The Lhx4 homeobox transcript in the rat pineal gland: Adrenergic regulation and impact on transcripts encoding melatonin‐synthesizing enzymes

Abstract

Homeobox genes generally encode transcription factors involved in regulating developmental processes. In the pineal gland, a brain structure devoted to nocturnal melatonin synthesis, a number of homeobox genes are also expressed postnatally; among these is the LIM homeobox 4 gene (Lhx4). We here report that Lhx4 is specifically expressed in the postnatal pineal gland of rats and humans. Circadian analyses revealed a fourfold rhythm in Lhx4 expression in the rat pineal gland, with rhythmic expression detectable from postnatal day 10. Pineal Lhx4 expression was confirmed to be positively driven by adrenergic signaling, as evidenced by in vivo modulation of Lhx4 expression by pharmacological (isoprenaline injection) and surgical (superior cervical ganglionectomy) interventions. In cultured pinealocytes, Lhx4 expression was upregulated by cyclic AMP, a second messenger of norepinephrine. By use of RNAscope technology, Lhx4 transcripts were found to be exclusively localized in melatonin-synthesizing pinealocytes. This prompted us to investigate the possible role of Lhx4 in regulation of melatonin-producing enzymes. By use of siRNA technology, we knocked down Lhx4 by 95% in cultured pinealocytes; this caused a reduction in transcripts encoding the melatonin-producing enzyme arylalkylamine N-acetyl transferase (Aanat). Screening the transcriptome of siRNA-treated pinealocytes by RNAseq revealed a significant impact of Lhx4 on the phototransduction pathway and on transcripts involved in development of the nervous system and photoreceptors. These data suggest that rhythmic expression of Lhx4 in the pineal gland is controlled via an adrenergic-cyclic AMP mechanism and that Lhx4 acts to promote nocturnal melatonin synthesis. K E Y W O R D Shomeobox, melatonin, pineal gland, RNA sequencing, RNAscope, siRNA 2 of 12 | HERTZ ET al.

The role of homeobox gene‐encoded transcription factors in regulation of phototransduction: Implementing the primary pinealocyte culture as a photoreceptor model

“…The transcriptomes of the photoreceptors of the retina and the pinealocyte of the pineal gland share a high degree of similarity. [8][9][10][11][12][15][16][17][18]20 Our systematic expression analyses in the current study confirm that the melatonin-producing enzymes and homeobox genes selected here are expressed in both the retina, pineal gland and cultured pinealocytes. However, while the phototransduction transcripts are known to be expressed in the retina and in the neonatal pineal gland, 6 the current study shows that genes representing all levels of the phototransduction cascade are expressed in the adult pineal gland.…”

“…We have previously shown that homeobox genes controlling rhythmic melatonin synthesis in the rat pineal gland exhibit 24 hour rhythms themselves. 20,31,36 To determine if rhythmic expression should be considered when analysing the role of homeobox genes in the postnatal retina, diurnal and circadian gene expression was analyzed in the retina and compared to that of the pineal gland (Figure 3; Tables 3,4). Our data confirmed a prominent 3-40-fold rhythmic expression of Aanat in both the retina and the pineal gland in LD and DD, as previously reported, 7,32 whereas minor and inconsistent rhythms were detected for acetylserotonin O-methyltransferase (Asmt) and tryptophan hydroxylase 1 (Tph1) transcripts encoding the other enzymes in melatonin synthesis (Figure 3A).…”

“…13,14 Large-scale screening efforts have identified a high degree of similarities between the mammalian retina and pineal gland transcriptomes 15 ; in this regard, the transcriptomes of the mature pineal gland and the retina also share a limited number of homeobox gene-encoded transcripts, including orthodenticle homeobox (Otx2), [16][17][18] cone-rod homeobox (Crx) [17][18][19] and LIM homeobox 4 (Lhx4). 20 Studies in knockout mice have shown that Otx2 and Crx are essential for retinal photoreceptor development, 16,21 but in both the retina and the pineal gland, expression of these transcription factors persists into adulthood. Recombinant CRX and OTX2 binds to, and in some cases also transactivate, promoters of photoreceptorspecific genes in reporter constructs, 19,22,23 but since these transcription factors also guide early developmental processes, evidence from in vivo biological systems is missing.…”

“…25,26 In addition, available immortalized cell lines related to photoreceptor cells express none or very few of the genes involved in phototransduction, 27,28 making it impossible to use them as a model for determining regulation of phototransduction. Prompted by a recent large-scale analyses of the role of homeobox genes in the pinealocyte of the pineal gland, 20,29 the purpose of the current study was to identify the possible role of homeobox gene-encoded transcription factors in regulation of phototransduction by implementing primary pinealocyte cultures as a cellular photoreceptor model.…”

Circadian regulation and molecular role of the Bsx homeobox gene in the adult pineal gland

“…However, as a special feature of the pineal gland, a number of homeobox genes continue to be expressed into adulthood or are even restricted to the postnatal gland . To this end, we have shown that certain pineal homeobox genes are involved in transcriptional regulation of the enzymes of the melatonin synthesis pathway and that rhythmic expression of these transcription factors is also controlled by the adrenergic input to the gland …”