed joints and other deformities. Moreover, similar changes were found in mice after injection of a serum from one anti-AChR antibody-negative mother who had had four AMC fetuses. Thus, we have confirmed the role of maternal antibodies in cases of AMC associated with maternal anti-AChR, and we have demonstrated the existence of pathogenic maternal factors in one other case. Importantly, this approach can be used to look at the effects of other maternal human antibodies on development of the fetus.

Functional analysis of the V gamma 3 promoter of the murine gamma delta T-cell receptor.

The initial day 14 wave of fetal thymocytes express a gamma delta T-cell receptor (TCR). This surface TCR is generated by preferential rearrangement of V gamma 3 and V delta 1 recombination segments. To delineate the role of regulatory sequences in this expression, we have analyzed the V gamma 3 promoter control region under the regulation of its cognate C gamma 1 enhancer. Transcription initiates 25 bases downstream from a TATTAA sequence at a consensus initiator motif. The minimal 5' promoter sequences supporting expression by transient analysis extend -243 nucleotides from the +1 start site. Three regulatory sequences in this region have been defined by deletion and mutagenesis: a consensus CTF/NF-1 site at -55, an Ets homology sequence at -65, and a degenerate, but crucial, SP-1 site at -100. The presence of additional sequences downstream of the start site which extend through the leader intron were necessary for expression. In contrast to other TCR or immunoglobulin variable regions, one or more strong upstream suppressor sequences resembling silencer elements have been observed. A 311-bp fragment, positions -586 to -897, exhibited strong repressing activity regardless of orientation when placed upstream of heterologous promoters.

Trophoblast cell-specific carcinoembryonic antigen cell adhesion molecule 9 is not required for placental development or a positive outcome of allotypic pregnancies.

The carcinoembryonic antigen (CEA) family consists of a large group of evolutionarily divergent glycoproteins. The secreted pregnancy-specific glycoproteins constitute a subgroup within the CEA family. They are predominantly expressed in trophoblast cells throughout placental development and are essential for a positive outcome of pregnancy, possibly by protecting the semiallotypic fetus from the maternal immune system. The murine CEA gene family member CEA cell adhesion molecule 9 (Ceacam9) also exhibits a trophoblast-specific expression pattern. However, its mRNA is found only in certain populations of trophoblast giant cells during early stages of placental development. It is exceptionally well conserved in the rat (over 90% identity on the amino acid level) but is absent from humans. To determine its role during murine development, Ceacam9 was inactivated by homologous recombination. Ceacam9(-/-) mice on both BALB/c and 129/Sv backgrounds developed indistinguishably from heterozygous or wild-type littermates with respect to sex ratio, weight gain, and fertility. Furthermore, the placental morphology and the expression pattern of trophoblast marker genes in the placentae of Ceacam9(-/-) females exhibited no differences. Both backcross analyses and transfer of BALB/c Ceacam9(-/-) blastocysts into pseudopregnant C57BL/6 foster mothers indicated that Ceacam9 is not needed for the protection of the embryo in a semiallogeneic or allogeneic situation. Taken together, Ceacam9 is dispensable for murine placental and embryonic development despite being highly conserved within rodents.

Abnormal deletions in the T-cell receptor delta locus of mouse thymocytes.

Separate genetic elements (V, D, and J) encode the variable regions of lymphocyte antigen receptors. During early lymphocyte differentiation, these elements rearrange to form contiguous coding segments (VJ and VDJ) for a diverse array of variable regions. Rearrangement is mediated by a recombinase that recognizes short DNA sequences (signals) flanking V, D, and J elements. Signals flank both the 5' and 3' sides of each D element, thereby allowing assembly of a functional VDJ gene. However, in rearrangements involving the D delta 2 and J delta 1 elements of the mouse T-cell receptor delta (TCR delta) locus, we unexpectedly found that the D delta 2 element and a portion of its 5' signal are often deleted. Approximately 50% of recovered D delta 2 to J delta 1 rearrangements from thymocytes of adult wild-type mice showed such deletions. An additional 20% of the rearrangements contained standard D delta 2-J delta 1 coding junctions but showed some loss of nucleotides from the 5' D delta 2 signal. This loss was clearly associated with another event involving a site-specific cleavage at the 5' signal/coding border of D delta 2 and rejoining of the modified signal and coding ends. The abnormal loss of D delta 2 and a portion of the 5' D delta 2 signal was infrequently observed in D delta 2-to-J delta 1 rearrangements recovered from neonatal mice. The possible basis and significance of this age-dependent phenomenon are discussed.

