Nathanael D. Pruett

Evidence that the satin hair mutant gene Foxq1 is among multiple and functionally diverse regulatory targets for Hoxc13 during hair follicle differentiation

It is increasingly evident that the molecular mechanisms underlying hair follicle differentiation and cycling recapitulate principles of embryonic patterning and organ regeneration. Here we used Hoxc13-overexpressing transgenic mice (also known as GC13 mice), known to develop severe hair growth defects and alopecia, as a tool for defining pathways of hair follicle differentiation. Gene array analysis performed with RNA from postnatal skin revealed differential expression of distinct subsets of genes specific for cells of the three major hair shaft compartments (cuticle, cortex, and medulla) and their precursors. This finding correlates well with the structural defects observed in each of these compartments and implicates Hoxc13 in diverse pathways of hair follicle differentiation. The group of medulla-specific genes was particularly intriguing because this included the developmentally regulated transcription factor-encoding gene Foxq1 that is altered in the medulladefective satin mouse hair mutant. We provide evidence that Foxq1 is a downstream target for Hoxc13 based on DNA binding studies as well as co-transfection and chromatin immunoprecipitation assays. Expression of additional medulla-specific genes down-regulated upon overexpression of Hoxc13 requires functional Foxq1 as their expression is ablated in hair follicles of satin mice. Combined, these results demonstrate that Hoxc13 and Foxq1 control medulla differentiation through a common regulatory pathway. The apparent regulatory interactions between members of the mammalian Hox and Fox gene families shown here may establish a paradigm for “cross-talk” between these two conserved regulatory gene families in different developmental contexts including embryonic patterning as well as organ development and renewal.

The Nude Mutant Gene Foxn1 Is a HOXC13 Regulatory Target during Hair Follicle and Nail Differentiation

Among the Hox genes, homeobox C13 (Hoxc13) has been shown to be essential for proper hair shaft differentiation, as Hoxc13 gene-targeted (Hoxc13(tm1Mrc)) mice completely lack external hair. Because of the remarkable overt phenotypic parallels to the Foxn1(nu) (nude) mutant mice, we sought to determine whether Hoxc13 and forkhead box N1 (Foxn1) might act in a common pathway of hair follicle (HF) differentiation. We show that the alopecia exhibited by both the Hoxc13(tm1Mrc) and Foxn1(nu) mice is because of strikingly similar defects in hair shaft differentiation and that both mutants suffer from a severe nail dystrophy. These phenotypic similarities are consistent with the extensive overlap between Hoxc13 and Foxn1 expression patterns in the HF and the nail matrix. Furthermore, DNA microarray analysis of skin from Hoxc13(tm1Mrc) mice identified Foxn1 as significantly downregulated along with numerous hair keratin genes. This Foxn1 downregulation apparently reflects the loss of direct transcriptional control by HOXC13 as indicated by our results obtained through co-transfection and chromatin immunoprecipitation (ChIP) assays. As presented in the discussion, these data support a regulatory model of keratinocyte differentiation in which HOXC13-dependent activation of Foxn1 is part of a regulatory cascade controlling the expression of terminal differentiation markers.

Krtap16, Characterization of a New Hair Keratin-associated Protein (KAP) Gene Complex on Mouse Chromosome 16 and Evidence for Regulation by Hoxc13

Intermediate filament (IF) keratins and keratin-associated proteins (KAPs) are principal structural components of hair and encoded by members of multiple gene families. The severe hair growth defects observed upon aberrant expression of certain keratin and KAP genes in both mouse and man suggest that proper hair growth requires their spatio-temporally coordinated activation. An essential prerequisite for studying these cis-regulatory mechanisms is to define corresponding gene families, their genomic organization, and expression patterns. This work characterizes eight recently identified high glycine/tyrosine (HGT)-type KAP genes collectively designated Krtap16-n. These genes are shown to be integrated into a larger KAP gene domain on mouse chromosome 16 (MMU16) that is orthologous to a recently described HGT- and high sulfur (HS)-type KAP gene complex on human chromosome 21q22.11. All Krtap16 genes exhibit strong expression in a narrowly defined pattern restricted to the lower and middle cortical region of the hair shaft in both developing and cycling hair. During hair follicle regression (catagen), expression levels decrease until expression is no longer detectable in follicles at resting stage (telogen). Since isolation of the Krtap16 genes was based on their differential expression in transgenic mice overexpressing the Hoxc13 transcriptional regulator in hair, we examined whether bona fide Hoxc13 binding sites associated with these genes might be functionally relevant by performing electrophoretic mobility shift assays (EMSAs). The data provide evidence for sequence-specific interaction between Hoxc13 and Krtap16 genes, thus supporting the concept of a regulatory relationship between Hoxc13 and these KAP genes.

