A transgenic mouse approach using bacterial artificial chromosomes (BAC) was used to identify regulatory regions that direct Müllerian duct expression for Amhr2 and Osterix (Osx, also known as Sp7). Amhr2 encodes the receptor that mediates anti-Müllerian hormone (AMH) signaling for Müllerian duct regression in male embryos. Amhr2 is expressed in the Müllerian duct mesenchyme of both male and female embryos. A ∼147-kb BAC clone containing the Amhr2 locus was used to generate transgenic mice. The transgene was able to rescue the block in Müllerian duct regression of Amhr2-null males, suggesting that the BAC clone contains regulatory sequences active in male embryos. Osx is expressed in the developing skeleton of male and female embryos but is also an AMH-induced gene that is expressed in the Müllerian duct mesenchyme exclusively in male embryos. Osx-Cre transgenic mice were previously generated using a ∼204-kb BAC clone. Crosses of Osx-Cre mice to Cre-dependent lacZ reporter mice resulted in reporter expression in the developing skeleton and in the Müllerian duct mesenchyme of male but not female embryos. Osx-Cherry transgenic mice were previously generated using a 39-kb genomic region surrounding the Osx locus. Osx-Cherry embryos expressed red fluorescence in the developing skeleton and Müllerian duct mesenchyme of male but not female embryos. In addition, female Osx-Cherry embryos ectopically expressing human AMH from an Mt1-AMH transgene activated red fluorescence in the Müllerian duct mesenchyme. These results suggest that the 39-kb region used to generate Osx-Cherry contains male-specific Müllerian duct mesenchyme regulatory sequences that are responsive to AMH signaling. These BAC transgenic mouse approaches identify two distinct regions that direct Müllerian duct mesenchyme expression and contribute fundamental knowledge to define a gene regulatory network for sex differentiation.

The LIM class of homeodomain protein 3 (LHX3) transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie complex pediatric syndromes featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. Here we characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in α-glycoprotein subunit -expressing cells secreting the TSHβ, LHβ, or FSHβ hormones and expressing the GATA2 and steroidogenic factor 1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module active in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression. Furthermore, these studies revealed significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice also provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system.

Lin-11, Isl-1, and Mec-3 (LIM)-homeodomain (HD)-class transcription factors are critical for many aspects of mammalian organogenesis. Of these, LHX3 is essential for pituitary gland and nervous system development. Pediatric patients with mutations in coding regions of the LHX3 gene have complex syndromes, including combined pituitary hormone deficiency and nervous system defects resulting in symptoms such as dwarfism, thyroid insufficiency, infertility, and developmental delay. The pathways underlying early pituitary development are poorly understood, and the mechanisms by which the LHX3 gene is regulated in vivo are not known. Using bioinformatic and transgenic mouse approaches, we show that multiple conserved enhancers downstream of the human LHX3 gene direct expression to the developing pituitary and spinal cord in a pattern consistent with endogenous LHX3 expression. Several transferable cis elements can individually guide nervous system expression. However, a single 180-bp minimal enhancer is sufficient to confer specific expression in the developing pituitary. Within this sequence, tandem binding sites recognized by the islet-1 (ISL1) LIM-HD protein are essential for enhancer activity in the pituitary and spine, and a pituitary homeobox 1 (PITX1) bicoid class HD element is required for spatial patterning in the developing pituitary. This study establishes ISL1 as a novel transcriptional regulator of LHX3 and describes a potential mechanism for regulation by PITX1. Moreover, these studies suggest models for analyses of the transcriptional pathways coordinating the expression of other LIM-HD genes and provide tools for the molecular analysis and genetic counseling of pediatric patients with combined pituitary hormone deficiency.

Precise manipulation of gene expression with temporal and spatial control is essential for functional analysis and determining cell lineage relationships in complex biological systems. The cyclic recombinase (Cre)-loxP system is commonly used for gene manipulation at desired times and places. However, specificity is dependent on the availability of tissue- or cell-specific regulatory elements used in combination with Cre. Here, we present CreLite, an optogenetically controlled Cre system using red light in developing zebrafish embryos.

Female mice homozygous for an engineered Gnrhr E90K mutation have reduced gonadotropin-releasing hormone signaling, leading to infertility. Their ovaries have numerous antral follicles but no corpora lutea, indicating a block to ovulation. These mutants have high levels of circulating estradiol and low progesterone, indicating a state of persistent estrus. This mouse model provided a unique opportunity to examine the lack of cyclic levels of ovarian hormones on uterine gland biology. Although uterine gland development appeared similar to controls during prepubertal development, it was compromised during adolescence in the mutants. By age 20 weeks, uterine gland development was comparable to controls, but pathologies, including cribriform glandular structures, were observed. Induction of ovulations by periodic human chorionic gonadotropin treatment did not rescue postpubertal uterine gland development. Interestingly, progesterone receptor knockout mice, which lack progesterone signaling, also have defects in postpubertal uterine gland development. However, progesterone treatment did not rescue postpubertal uterine gland development. These studies indicate that chronically elevated levels of estradiol with low progesterone and therefore an absence of cyclic ovarian hormone secretion disrupts postpubertal uterine gland development and homeostasis.

Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.

Dlx5 and Dlx6 encode distal-less homeodomain transcription factors that are present in the genome as a linked pair at a single locus. Dlx5 and Dlx6 have redundant roles in craniofacial, skeletal, and uterine development. Previously, we performed a transcriptome comparison for anti-Müllerian hormone (AMH)-induced genes expressed in the Müllerian duct mesenchyme of male and female mouse embryos. In that study, we found that Dlx5 transcripts were nearly seven-fold higher in males compared to females and Dlx6 transcripts were found only in males, suggesting they may be AMH-induced genes. Therefore, we investigated the role of Dlx5 and Dlx6 during AMH-induced Müllerian duct regression. We found that Dlx5 was detected in the male Müllerian duct mesenchyme from E14.5 to E16.5. In contrast, in female embryos Dlx5 was detected in the Müllerian duct epithelium. Dlx6 expression in Müllerian duct mesenchyme was restricted to males. Dlx6 expression was not detected in female Müllerian duct mesenchyme or epithelium. Genetic experiments showed that AMH signaling is necessary for Dlx5 and Dlx6 expression. Müllerian duct regression was variable in Dlx5 homozygous mutant males at E16.5, ranging from regression like controls to a block in Müllerian duct regression. In E16.5 Dlx6 homozygous mutants, Müllerian duct tissue persisted primarily in the region adjacent to the testes. In Dlx5-6 double homozygous mutant males Müllerian duct regression was also found to be incomplete but more severe than either single mutant. These studies suggest that Dlx5 and Dlx6 act redundantly to mediate AMH-induced Müllerian duct regression during male differentiation.