Biological functions of tumor necrosis factor cytokines and their receptors.

Tumor necrosis factor (TNF; formerly known as TNFalpha) and lymphotoxin (LT)alpha, originally characterized by their ability to induce tumor cell apoptosis and cachexia, are now considered as central mediators of a broad range of biological activities. These activities encompass beneficial effects for the host in inflammation and in protective immune responses against a variety of infectious pathogens. TNF family members on the other hand also exert host-damaging effects in sepsis, in tumor cachexia as well as in autoimmune diseases. In addition, the essential roles of the core members of the TNF superfamily, LTalpha, LTbeta, TNF, and LIGHT as well as their receptors during the organogenesis of secondary lymphoid organs and the maintenance of the architecture of lymphatic tissues now becomes appreciated. The elucidation of the biological functions of these cytokines and their specific cell surface receptors has been crucially advanced by the study of gene-targeted mouse strains. This presentation summarizes the roles of TNFR and TNF-like cytokines in infection, sepsis and autoimmunity as well as the pivotal involvement of these molecules in the development of secondary lymphoid organs.

Peyer's patch organogenesis as a programmed inflammation: a hypothetical model.

Gene-knock-out studies implicate roles of lymphotoxin (LT) alphabeta and LT betaR in the initial phase of Peyer's patch (PP) organogenesis. We recently identified the requirement of IL-7R alpha/gamma c/Jak3 signal in LT alphabeta production of IL-7R alpha+ cells. These observations lead us to a hypothetical model for PP organogenesis with three cellular components. The first is the producer of the ligand for IL-7R alpha, which then stimulate the IL-7R alpha+ cells to produce LT alphabeta activating the LT betaR+ cells to form an organizing center for PP organogenesis. This model is similar to that of inflammation, suggesting that PP organogenesis is a programmed version of inflammation.

Expression of epithelial antigens Exo-1 and EPM-1 in human epidermal keratinocyte maturation and benign and malignant neoplasia.

Exo-1, a polar neutral glycolipid, and EPM-1, a high molecular weight glycoprotein, are developmental antigens of human epithelial cells, initially described as components both on the cell surface and in secretions of gastrointestinal epithelial and respective tumors. In order to assess the biological significance of both antigens for epithelial cell differentiation and neoplastic transformation, their expression during human skin development and benign and malignant neoplasia was analyzed in fresh frozen tissue specimens of skin biopsies and of human epidermal keratinocytes growing in experimental model systems. Antigen expression was assessed immunohistochemically with specific monoclonal antibodies. During fetal development Exo-1 was temporarily expressed in intermediate cells but was absent in normal adult human skin. Exo-1 expression reemerged in neoplasias, both benign and malignant, but was restricted to spinous-like differentiated cells. Similarly, Exo-1 was not expressed in transplants of normal keratinocytes mimicking the normal epidermis but was clearly visible in differentiated areas of transplants of malignantly transformed keratinocytes. EPM-1 appeared first in basal epidermal cells in the second half of gestation and remained detectable in the stratum basale of adult skin. While squamous cell carcinomas continued to express EPM-1, it was not detectable in basal cell epitheliomas and in normal epidermis after invasion by neuroectodermal tumor cells. In experimental models, EPM-1 was present in the basal layers of normal human keratinocytes and of transformed keratinocytes with benign growth characteristics whenever a well stratified and keratinized epidermis-like epithelium had formed in transplants. In transformed keratinocytes with malignant growth behavior, EPM-1 was expressed irregularly, as in squamous cell carcinomas in situ. Thus, expression of Exo-1 is a marker for an early embryonic differentiation pathway of human keratinocytes and in adult tissue reveals abnormal differentiation associated with certain stages of hyperproliferation. EPM-1 expression is part of developmental programs and is influenced by microenvironmental interactions and alterations of tissue homeostasis.

Differentiation-dependent expression of I and sialyl I antigens in the developing lung of human embryos and in lung cancers.