Hoxc12 expression pattern in developing and cycling murine hair follicles

We examine the Hoxc12 RNA expression pattern during both hair follicle morphogenesis and cycling in direct comparison to its only upstream neighbor, Hoxc13. Expression of both genes is restricted to the epidermal part of the follicle excluding the outer root sheath and interfollicular epidermis in a distinct stage-dependent and cyclical manner. During the progressive growth phase (anagen) of developing and cycling follicles, the distinct proximo-distal expression domain of Hoxc12 overlaps only proximally, at the upper-most region of the bulb, with the more proximally restricted Hoxc13 domain. This arrangement of the expression domains of the two genes along the proximal-toward-distal axis of increasing follicular differentiation correlates with the sequential expression of first Hoxc13 and then Hoxc12. This indicates a reversal of the typical temporal colinearity of Hox gene activation otherwise observed along the anterior-posterior morphogenetic axis of the embryo (review: Cell 78 (1994) 191).

Changing topographic Hox expression in blood vessels results in regionally distinct vessel wall remodeling

Summary The distinct topographic Hox expression patterns observed in vascular smooth muscle cells (VSMCs) of the adult cardiovascular system suggest that these transcriptional regulators are critical for maintaining region-specific physiological properties of blood vessels. To test this proposition, we expanded the vascular Hoxc11 expression domain normally restricted to the lower limbs by utilizing an innovative integrated tetracycline regulatory system and Transgelin promoter elements to induce Hoxc11 expression universally in VSMCs of transgenic mice. Ectopic Hoxc11 expression in carotid arteries, aortic arch and descending aorta resulted in drastic vessel wall remodeling involving elastic laminae fragmentation, medial smooth muscle cell loss, and intimal lesion formation. None of these alterations were observed upon induction of Hoxc11 transgene expression in the femoral artery, i.e. the natural Hoxc11 activity domain, although this vessel was greatly enlarged, comparable to the topographically restricted vascular changes seen in Hoxc11−/− mice. To begin defining Hoxc11-controlled pathways of vascular remodeling, we performed immunolabeling studies in conjunction with co-transfection and chromatin immunoprecipitation (ChIP) assays using mouse vascular smooth muscle (MOVAS) cells. The results suggest direct transcriptional control of two members of the matrix metalloproteinase (Mmp) family, including Mmp2 and Mmp9 that are known as key players in the inception and progression of vascular remodeling events. In summary, the severe vascular abnormalities resulting from the induced dysregulated expression of a Hox gene with regional vascular patterning functions suggest that proper Hox function and regulation is critical for maintaining vascular functional integrity.

Dysregulated expression of sterol O-acyltransferase 1 (Soat1) in the hair shaft of Hoxc13 null mice

The cholesterol-metabolizing enzyme sterol O-acetyltransferase (SOAT1) is implicated in an increasing number of biological and pathological processes in a number of organ systems, including the differentiation of the hair shaft. While the functional and regulatory mechanisms underlying these diverse functional roles remain poorly understood, the compartment of the hair shaft known as medulla, affected by mutations in Soat1, may serve as a suitable model for defining some of these mechanisms. A comparative analysis of mRNA and protein expression patterns of Soat1/SOAT1 and the transcriptional regulator Hoxc13/HOXC13 in postnatal skin of FVB/NTac mice indicated co-expression in the most proximal cells of the differentiating medulla. This finding combined with the significant downregulation of Soat1 expression in postnatal skin of both Hoxc13 gene-targeted and transgenic mice based on previously reported DNA microarray results suggests a potential regulatory relationship between the two genes. Non-detectable SOAT1 expression in the defective hair follicle medulla of Hoxc13(tm1Mrc) mice and evidence for binding of HOXC13 to the Soat1 upstream control region obtained by ChIP assay suggests that Soat1 is a downstream regulatory target for HOXC13 during medulla differentiation.