The localization of two carbohydrate antigens, I and sialyl I antigens, in the lungs of developing human embryos was investigated using specific monoclonal antibodies and compared with the distribution patterns of the known embryonic antigen, stage-specific embryonic antigen-1 (Lex hapten). When the future bronchi were actively developing from the bronchial buds in the lungs of 50- to 53-day-old embryos, the immature bronchial bud cells were I-, Lex+, while the fully differentiated epithelial cells of the larger bronchus were I+, Lex-. When the bronchiolar bud cells matured into bronchiolar epithelial cells in the lung of a 12-week-old embryo, the immature bronchiolar bud cells were I-,Lex+, while the fully differentiated epithelial cells of the bronchioles were I+,Lex-. Sialylated forms of the antigens finally appeared in the lungs of 18-week-old embryos, when the terminal bud cells actively proliferated and underwent the differentiation process into epithelial cells of alveoli and alveolar ducts. The immature terminal bud cells at this stage were I-, sialyl I-, Lex+, sialyl Lex-i+, while the fully differentiated alveolar epithelial cells were I+, sialyl I+, Lex-, sialyl Lex-i-. After 8 months, the flattened mature alveolar epithelial cells were strongly positive for both I and sialyl I antigens, the strong expression of which continued after birth and even into the adult stage. These distribution patterns indicate that the I and sialyl I antigens are specific markers for the differentiated type cells in each stage of development, while Lex and related embryonic antigens were specific to the immature bud cells in every stage. The above-described differentiation-dependent expression patterns of these antigens seem to be reflected in the distribution of these antigens in human lung cancers, i.e., I and sialyl I antigens were expressed in lung cancer cells more weakly than in normal lung cells, while the Lex and sialyl Lex-i were expressed in cancer cells much more strongly than in normal lung cells. This was further reflected in the serum levels of these antigens in the patients with respiratory disorders. The distribution pattern of the serum levels of these antigens in patients with lung cancers showed sialyl Lex-i greater than sialyl I, indicating that these serum antigens originated from the lung cancer lesion where sialyl Lex-i is much more dominant than sialyl I antigen.(ABSTRACT TRUNCATED AT 400 WORDS)

Maternal/fetal interactions: the role of the MHC class I molecule HLA-G.

The unique pattern of class I major histocompatibility complex (MHC) antigen expression seen at the human maternal/fetal interface is thought to be vital for fetal well-being. The lack of polymorphic class I and II MHC antigens on trophoblasts, the only fetal tissue in direct contact with the mother, is likely to be at least a part of the explanation of fetal evasion of allograft rejection. The recent observation that the HLA-G-encoded class I MHC molecule is present on certain subpopulations of cytotrophoblasts suggests that this nonpolymorphic molecule may have a role in the maternal/fetal immune response. Although no experimental evidence exists to support a particular function for HLA-G, reasoned speculation about the possible roles of this nonpolymorphic class I molecule is possible. Data derived from sequence analysis, analysis of HLA-G expression patterns, analysis of the extraembryonic expression patterns of other genes, and analysis of decidual lymphocyte phenotype and function provide insight into the possible functions of HLA-G at the maternal/fetal interface and are considered here.

Antibody repertoire development in fetal and neonatal piglets. V. VDJ gene chimeras resembling gene conversion products are generated at high frequency by PCR in vitro.

The recovery of VDJ rearrangement is most often accomplished by PCR amplification of DNA extracted from mixtures of B-cells. Using this procedure in swine, VDJs containing chimeric V(H) genes that resemble gene-conversion products, are frequently encountered. To examine whether these chimeras could be the result of PCR artifacts, we used different combinations of swine VDJ templates, each having unique CDR1, CDR2 and D(H) segments, to generate >2600 clones. Using equal amounts of two templates and 30 cycles of PCR, up to 45% of the resultant clones were VDJ chimeras. The frequency of chimeras was independent of the specific VDJ template and the chimeras were generated regardless of whether Taq-, Pfu- or mixtures of Taq- and Pfu-polymerases were employed or whether PCR extension time was prolonged six-fold. The frequency of generating chimeras was dependent on the ratio of the two target DNAs although even ratios approximately 1:10 generated approximately 10% chimeric VDJs. Chimeras could be generated using only 10 cycles of PCR or using the initial template DNAs diluted as much as 1:10000. Of the 279 chimeric VDJs generated, 61% of the crossovers occurred in FR3, 21% in FR2 and 18% in both FR2 and FR3. We interpret these results to mean that in vivo gene conversion in this species can only be unambiguously proven when the VDJs from individual B-cells are bearing a single VDJ rearrangement amplified and sequenced or when VDJs are cloned without the use of PCR.

Purification of immunologically active recombinant 21.5 kDa isoform of human myelin basic protein.

We have designed and expressed in bacteria a recombinant fetal form of human myelin basic protein (21.5 kDa isoform; rhMBP21.5), a candidate autoantigen in multiple sclerosis. An exon 2 insertion, carboxy-terminal histidine tag and preferred bacterial codons differentiate the MBP21.5 gene from that encoding the adult, brain-derived form of human MBP (18.5 kDa isoform; hMBP18.5). MBPs were expressed at high levels in E. coli and extracted from whole cells by simultaneous acid solubilization and mechanical disruption. A nearly two-fold increase in recombinant protein was detected in strains harboring MBP genes with bacterial preferred codons compared to genes containing human codons. The recombinant molecules were purified in two steps, first by reversed-phase chromatographic separation and then by metal affinity chromatography. Dimeric forms of recombinant MBP21.5 were detected under physiological conditions, however, substitution of a serine for the single cysteine at amino acid residue 81 resulted in only monomer formation. All forms of recombinant MBPs induced proliferative responses of human T lymphocytes specific for epitopes in MBP18.5 kDa. In contrast, human T cell lines that recognize an exon 2-encoded epitope of MBP responded to the 21.5 kDa isoform of MBP, but not the 18.5 kDa isoform.

CD5 antigen-positive B lymphocytes in human B cell ontogeny during fetal development and after autologous bone marrow transplantation.

We examined bone marrow and peripheral blood specimens from pediatric acute lymphoblastic leukemia (ALL) patients after autologous bone marrow transplantation (BMT) as well as fetal lymphohematopoietic tissues for the presence of CD5+ B lymphocytes. CD5+ B lymphocytes represented 23.6 +/- 0.7% of CD19+ fetal spleen B lineage lymphoid cells, 24.2 +/- 2.5% of CD19+ fetal bone marrow B lineage lymphoid cells and 18.1 +/- 1.7% of CD19+ fetal liver B lineage lymphoid cells. By comparison, in normal pediatric bone marrow samples, only 1.5 +/- 0.3% of lymphoid cells and 4.3 +/- 0.2% of CD19+ B lineage lymphoid cells expressed CD5 antigen. Similarly, very few CD5+CD19+ cells (< or = 2% of lymphoid cells) were found in day 30 and day 100 post-BMT bone marrow or peripheral blood specimens from B lineage ALL patients undergoing autologous BMT using autografts purged ex vivo with B43(anti-CD19)-PAP immunotoxin plus 4-hydroperoxycyclophosphamide (4-HC). Two bone marrow samples that were obtained and analyzed 1 year post-BMT contained only 2 to 4% CD5+CD19+ cells, accounting for 5 to 7% of the total CD19+ population. The fraction of CD5+ B lymphocytes in post-BMT bone marrow samples was not greater than the fraction of CD5+ B lymphocytes in normal healthy bone marrow.

The CD44 protein family.

The designation CD44 describes a group of type I transmembrane proteins which share N-terminal and C-terminal sequences. These molecules differ in the central extracellular domain by the use of sequences encoded by ten variant exons which may be completely absent or included in various combinations and by cell type specific addition of glycosaminoglycan and carbohydrate moieties. Expression of variant proteins is observed in normal tissues such as on keratinocytes, dendritic cells and activated lymphocytes in the adult organism and on morphogenetically active epithelium such as the apical ectodermal ridge (AER) in the embryo. Certain CD44 proteins expressed on the AER can act as low affinity fibroblast growth factor receptors and are vital for epithelial-mesenchymal cell communication. CD44 variant proteins have also been implicated in tumour growth and metastasis and we speculate that CD44 mediated growth factor presentation may also be decisive in metastasis formation. Molecular strategies designed to block growth factor presentation by CD44 may aid in the therapy of metastatic cancer.

B-cell development and differentiation.

B-cell development is characterized by antigen-independent phases in fetal hematopoietic organs and adult bone marrow, which lead to naive recirculating B cells in the periphery. The diversity of the naive (primary) B-cell repertoire resides in somatic events where minigenes from a large available repertoire are imprecisely rearranged and encode antigen-binding sites of antibodies. Naive B cells expand upon antigenic stimulation, acquire somatic mutations leading to high-affinity antibodies, and switch immunoglobulin (Ig) isotype in peripheral lymphoid organs. This immune reaction leads to the generation of memory B cells, which are long-lived and capable of rapid generation of specific high-affinity antibodies upon recall antigenic stimulation, as well as plasma cells, which constitutively secrete a large amount of antibodies.

Immunocytochemical analysis of a novel carbohydrate differentiation antigen (CDA-3C2) associated with olfactory and otic systems during embryogenesis in the rat.

Carbohydrate differentiation antigens are known to display specific patterns of expression during mammalian development and are thought to participate in significant morphogenetic events. In the present study, two monoclonal antibodies that react with a novel carbohydrate differentiation antigen (CDA-3C2) were used to analyze, by light microscopy, the spatiotemporal distribution of this unique high molecular weight antigen during embryogenesis in the rat. Correlative analysis of the development of peripheral neural structures, in which CDA-3C2 was expressed, was carried out with an anti-neurofilament antibody. Enzymatic digestion, combined with Western blots, reveal that the CDA-3C2 epitope is a carbohydrate which is carried on a high molecular weight glycoprotein with a mass of greater than 1 million Daltons. Characteristic of carbohydrate antigens, immunoreactivity was found in several distinct cellular patterns: only along the apical border of cells, along lateral and basal membranes of cells, and extracellular-like staining in the mesenchyme. During neurulation, CDA-3C2 showed differential staining in the ectoderm, distinguishing lateral from neural regions. Following closure of the neural tube, there was a striking specificity of expression of CDA-3C2 in the periphery, found almost exclusively in olfactory and otic epithelial structures. While CDA-3C2 is found in placode-derived tissues that subserve sensory transduction, it appears to be primarily associated with the supportive cells (and their secretions) in both otic and olfactory regions and less so with the sensory cells. The data suggest that a unique carbohydrate antigen on a large macromolecule may play a role in neurulation and/or morphogenesis of the placode-derived otic and olfactory structures.

Immunoelectron microscopic analysis of a novel carbohydrate differentiation antigen (CDA-3C2) in the developing rat olfactory and otic systems.

A carbohydrate differentiation antigen (CDA-3C2) exhibits a highly specific and restricted pattern of expression during rat embryogenesis. In the periphery of the embryo, this antigen is associated transiently with the lateral ectoderm but is retained only in the olfactory and otic epithelium throughout morphogenesis. At the light microscopic level, CDA-3C2 immunoreactivity appears mostly along cell periphery and in the extracellular matrix. The aim of the present study was to determine the specific cellular and subcellular distribution of CDA-3C2 in vivo in order to identify potential sites of cellular and tissue function of the antigen during embryogenesis. There was a strikingly similar subcellular distribution of CDA-3C2 in the developing otic and olfactory systems, found mostly along cell membranes, microvillar projections and acellular secretions of the epithelium. Mature sensory components of the epithelia were not immunoreactive, whereas supportive cells and their secreted structures were densely stained. The highly coincident nature of CDA-3C2 in both sensory epithelia suggests that this carbohydrate epitope, and possibly its carrier macromolecule, participate in a morphogenetic function common to these two sensory epithelia.

Transient expression of GABA immunoreactivity in the developing rat spinal cord.

The development of GABAergic neurons in the spinal cord of the rat has been investigated by immunocytochemical staining of frozen sections with anti-gamma-aminobutyric acid (GABA) antiserum. In the cervical cord, GABA-immunoreactive fibers first appeared at embryonic day (E) 13 in the presumptive white matter within the ventral commissure, ventral funiculus, and dorsal root entrance zone, and in the ventral roots. There were no GABA-immunoreactive cell bodies detected at this age. By E14, motoneurons, the earliest generated spinal cells, were the first cell population to become GABA-immunoreactive at the cell body level. Thereafter, GABA-immunoreactive neurons increased progressively in number and extended from ventral to dorsal regions. GABA-immunoreactive relay neurons within lamina I of the dorsal horn were initially detected at E17. Interneurons in the substantia gelatinosa, the latest generated cells in the spinal cord, were also the last to express the GABA immunoreactivity at E18. Immunoreactive neurons peaked in intensity and extent at E18 and 19. GABA immunoreactivity was only detectable in neurons within the intermediate and marginal zones 1-3 days after they withdrew from the cell cycle. This contrasts to glutamate decarboxylase immunoreactivity, which is detected in precursor cells in the ventricular zone prior to, or during, withdrawal from the cell cycle. Toward the end of gestation, GABA immunoreactivity declined in intensity and extent. This regression began in the ventral horn of the cervical region and ended in the dorsal horn of the lumbosacral region. During the first week after birth, immunoreactivity in motoneurons and in many other neurons within the ventral horn, intermediate gray, and deeper layers of the dorsal horn disappeared, and only in those neurons predominantly within the superficial layers of the dorsal horn did it persist into adulthood. Thus, the expression and regression of GABA immunoreactivity in the spinal cord followed ventral-to-dorsal, rostral-to-caudal, and medial-to-lateral gradients. These observations indicate that the majority of embryonic spinal neurons pass through a stage of transient expression of GABA immunoreactivity. The functional significance of this transient expression is unknown, but it coincides with the period of intense neurite growth of motoneurons, sensory neurons, and interneurons, and of neuromuscular junction formation, suggesting that the transient presence of GABA may play an important role in the differentiation of sensorimotor neuronal circuits.